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Viruses
Volume 13
Issue 2
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Viruses, Volume 13, Issue 2 (February 2021) – 213 articles

Cover Story (view full-size image): Clonal expansion of hepatocytes, a risk factor for the development of hepatocellular carcinoma (HCC), occurs in the liver during chronic hepatitis B virus infection. Hepatocytes are self-renewing, and the emergence of HCC from hepatocytes involves initiation (genetic) and promotion (cell proliferation). Genetic changes are due to random integration of HBV DNA and oxidative DNA damage caused by chronic inflammation. Promotion is due to replacement of hepatocytes killed by the immune response leading to loss of some hepatocyte lineages and expansion of others, including hepatocytes that no longer sustain virus replication. Shown here, as an example, is a focus of woodchuck hepatitis virus (WHV) core antigen-negative hepatocytes in the liver of a woodchuck with chronic WHV infection. View this paper
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18 pages, 4427 KiB  
Article
Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy
by Valeria Morante, Martina Borghi, Iole Farina, Zuleika Michelini, Felicia Grasso, Alessandra Gallinaro, Serena Cecchetti, Antonio Di Virgilio, Andrea Canitano, Maria Franca Pirillo, Roberta Bona, Andrea Cara and Donatella Negri
Viruses 2021, 13(2), 355; https://doi.org/10.3390/v13020355 - 23 Feb 2021
Cited by 19 | Viewed by 3656
Abstract
Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine [...] Read more.
Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy. Full article
(This article belongs to the Special Issue Lentiviral Vectors)
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Figure 1

Figure 1
<p>Therapeutic efficacy of integrase-defective lentiviral vectors (IDLV)-ovalbumin (OVA) vaccination in E.G7-OVA-bearing mice. (<b>a</b>) Scheme of the experiment: 14 C57BL/6 mice were inoculated s.c. with 3.5 × 10<sup>6</sup> E.G7-OVA cells/mouse. Mice with 7–9 mm diameter tumor mass were vaccinated with 3 × 10<sup>6</sup> RT/mouse of IDLV-OVA (<span class="html-italic">n</span> = 7) or left untreated (Naïve, <span class="html-italic">n</span> = 7). Tumor growth, survival, and immune response were monitored over time. Tumor-free mice were injected a second time with tumor cells at 175 days and monitored up to the end of the experiment (300 days). (<b>b</b>) Tumor growth after the first tumor injection is shown. Mice were sacrificed when the tumor diameter reached 15 mm or an ulceration of tumor was observed. (<b>c</b>,<b>d</b>) Kaplan–Meier survival curves are shown. Survival was monitored up to 175 days after the first tumor injection (Log-rank Mantel-Cox test) and up to 125 days from the second tumor injection (Log-rank Mantel-Cox test). (<b>e</b>) Kinetics of OVA-specific T cell response in IDLV-OVA vaccinated mice, after E.G7-OVA injection (red arrows). Blood cells were collected at the indicated time points and stimulated with the H-2Kb restricted OVA 8mer peptide (SIINFEKL). Data are expressed as specific spot forming cells (SFC) per 10<sup>6</sup> cells. Error bars indicate the standard deviation among the animals from the same group. (<b>f</b>) Polyfunctional OVA-specific CD8+ T cells. Vaccinated and tumor-free mice were sacrificed at 300 days and splenocytes were used to evaluate the magnitude and quality of OVA-specific CD8+ T cell response by intracellular cytokine staining (ICS). A representative experiment is shown. CD8+ T cells were analyzed in splenocytes stimulated with OVA 8mer peptide (OVApep). The percentage of CD8+ T cells producing IFNγ and TNFα and expressing CD107a is indicated within the quadrants.</p> Full article ">Figure 2
<p>Therapeutic efficacy of IDLV-OVA vaccination in B16OVA-bearing mice. (<b>a</b>) Scheme of the experiment. C57BL/6 mice were s.c. injected with 2 × 10<sup>5</sup> B16OVA cells/mouse. After 12 days, groups of mice were vaccinated either with 10 × 10<sup>6</sup> RT/mouse of IDLV-OVA or IDLV expressing an unrelated antigen (mock) or left untreated (Naïve). (<b>b</b>) OVA-specific T cell response was generated in all IDLV-OVA immunized mice, as evaluated by IFNγ ELISpot measured two weeks after immunization in blood cells (left panel) and at sacrifice in splenocytes (right panel). Cells were collected and stimulated with H-2Kb restricted OVA 8mer peptide (SIINFEKL). Data are expressed as spot forming cells (SFC) per million cells. (<b>c</b>) Tumor growth. All groups developed a tumor mass measured until the end of the experiment. The delay of tumor growth in IDLV-OVA immunized mice is highlighted with the red rectangle. Mock and Naïve groups were sacrificed within 18 days from tumor injection. (<b>d</b>) Kaplan–Meier survival curve. Mice with tumor diameter &gt;15 mm or a serious ulceration were sacrificed. (Log-rank Mantel-Cox test, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 3
<p>Confocal laser scanning microscopy (CLSM) analyses on mice tissue sections. Representative images of tumor from Naïve and IDLV-OVA mice are shown. Tissue sections 8 µm thick were stained for MHC Class I (green), OVA (green), or CD3 (red) as indicated (left columns) and for DAPI as nuclear staining (blue, right columns). Images represent a 3D reconstruction of 30–40 single Z-stack. Results from one representative experiment are shown for each analysis. Scale bars are 40 µm.</p> Full article ">Figure 4
<p>IDLV expressing mTRP2 as a self-antigen induces low specific T cells. (<b>a</b>) Expression of mTRP2 in cell lines. Mock Lenti-X cells, pTY2CMVmTRP2W-transfected Lenti-X cells and B16F10 cells were fixed and stained with anti-TRP2 antibody (green) and DAPI (blue) and analyzed by CLSM. Images represent a 3D reconstruction from 14 single optical sections. Results from one representative experiment are shown for each analysis. Scale bars are shown for each figure. (<b>b</b>) Pilot study of immunogenicity. C57Bl6 mice (<span class="html-italic">n</span> = 3) were immunized once with 10 × 10<sup>6</sup> RT units/mouse of either IDLV-mTRP2 or LV-mTRP2. The TRP2-specific T cell-mediated immune response was evaluated by IFNγ-ELISpot assay at two and four weeks in blood. Blood cells were stimulated with H-2Kb restricted TRP2 9mer peptide (SVYDFFVWL). Data are expressed as specific spot forming cells (SFC) per million cells. Box plots show mean ± SEM and single values from each immunized mouse.</p> Full article ">Figure 5
<p>Expression of different TRP2 fusion proteins by Western blot. (<b>a</b>) Cell lysates of Lenti-X transfected with transfer vector expressing either mTRP2 (predicted size 70 kDa) or mTRP2 fused with CRT (Calreticulin, predicted size 123 kDa), hIi (human invariant chain, predicted size 109 kDa), or mIi (murine invariant chain, predicted size 106 kDa). Lenti-X cells transfected with mTRP2 wild type (5 × 10<sup>4</sup> cells, lane 1) and GFP transfer vectors (lanes 2 and 3) were used as positive and negative control, respectively. The assay was performed using 1.6 × 10<sup>5</sup> (lane 2–4–6–8) or 3.2 × 10<sup>5</sup> cells (lane 3–5–7–9). Red boxes indicate the band with the correct molecular weight. (<b>b</b>) Detection of mTRP2 in viral vector preparations. 1.5 × 10<sup>6</sup> RT units of LV-mTRP2 (lane 1), IDLV-GFP (lane 2), IDLV-mTRP2 (lane 3), IDLV-CRT-mTRP2 (lane 4), IDLV-hIi-mTRP2 (lane 5), IDLV-mIi-mTRP2 (lane 6) were analyzed.</p> Full article ">Figure 6
<p>Expression of murine and human TRP2 evaluated by Western blot. Left panel: Detection of TRP2 in cell lysates of Lenti-X cells (1 × 10<sup>5</sup>) transfected with plasmids expressing either mTRP2 or hTRP2. Lenti-X and B16F10 cell lysates were used as negative and positive control of TRP2 expression, respectively. Right panel: Detection of TRP2 in concentrated vector preparations (3 × 10<sup>6</sup> RT/lane). The rabbit anti-TRP2 polyclonal antibody used to detect the expression of TRP2 recognizes both human and murine TRP2 proteins.</p> Full article ">Figure 7
<p>Antitumor efficacy of lentiviral vectors expressing either murine or human TRP2. (<b>a</b>) Scheme of the experiment. C57BL/6 mice (<span class="html-italic">n</span> = 9–11) were immunized with vectors expressing TRP2, OVA (Mock) or left untreated. After 30 days all mice were s.c. injected with 5 × 10<sup>4</sup> B16F10 cells/mouse. (<b>b</b>) Tumor growth was monitored over time. All groups developed a tumor mass measured until the end of the experiment. Mock and Naïve groups were sacrificed within 32 days from tumor injection. (<b>c</b>) Survival was monitored over time and Kaplan–Meier survival curve is shown. Mice with tumor diameter &gt;15 mm or a serious ulceration were sacrificed. (Log-rank Mantel-Cox test, *** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 8
<p>Kinetics of TRP2-specific immune responses in mice immunized with IDLV delivering either murine or human TRP2 and challenged with B16F10 tumor. C57BL/6 mice (<span class="html-italic">n</span> = 9–11) immunized with vectors expressing TRP2, OVA, or left untreated were s.c. injected with 5 × 10<sup>4</sup> B16F10 cells/mouse after 30 days from immunization, as depicted in <a href="#viruses-13-00355-f007" class="html-fig">Figure 7</a>a. (<b>a</b>) TRP2-specific T cell response was evaluated by IFNγ ELISPOT, measured at different time points in blood. Cells were collected and stimulated with H-2Kb restricted TRP2 9mer peptide (SVYDFFVWL). Data are expressed as mean spot forming cells (SFC) per 10<sup>6</sup> cells, bars represent standard error among animals from the same group. (<b>b</b>) Anti-mTRP2 IgG antibodies were analyzed in plasma of immunized animals by ELISA. Data are expressed as mean of endpoint titers and bars represent standard error among animals from the same group. Comparison among groups was evaluated using the Mann–Whitney test, as indicated by <span class="html-italic">p</span> values shown in the tables under the graphs. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; **** <span class="html-italic">p</span> &lt; 0.0001. NA: The comparison was not performed since only one animal from IDLVmTRP2 group was alive at day 42.</p> Full article ">Figure 9
<p>Confocal laser scanning microscopy (CLSM) analyses on mice tissue sections. Representative images of tumor from naïve, LV-mTRP2, IDLV-mTRP2, and IDLV-hTRP2 mice are shown. Tissue sections 8 µm thick were stained for MHC–I (green), TRP2 (green), or CD3 (red) as indicated (left columns) and for DAPI as nuclear staining (blue, right columns). Images represent a 3D reconstruction of 30–40 single Z-stack. Results from one representative experiment are shown for each analysis. Scale bars are indicated.</p> Full article ">
11 pages, 1940 KiB  
Article
Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro
by Sébastien Pasquereau, Zeina Nehme, Sandy Haidar Ahmad, Fadoua Daouad, Jeanne Van Assche, Clémentine Wallet, Christian Schwartz, Olivier Rohr, Stéphanie Morot-Bizot and Georges Herbein
Viruses 2021, 13(2), 354; https://doi.org/10.3390/v13020354 - 23 Feb 2021
Cited by 91 | Viewed by 7058
Abstract
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug [...] Read more.
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19. Full article
(This article belongs to the Special Issue Drug-Repositioning Opportunities for Antiviral Therapy)
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Figure 1

Figure 1
<p>Inhibition of replication of high HCoV-229E viral load (Red), measured by plaque forming unit (PFU) assay, coupled with MTT toxicity assay (Blue) for Lopinavir/Ritonavir (<b>A</b>), Chloroquine (<b>B</b>), Resveratrol (<b>C</b>), Disulfiram (<b>D</b>), Loperamide (<b>E</b>), Rapamycin (<b>F</b>) and valproic acid (VPA) (<b>G</b>). Cells were treated by compounds at the time of infection with HCoV-229E (1 multiplicity of infection (MOI)). PFU and MTT assays were performed after 48 h. Uninfected cells were used for normalization of MTT assay. Untreated cells infected with HCoV-229E were used for normalization of PFU assay.</p> Full article ">Figure 2
<p>Inhibition of replication of low HCoV-229E viral load (0.01 MOI) by drugs. (<b>A</b>) No pretreatment (Blue) or 3-h pretreatment of cells (Red). (<b>B</b>) Combination of drugs. The cells were treated with Lopinavir/Ritonavir (LR), Chloroquine (Ch), Resveratrol (Re) or left untreated (UT) for 48 h. The viral replication was measured by PFU assay.</p> Full article ">Figure 3
<p>Inhibition of replication of high HCoV-229E viral load (1 MOI) by Resveratrol (<b>A</b>), Lopinavir/Ritonavir (<b>B</b>) and Chloroquine (<b>C</b>) in pre-treated (3 h, Red) or post-treated (4 h, Blue) MRC5 cells. Viral replication was measured by PFU assay after 48 h.</p> Full article ">Figure 4
<p>(<b>A</b>) Toxicity assay for Lopinavir/Ritonavir, Chloroquine and Resveratrol in Vero E6 cells, after 48 h. (<b>B</b>) Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in Vero E6 cells treated with Resveratrol as measured by qRT-PCR after 48 h. <b>*</b> Cytotoxicity impaired the evaluation of the anti-SARS-CoV-2 effect of Lopinavir/Ritonavir and Chloroquine.</p> Full article ">
30 pages, 4963 KiB  
Review
Recent Advances in Bunyavirus Glycoprotein Research: Precursor Processing, Receptor Binding and Structure
by Ruben J. G. Hulswit, Guido C. Paesen, Thomas A. Bowden and Xiaohong Shi
Viruses 2021, 13(2), 353; https://doi.org/10.3390/v13020353 - 23 Feb 2021
Cited by 45 | Viewed by 6768
Abstract
The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a [...] Read more.
The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry. Full article
(This article belongs to the Special Issue Bunyavirus 2020)
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Figure 1
<p>Families belonging to the <span class="html-italic">Bunyavirales</span> order. Polymerase sequences of a representative set of bunyaviruses were aligned using MAFFT (Multiple Alignment using Fast Fourier Transform) [<a href="#B20-viruses-13-00353" class="html-bibr">20</a>], the alignment was trimmed using trimAI [<a href="#B21-viruses-13-00353" class="html-bibr">21</a>] and a phylogenetic tree created in PhyML [<a href="#B22-viruses-13-00353" class="html-bibr">22</a>] using the Smart Model Selection option [<a href="#B23-viruses-13-00353" class="html-bibr">23</a>]. The unrooted tree was displayed using iTOL (interactive Tree of Life) [<a href="#B24-viruses-13-00353" class="html-bibr">24</a>]. Asterisks indicate main branches for which bootstrap support was &lt;0.7. The dataset used for the phylogeny includes all currently assigned bunyavirus families [<a href="#B1-viruses-13-00353" class="html-bibr">1</a>], the grouping of which is broadly in line with that presented in a recently published tree [<a href="#B25-viruses-13-00353" class="html-bibr">25</a>]. The numbered, black lines in the figure denote bunyavirus species currently not assigned to a family [Wuhan spider virus (<b>1</b>), Laurel lake virus (<b>2</b>), Hubei blood fluke virus 2 (<b>3</b>), Hubei blood fluke virus 1 (<b>4</b>), Mothra virus (<b>5</b>), Shistocephalus solidus bunya-like virus (<b>6</b>)]. Common organisms from which the viruses were isolated are indicated by symbols and viruses known to be capable of zoonotic transmission to humans are annotated with red symbols (the panel at the right denotes what the symbols represent).</p> Full article ">Figure 2
<p>Genomic structure and coding strategies for the viral genomic RNA segments coding for viral glycoprotein precursors (GPCs) from well-studied families within the <span class="html-italic">Bunyavirales</span> order. Genomic RNAs (3’ to 5’) are represented by black lines (the numbers of nucleotides and the amino acid of GPC are given above). mRNAs are shown as arrows (♦ indicates host-derived primer sequence at 5’ end by cap-snatching mechanism). Gene products are presented by bars with their approximate size shown underneath. The sites for cleavage are represented by “<b><span style="color:red">|</span></b>”. Virus abbreviations: HTNV, Hantaan virus; HEBV, Herbet virus; UUKV, Uukuniemi virus; EMARaV, European mountain ash ringspot-associated virus; BUNV, Bunyamwera virus; RVFV, Rift Valley fever virus; FERV, Fermo virus; CCHFV, Crimean-Congo haemorrhagic fever virus; TSWV, Tomato spotted wilt virus; RSV, rice stripe virus; LASV, Lassa virus. (1) The M segments of hantaviruses (<span class="html-italic">Hantaviridae</span>) [<a href="#B33-viruses-13-00353" class="html-bibr">33</a>], herbeviruses (<span class="html-italic">Peribunyaviridae</span>) [<a href="#B34-viruses-13-00353" class="html-bibr">34</a>], Uukuviruses (e.g., UUKV) (<span class="html-italic">Phenuiviridae</span>) [<a href="#B35-viruses-13-00353" class="html-bibr">35</a>], and the RNA2 segment of the emaraviruses (<span class="html-italic">Fimoviridae</span>) [<a href="#B36-viruses-13-00353" class="html-bibr">36</a>] encode GPCs containing two structural glycoproteins, Gn and Gc. (2) The M segments of orthobunyaviruses (<span class="html-italic">Peribunyaviridae</span>) [<a href="#B11-viruses-13-00353" class="html-bibr">11</a>] encode three proteins, with an NSm located between Gn and Gc in the precursor protein. (3) The M segments of phleboviruses (e.g., RVFV) (<span class="html-italic">Phenuiviridae</span>) [<a href="#B37-viruses-13-00353" class="html-bibr">37</a>] and orthophasmaviruses (e.g., Ferak virus [FRKV] and jonchet virus, [JONV] (<span class="html-italic">Phasmaviridae</span>) encode GPCs containing three proteins: Gn and Gc, and an N-terminal NSm [<a href="#B38-viruses-13-00353" class="html-bibr">38</a>,<a href="#B39-viruses-13-00353" class="html-bibr">39</a>]. (4) The M segment of nairoviruses (e.g., CCHFV) (<span class="html-italic">Nairoviridae</span>) encodes a GPC with five proteins: Gn and Gc, and three non-structural proteins; Mucin like protein/domain (MLD), GP38, and NSm [<a href="#B40-viruses-13-00353" class="html-bibr">40</a>,<a href="#B41-viruses-13-00353" class="html-bibr">41</a>]. The M segments of other members of the family encode precursors for two to four proteins whose exact nature has yet to be confirmed experimentally [<a href="#B42-viruses-13-00353" class="html-bibr">42</a>]. (5) The ambisense M segments of orthotospoviruses (<span class="html-italic">Tospoviridae</span>) [<a href="#B29-viruses-13-00353" class="html-bibr">29</a>] and RNA2 segments of tenuiviruses (<span class="html-italic">Phenuiviridae</span>) encode GPCs containing Gn and Gc in the antigenomic sense, and an NSm in the genomic sense [<a href="#B43-viruses-13-00353" class="html-bibr">43</a>]. (6) The ambisense S segments of members of the <span class="html-italic">Hartmanivirus</span>, <span class="html-italic">Mammarenavirus</span> and <span class="html-italic">Reptarenavirus</span> genera (<span class="html-italic">Arenaviridae</span> family) encode a so-called stable signal peptide (SSP) and the glycoproteins GP1 and GP2 in the genomic sense, and a nucleocapsid protein in the antigenomic sense [<a href="#B26-viruses-13-00353" class="html-bibr">26</a>]. Similarly, the M segment of Wēnlǐng frogfish arenaviruses (<span class="html-italic">Antennavirus</span> genus) encode their GPCs in the genomic sense and an unknown protein in the anti-genomic sense [<a href="#B44-viruses-13-00353" class="html-bibr">44</a>].</p> Full article ">Figure 3
<p>Current models of bunyaviral GPC processing for selected peribunyaviruses, hantaviruses, phenuiviruses, nairoviruses, and arenaviruses. GPC polyproteins are shown for BUNV (<b>A</b>), HTNV (<b>B</b>), RVFV (<b>C</b>), CCHFV (<b>D</b>) and LASV (<b>E</b>). The signal peptide (SP) and stable SP (SPP, for LASV) are represented in orange. Transmembrane domains (TM) are shown as black boxes. The cleavage sites for signal peptidase (SPase) and signal peptide peptidase (SPP) are marked by scissors and an arrowhead (➣), respectively. The SKI-1/S1P cleavage site is marked by a downward arrow. Nonstructural proteins (NSm and GP38) are shown as grey boxes and the CCHFV mucin domain as a grey and white box. The pre-Gc N-terminal 41 residues that are processed at the RKPR/RKPL motif by a SKI-1/S1P-like protease are shown as green box.</p> Full article ">Figure 4
<p>Envelope glycoprotein ultrastructure of orthobunya-, hanta-, phlebo- and arenavirus particles. (<b>A</b>) <span class="html-italic">Left panel</span>: an EM reconstruction of an orthobunyaviral envelope glycoprotein (in grey; EMD-2352<sup>7</sup>) obtained using Bunyamwera (BUNV) particles, to which the trimeric BUNV Gc head domain structure (PDB: 6H3V<sup>8</sup>) was fitted, in side view. The center of the tripodal organization is linked together by the membrane-distal, N-terminal extension of the Gc. The different regions of the Gn-Gc spike complex (head, stalk, floor) are indicated on the left with corresponding heights. The head and stalk regions make up the N-terminal extension of the peribunyaviral Gc glycoprotein, whereas the floor region is thought to represent the Gn ectodomain and the fusogenic C-terminal region of the Gc ectodomain. <span class="html-italic">Middle panel</span>: zoomed-in surface representation of the Gc head domain trimer. Each of the protomers is colored in a different shade of blue. <span class="html-italic">Right panel</span>: Ribbon presentation of the middle panel. (<b>B</b>) Top views of the three upper panels. <span class="html-italic">Left panel</span>: the different threefold symmetry axes are indicated by open or solid triangles. (<b>C</b>) <span class="html-italic">Left panel</span>: EM density corresponding to the region of a phleboviral envelope Gn-Gc complex that exhibits five-fold symmetry (in grey; EMD-4201 [<a href="#B151-viruses-13-00353" class="html-bibr">151</a>]), obtained using Rift Valley fever virus (RVFV) particles. RVFV Gn and Gc crystal structures were fitted into this this pentameric RVFV envelope glycoprotein capsomer (PDB: 6F9F [<a href="#B151-viruses-13-00353" class="html-bibr">151</a>]), in side view. N-terminal regions of the Gn (membrane distal) are indicated in shades of purple and pink and the Gc ectodomain (membrane proximal) in shades of blue. Glycoprotein EM density is shown in grey. <span class="html-italic">Middle panel</span>: surface representation of the pentameric organization of Gn/Gc heterodimers. <span class="html-italic">Right panel</span>: Ribbon presentation of the middle panel. (<b>D</b>) Top views of the three upper panels. <span class="html-italic">Left panel</span>: the fivefold symmetry axis is indicated by a solid pentagon. (<b>E</b>) <span class="html-italic">Left panel</span>: the EM density of a hantaviral envelope Gn-Gc complex (in grey; EMD-11236, [<a href="#B154-viruses-13-00353" class="html-bibr">154</a>]) obtained using Tula virus (TULV) particles to which a model of tetrameric (Gn-Gc)<sub>4</sub> ANDV spike (PDB: 6ZJM, [<a href="#B154-viruses-13-00353" class="html-bibr">154</a>]) was fitted, in side view. ANDV Gn is indicated in shades of purple and pink and ANDV Gc in shades of blue. <span class="html-italic">Middle panel</span>: surface representation of the tetrameric organization of Gn/Gc heterodimers. <span class="html-italic">Right panel</span>: ribbon presentation of the middle panel. (<b>F</b>) Top views of the three upper panels. <span class="html-italic">Left panel</span>: the fourfold symmetry axis is indicated by a solid square. (<b>G</b>) <span class="html-italic">Left panel</span>: EM density of a trimeric arenavirus envelope glycoprotein spike (in grey; EMD-3290 [<a href="#B155-viruses-13-00353" class="html-bibr">155</a>]) obtained using Lassa virus (LASV) particles to which the trimeric LASV GP1-GP2 envelope glycoprotein (PDB: 5VK2 [<a href="#B156-viruses-13-00353" class="html-bibr">156</a>]) was fitted, in side view. GP1 protomers (membrane distal) are indicated in shades of purple and pink and GP2 protomers (membrane proximal) in shades of blue. Glycoprotein EM density is shown in grey. <span class="html-italic">Middle panel</span>: zoomed-in surface representation of the trimeric organization of LASV GP1/GP2 heterodimers. <span class="html-italic">Right panel</span>: Ribbon presentation of the middle panel. (<b>H</b>) Top views of the three upper panels. <span class="html-italic">Left panel</span>: the threefold symmetry axis is indicated by a solid triangle. In all structural representations crystallographically observed glycans are shown as white sticks. In the case of the RVFV Gn-Gc pentameric assembly, glycan chains were modelled onto N-linked glycosylation sites. The position of the viral membrane is shown as a yellow surface or two yellow dashed lines. To emphasize the protein components of the reconstructions, lipid bilayer EM densities were rendered at a lower sigma level than the protein surfaces. A color legend is shown on the top right-hand side of each panel.</p> Full article ">Figure 5
<p>Known structural features of bunyaviral Gn and Gc envelope proteins. (<b>A</b>) The Gn envelope glycoprotein displays limited structural similarity across bunyavirus families. Five crystal structures of Gn ectodomain regions of different bunyaviruses are shown. <span class="html-italic">Upper panel</span>: cartoon representation of the Gn N-terminal region of the ectodomain of the New World orthohantavirus ANDV (PDB: 6Y6P [<a href="#B154-viruses-13-00353" class="html-bibr">154</a>]), which displays a four-domain architecture (domain A, deep purple; a β-ribbon domain, purple-brown; domain B, warm pink; and a domain reminiscent of the alphavirus E3 protein, white). <span class="html-italic">Second panel</span>: cartoon representation of the Gn N-terminal region of the ectodomain of the Old World orthohantavirus PUUV (PDB: 5FXU [<a href="#B165-viruses-13-00353" class="html-bibr">165</a>]). <span class="html-italic">Third panel</span>: cartoon representation of the N-terminal region of the Gn ectodomain from RVFV (PDB: 6F8P [<a href="#B151-viruses-13-00353" class="html-bibr">151</a>]). <span class="html-italic">Fourth panel</span>: cartoon representation of the N-terminal region of the Gn ectodomain from SFTSV (PDB: 5Y10 [<a href="#B166-viruses-13-00353" class="html-bibr">166</a>]). Interestingly, SFTSV Gn contains a region reminiscent of the E3-like domain observed in hantavirus Gn proteins. <span class="html-italic">Bottom panel</span>: cartoon representation of the Gn ectodomain from TSWV (PDB: 6Y9L [<a href="#B167-viruses-13-00353" class="html-bibr">167</a>]). TSWV Gn displays a largely conserved three-domain architecture in which domain B is reduced to a β-hairpin. The C-terminal domain (CTD) comprises a β-sandwich domain (cyan) (please see <a href="#viruses-13-00353-f006" class="html-fig">Figure 6</a>). (<b>B</b>) Structurally characterized bunyaviral Gc fusion proteins display a conserved class II fusion protein architecture (domain I, light blue; domain II, slate blue; domain III, sky blue). Four crystal structures of the Gc ectodomain of different bunyaviruses are shown in a putative pre-fusion conformation (except SFTSV Gc for which a post-fusion state was determined). The dashed grey arrow indicates the movement of domain III between putative pre- and post-fusion conformations. Fusion loop(s) are indicated in bright orange. <span class="html-italic">Top panel</span>: crystal structure of the ANDV New World orthohantavirus Gc protein ectodomain in its pre-fusion conformation (PDB: 6Y5F [<a href="#B154-viruses-13-00353" class="html-bibr">154</a>]). <span class="html-italic">Second panel</span>: crystal structure of the Old World orthohantavirus PUUV Gc protein ectodomain in its pre-fusion conformation (PDB: 7B09 [<a href="#B168-viruses-13-00353" class="html-bibr">168</a>]). <span class="html-italic">Third panel</span>: crystal structure of the RVFV phlebovirus Gc protein ectodomain in its pre-fusion conformation (PDB: 4HJ1 [<a href="#B160-viruses-13-00353" class="html-bibr">160</a>]). <span class="html-italic">Bottom panel</span>: crystal structure of the SFTSV Gc protein ectodomain in its post-fusion conformation (PDB: 5G47 [<a href="#B169-viruses-13-00353" class="html-bibr">169</a>]). Note that the position of domain III has shifted from the tip of domain I in pre-fusion conformations towards the border of domains I and II in this post-fusion state. In all structural representations crystallographically observed glycans are shown as white sticks.</p> Full article ">Figure 6
<p>The C-terminal regions of ANDV Gn, TSWV Gn, and alphavirus E2 ectodomains have been shown to contain a seven-stranded β-sandwich fold. Rainbow cartoon representation of the aligned C-terminal β-sandwich domains of ANDV Gn, TSWV Gn and CHIKV E2. (<b>A</b>) The N-terminus of the displayed ANDV Gn structure (PDB: 6YRB, [<a href="#B154-viruses-13-00353" class="html-bibr">154</a>]) connects to the multi-domain globular region presented in <a href="#viruses-13-00353-f005" class="html-fig">Figure 5</a> and the C-terminus connects to an α-helical hairpin (grey cartoon), also known as the ‘base’ domain. (<b>B</b>) The N-terminus of the displayed TSWV Gn structure (PDB: 6Y9L, [<a href="#B167-viruses-13-00353" class="html-bibr">167</a>]) connects to the multi-domain globular region (grey cartoon, also presented in <a href="#viruses-13-00353-f005" class="html-fig">Figure 5</a>) and the C-terminus connects to the transmembrane domain. (<b>C</b>) The N-terminus of the displayed CHIKV E2 structure (PDB: 3N43, [<a href="#B170-viruses-13-00353" class="html-bibr">170</a>]) connects to a multi-domain globular region (partially shown by grey cartoon) and the C-terminus connects to the transmembrane domain. The N- and C-termini of the β-sandwich folds are colored blue and red, respectively, and the N- and C-termini of the structures are indicated.</p> Full article ">Figure 7
<p>Crystal structures of the orthobunyavirus Gc N-terminal region, CCHFV GP38, and arenavirus glycoproteins. (<b>A</b>) Crystal structure of the multi-domain N-terminal region of SBV Gc (head domain, light blue; stalk domain I, violet purple; stalk domain II, blue white) (PDB: 6H3S [<a href="#B153-viruses-13-00353" class="html-bibr">153</a>]). The N- terminus of the protein is indicated, as is the C-terminus that connects to the Gc protein. (<b>B</b>) Crystal structure of CCHFV GP38 protein (PDB: 6VKF [<a href="#B175-viruses-13-00353" class="html-bibr">175</a>]). The N- and C-termini of the protein are indicated. (<b>C</b>) Single protomer of the trimeric LASV GP crystal structure (PDB: 5VK2 [<a href="#B156-viruses-13-00353" class="html-bibr">156</a>]). The N- and C-termini of the GP1 and GP2 components are indicated. (<b>D</b>) Crystal structure of the trimeric post-fusion state of LASV GP2 (PDB: 5OMI [<a href="#B176-viruses-13-00353" class="html-bibr">176</a>]). N- and C-termini of the different GP2 protomers are indicated. In all structural representations crystallograhpically observed glycans are shown as white sticks.</p> Full article ">Figure 8
<p>Diverse class II fusion protein architectures converge on an evolutionary conserved trimeric post-fusion conformation. Schematic representation of class II fusion proteins in their pre-fusion oligomeric state. The schematic assemblies are based on crystal structures and/or cryoEM reconstructions (<span class="html-italic">Peribunyaviridae</span>: BUNV (EMD-2352 [<a href="#B163-viruses-13-00353" class="html-bibr">163</a>]) and SBV (PDB: 6H3V [<a href="#B153-viruses-13-00353" class="html-bibr">153</a>]); <span class="html-italic">Hantaviridae</span>: TULV (EMD-3364 [<a href="#B165-viruses-13-00353" class="html-bibr">165</a>]); <span class="html-italic">Phenuiviridae</span>: RVFV (EMD-4201 and PDB: 6F9F [<a href="#B151-viruses-13-00353" class="html-bibr">151</a>]); <span class="html-italic">Flaviviridae</span>: (PDB: 4UTC [<a href="#B189-viruses-13-00353" class="html-bibr">189</a>]); <span class="html-italic">Togaviridae</span>: Alphavirus eastern equine encephalitis virus (EEEV; PDB: 6MX4 [<a href="#B190-viruses-13-00353" class="html-bibr">190</a>]), Rubivirus RUBV (PDB: 4ADJ[<a href="#B191-viruses-13-00353" class="html-bibr">191</a>]); <span class="html-italic">C. elegans</span> (PDB: 4OJC [<a href="#B149-viruses-13-00353" class="html-bibr">149</a>]). The elongated structures of class II fusion proteins are shown as blue shapes (Gc for members of the <span class="html-italic">Bunyavirales</span>, E1 for <span class="html-italic">Togaviridae</span>, E for <span class="html-italic">Flaviviridae</span> and EFF-1 for the cellular <span class="html-italic">C. elegans</span> protein). Putative fusion protein stabilizing entities present on mature viral particles, are shown as purple shapes and have been hypothesized to prevent premature fusion activation (<span class="html-italic">Bunyavirales</span>: Gn, <span class="html-italic">Togaviridae</span>: E2). The E3 protein has been shown to be present in some alphavirus particles [<a href="#B170-viruses-13-00353" class="html-bibr">170</a>] but is omitted from this representation for clarity. The level of symmetry of each of the protein assemblies is indicated by symmetry symbols at the bottom right-hand corner. The approximate position of the fusion loop(s) is indicated with an asterisk (*) for each panel. In the case of peribunyaviruses, the exact location of the fusion loop (white asterisk) within the Gc protein is currently not known, but was inferred from the location of the N-terminal extensions within the tripodal EM reconstruction [<a href="#B153-viruses-13-00353" class="html-bibr">153</a>,<a href="#B163-viruses-13-00353" class="html-bibr">163</a>] and the C-terminal positioning of Gc transmembrane domains. Note that, although <span class="html-italic">C. elegans</span> EFF-1 (epithelial fusion failure 1) protein presents a class II fusogen architecture, it does not contain a fusion loop. Fusion is believed to be initiated by trimerization of the plasma membrane anchored EFF-1 ectodomains protruding in the extracellular space [<a href="#B149-viruses-13-00353" class="html-bibr">149</a>]. The grey region of the column shown for <span class="html-italic">Peribunyaviridae</span> represents the N-terminal extension of the Gc fusion protein, which has not been observed in other bunyavirus glycoproteins. The pre-fusion oligomeric state of EFF-1 has been observed to be monomeric on the plasma membrane [<a href="#B192-viruses-13-00353" class="html-bibr">192</a>]. The pre-fusion oligomeric state of rubella virus E1 on the virus membrane is currently unknown and therefore represented as a protomer of an unknown oligomeric assembly. The fusion proteins of alpha- (e.g., Semliki Forest virus (SFV), chikungunya virus (CHIKV)) and flaviviruses (e.g., dengue virus (DENV), zika virus (ZIKV)) are structurally related despite a lack of detectable sequence conservation and are therefore positioned next to each other in the diagram. Similarly, phenuivirus Gc has been shown to be genetically more closely related to the fusion envelope (E) proteins of flaviviruses than to those of other genera in its own order [<a href="#B152-viruses-13-00353" class="html-bibr">152</a>]. These proteins are placed next to each other to represent this predicted relationship. The box depicting the cellular EFF-1 protein is colored in yellow as to oppose the boxes in different shades of blue which all contain viral fusion proteins.</p> Full article ">
17 pages, 2629 KiB  
Article
Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies
by Harrison C. Bergeron, Jackelyn Murray, Ana M. Nuñez Castrejon, Rebecca M. DuBois and Ralph A. Tripp
Viruses 2021, 13(2), 352; https://doi.org/10.3390/v13020352 - 23 Feb 2021
Cited by 24 | Viewed by 5966
Abstract
Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the [...] Read more.
Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine. Full article
(This article belongs to the Special Issue Respiratory Syncytial Virus)
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Figure 1

Figure 1
<p>Rational design and expressiom of RSV G protein immunogens. (<b>A</b>) RSV G protein central conserved domain (CCD) (cyan) with CX3C motif highlighted, and sites of anti-G protein mAb binding: 2D10 (orange), 3G12 (yellow), and 3D3 (magenta). Serine 177 mutations modeled with (<b>B</b>) glutamine and (<b>C</b>) arginine. (<b>D</b>) Coomassie-stained SDS-PAGE gel of RSV G protein immunogens at ~90kDa. Lane 1: wild-type, lane 2: CX4C, lane 3: 177R, lane 4: 177Q.</p> Full article ">Figure 2
<p>Outline of vaccination and challenge scheme. Mice were i.p. vaccinated with 10 μg/immunogen + 10 μg MPLA on days 0, 28, and 60. Mice were i.n. challenged (D72) with 10<sup>6</sup> PFU RSV/A2 and sera were collected prior to and 7 days post-challenge (D79).</p> Full article ">Figure 3
<p>Immunogen vaccination induces anti-RSV IgG. Antibody levels were detected by indirect anti-RSV ELISA specific for total IgG (<b>A</b>,<b>D</b>); IgG1 (<b>B</b>,<b>E</b>); IgG2a (<b>C</b>,<b>F</b>) on days 0 (<b>A–C</b>) and day 7 (<b>D–F</b>) post-challenge. Graphs are representative of three independent experiments. Data represents the mean value of 3 experiments subtracted from the background. Bars represent the mean OD450 + SEM. <span class="html-italic">n</span>= 3–5 animals per group. Data were analyzed by one-way ANOVA tests where * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 compared to adjuvant-only group.</p> Full article ">Figure 4
<p>RSV G protein enriched sera. Antibody levels were detected by indirect anti-RSV IgG ELISA. Sera from <span class="html-italic">n</span> = 3–5 animals/group were panned against F protein to remove anti-F Abs prior to analysis by indirect ELISA. Bars represent the mean + SEM. Data were analyzed by one-way ANOVA tests where * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001 compared to adjuvant only group in three independent experiments.</p> Full article ">Figure 5
<p>CX3CR1.293 cells abundantly express CX3CR1. Representative histogram (<b>A</b>) and percentages (<b>B</b>) of CX3CR1 expression in 293 (black) and CX3CR1.293 (gray) cells after staining with anti-CX3CR1-Alexa647. 20,000 events were collected. Bars represent the mean of three independent experiments + SEM where * <span class="html-italic">p</span> &lt; 0.001 by two-tailed T test.</p> Full article ">Figure 6
<p>FKN and G protein CX3C binding to CX3CR1 in the presence or absence of heparin. 20 nM FKN (CX3CL1) (<b>A</b>) or 500 nM G protein (<b>B</b>) was pre-incubated with or without 5 μg/mL heparin to block non-specific binding. FKN binding was observed using Streptavidin-PE. RSV G protein was observed using anti-G protein mAb (clone 130-5F) followed by secondary anti-mouse conjugated to AlexaFluor-488. 20,000 events were collected. Bars represent the mean of three independent experiments + SEM analyzed by one-way ANOVA where ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001, ns (no significance).</p> Full article ">Figure 7
<p>RSV G protein CX3C-CX3CR1 binding is inhibited by serum IgG from G protein immunogen vaccinated mice. Purified serum IgG from G protein immunogen or adjuvant vaccinated mice was examined for the ability to inhibit (<b>A</b>) FKN or (<b>B</b>) G protein CX3C binding to CX3CR1. IgG was co-incubated with FKN or G protein and 5 μg/mL heparin to block non-specific binding. Ligand-specific binding and percent inhibition was determined by: [1 − (% Alexa Fluor-488+ CX3CR1.293<sup>+</sup> cells treated with ligand + antibody mixture) / (% Alexa Fluor-488+ CX3CR1.293 cells treated with ligand + normal mouse IgG)] × 100, as previously described. Bars represent mean of at least three independent experiments + SEM. Analysis by one-way ANOVA compared to adjuvant-only control where, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 **** <span class="html-italic">p</span> &lt; 0.00001.</p> Full article ">Figure 8
<p>Disease markers in challenged mice. Mice were vaccinated with G protein immunogens and challenged with 10<sup>6</sup> PFU RSV/A2. (<b>A</b>) Mice were weighed daily from days 0–12 post-challenge. Each shape represents the mean weight change from starting weight in each group each day. (<b>B</b>) Pulmonary leuokcyte infiltrates were collected seven days post-challenge. Graph indicates the mean BAL cells/mL + SEM. * <span class="html-italic">p</span> &lt; 0.05 by one-way ANOVA compared to adjuvant-only group. (<b>C</b>) BAL cells were pooled and analyzed by flow cytometry. Percent granulocytes were determined using SSC<sup>hi</sup> singlets. Twenty-thousand events were collected on LSR-II. (<b>D</b>) Lungs of challenged mice were collected on days 3 and 7 post-challenge and evaluated for RSV M protein transcripts by qRT-PCR. Data represent the mean of triplicate experiments. Standard curve was generated with a known concentration of RSV/A2 and two-way ANOVA was performed.</p> Full article ">
11 pages, 1186 KiB  
Article
BK Polyomavirus Micro-RNAs: Time Course and Clinical Relevance in Kidney Transplant Recipients
by Baptiste Demey, Véronique Descamps, Claire Presne, Francois Helle, Catherine Francois, Gilles Duverlie, Sandrine Castelain and Etienne Brochot
Viruses 2021, 13(2), 351; https://doi.org/10.3390/v13020351 - 23 Feb 2021
Cited by 19 | Viewed by 2925
Abstract
Background: Kidney transplant recipients (KTRs) are exposed to a high risk of BK polyomavirus (BKPyV) replication, which in turn may lead to graft loss. Although the microRNAs (miRNAs) bkv-miR-B1-3p and bkv-miR-B1-5p are produced during the viral cycle, their putative value as markers of [...] Read more.
Background: Kidney transplant recipients (KTRs) are exposed to a high risk of BK polyomavirus (BKPyV) replication, which in turn may lead to graft loss. Although the microRNAs (miRNAs) bkv-miR-B1-3p and bkv-miR-B1-5p are produced during the viral cycle, their putative value as markers of viral replication has yet to be established. In KTRs, the clinical relevance of the changes over time in BKPyV miRNA levels has not been determined. Methods: In a retrospective study, we analyzed 186 urine samples and 120 plasma samples collected from 67 KTRs during the first year post-transplantation. Using a reproducible, standardized, quantitative RT-PCR assay, we measured the levels of bkv-miR-B1-3p and bkv-miR-B1-5p (relative to the BKPyV DNA load). Results: Detection of the two miRNAs had low diagnostic value for identifying patients with DNAemia or for predicting DNAuria during follow-up. Seven of the 14 KTRs with a sustained BKPyV infection within the first year post-transplantation showed a progressive reduction in the DNA load and then a rapid disappearance of the miRNAs. DNA and miRNA loads were stable in the other seven KTRs. Conclusions: After the DNA-based diagnosis of BKPyV infection in KTRs, bkv-miR-B1-3p and bkv-miR-B1-5p levels in the urine might be valuable markers for viral replication monitoring and thus might help physicians to avoid an excessive reduction in the immunosuppressive regimen. Full article
(This article belongs to the Special Issue BK Virus and Transplantation)
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<p>Receiver operating characteristic (ROC) curves for urine levels of bkv-miR-B1-3p (<b>a</b>), bkv-miR-B1-5p (<b>b</b>) and BKPyV DNA (<b>c</b>) in KTRs with (patients) vs. without (controls) BKPyV DNAemia at the time of sampling. AUC: area under the curve.</p> Full article ">Figure 2
<p>Changes over time of BKPyV markers (DNA, bkv-miR-B1-3p, bkv-miR-B1-5p) in urine (U) and plasma (P) among 14 patients who developed sustained BKPyV infection during the year after kidney transplantation. (<b>A</b>) Patients with a fall in the DNA load after immunosuppressant dose reduction. (<b>B</b>) Patients with a stable DNA load after immunosuppressant dose reduction.</p> Full article ">Figure 2 Cont.
<p>Changes over time of BKPyV markers (DNA, bkv-miR-B1-3p, bkv-miR-B1-5p) in urine (U) and plasma (P) among 14 patients who developed sustained BKPyV infection during the year after kidney transplantation. (<b>A</b>) Patients with a fall in the DNA load after immunosuppressant dose reduction. (<b>B</b>) Patients with a stable DNA load after immunosuppressant dose reduction.</p> Full article ">
12 pages, 1024 KiB  
Review
Antiviral Bioactive Compounds of Mushrooms and Their Antiviral Mechanisms: A Review
by Dong Joo Seo and Changsun Choi
Viruses 2021, 13(2), 350; https://doi.org/10.3390/v13020350 - 23 Feb 2021
Cited by 64 | Viewed by 9914
Abstract
Mushrooms are used in their natural form as a food supplement and food additive. In addition, several bioactive compounds beneficial for human health have been derived from mushrooms. Among them, polysaccharides, carbohydrate-binding protein, peptides, proteins, enzymes, polyphenols, triterpenes, triterpenoids, and several other compounds [...] Read more.
Mushrooms are used in their natural form as a food supplement and food additive. In addition, several bioactive compounds beneficial for human health have been derived from mushrooms. Among them, polysaccharides, carbohydrate-binding protein, peptides, proteins, enzymes, polyphenols, triterpenes, triterpenoids, and several other compounds exert antiviral activity against DNA and RNA viruses. Their antiviral targets were mostly virus entry, viral genome replication, viral proteins, and cellular proteins and influenced immune modulation, which was evaluated through pre-, simultaneous-, co-, and post-treatment in vitro and in vivo studies. In particular, they treated and relieved the viral diseases caused by herpes simplex virus, influenza virus, and human immunodeficiency virus (HIV). Some mushroom compounds that act against HIV, influenza A virus, and hepatitis C virus showed antiviral effects comparable to those of antiviral drugs. Therefore, bioactive compounds from mushrooms could be candidates for treating viral infections. Full article
(This article belongs to the Special Issue Antivirals for Newly Emerging Viral Diseases of Global Importance)
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<p>Inhibitory stages of bioactive compounds against herpes simplex virus.</p> Full article ">Figure 2
<p>Inhibitory mechanisms of antiviral bioactive compounds against non-enveloped viruses.</p> Full article ">
20 pages, 5269 KiB  
Article
Dengue Virus Serotype 2 Intrahost Diversity in Patients with Different Clinical Outcomes
by Maria Celeste Torres, Marcos Cesar Lima de Mendonça, Cintia Damasceno dos Santos Rodrigues, Vagner Fonseca, Mario Sergio Ribeiro, Ana Paula Brandão, Rivaldo Venâncio da Cunha, Ana Isabel Dias, Lucy Santos Vilas Boas, Alvina Clara Felix, Maira Alves Pereira, Luzia Maria de Oliveira Pinto, Anavaj Sakuntabhai, Ana Maria Bispo de Filippis and on behalf of ZikAction Consortium
Viruses 2021, 13(2), 349; https://doi.org/10.3390/v13020349 - 23 Feb 2021
Cited by 16 | Viewed by 3990
Abstract
Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. [...] Read more.
Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus’s dynamics within its human host. Full article
(This article belongs to the Special Issue Endemic Arboviruses)
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<p>Maximum likelihood phylogenetic tree of DENV-2 polyprotein. It was constructed in RaxML v8.2.8 under GTR + I+G substitution model (General Time Reversible with gamma distribution and invariant sites), and 1000 bootstrap replicates. Main nodes with more than 70% of replicate trees of the bootstrap test for which the taxa clustered together are denoted with a black star. Brazilian sequences obtained in this study are represented in the tree with an orange circle. Gt: genotype; SA: South America; CA: Central America; and BR1-4: Brazilian lineages 1 to 4.</p> Full article ">Figure 2
<p>Characteristics of the intrahost viral population of cases grouped by clinical category or immune response. (<b>A</b>) Variability along the viral genome according to the patients’ clinical outcome. The total number of iSNVs located in each gene along the viral genome was normalized by each gene’s length (total nucleotide positions) and the median value for each group plotted in the graph, with error bars representing the interquartile ranges. (*) Statistically-supported differences among clinical groups. (<b>B</b>) iSNV/LVs frequency distribution along the viral genome according to the clinical category. The dotted line represents the median frequency among all iSNV/LVs found within each group. (<b>C</b>) Percentage of synonymous, non-synonymous iSNVs, and LVs for each gene. Clinical categories are represented separately. The total amount of each variant class per gene was summed and then normalized by group size. (<b>D</b>–<b>F</b>) The same analysis was performed for cases grouped by patients’ immune response. Genome/polyprotein schemes in graphs A, C, D, and F are not scaled to genes real size, as are the schemes in graphs B and E. DF: dengue fever cases, WS: dengue with warning signs, SD: severe dengue cases, P: primary cases, S: secondary cases, SS: synonymous variants, NS: Non-synonymous variants, and LV: insertion/deletion variants.</p> Full article ">Figure 2 Cont.
<p>Characteristics of the intrahost viral population of cases grouped by clinical category or immune response. (<b>A</b>) Variability along the viral genome according to the patients’ clinical outcome. The total number of iSNVs located in each gene along the viral genome was normalized by each gene’s length (total nucleotide positions) and the median value for each group plotted in the graph, with error bars representing the interquartile ranges. (*) Statistically-supported differences among clinical groups. (<b>B</b>) iSNV/LVs frequency distribution along the viral genome according to the clinical category. The dotted line represents the median frequency among all iSNV/LVs found within each group. (<b>C</b>) Percentage of synonymous, non-synonymous iSNVs, and LVs for each gene. Clinical categories are represented separately. The total amount of each variant class per gene was summed and then normalized by group size. (<b>D</b>–<b>F</b>) The same analysis was performed for cases grouped by patients’ immune response. Genome/polyprotein schemes in graphs A, C, D, and F are not scaled to genes real size, as are the schemes in graphs B and E. DF: dengue fever cases, WS: dengue with warning signs, SD: severe dengue cases, P: primary cases, S: secondary cases, SS: synonymous variants, NS: Non-synonymous variants, and LV: insertion/deletion variants.</p> Full article ">Figure 3
<p>Interhost frequency of iSNV/LVs consistently detected among samples. On this graph, only those repNS-iSNV + UTR-iSNVs found in at least two different samples were considered for analysis. From left to right, F: DF cases, W: WS cases, S: SD cases; <span class="html-italic">p</span>: primary infection, and S: secondary infection. Colored-scale indicates the percentage of positive samples, with color intensity and numeric scale increasing with interhost frequency. Colored arrows indicate the most relevant variants within subgroups 2 (green), 3 (orange), and 4 (grey).</p> Full article ">Figure 4
<p>Natural selection strength assessment. The strength of host selection on virus populations was compared between clinical categories. Left: dN/dS ratio over all coding positions. dN/dS ratio of 1, is interpreted as evidence for neutral evolution (dotted line). dN/dS &gt; 1 represents positive selection, while dN/dS &lt; 1 represents a negative (purifying) selection. Right: Number of accumulated iLVs. In all cases, each dot represents a sample and the lines are the median with the IQR. (*) <span class="html-italic">p</span> &lt; 0.01; (****) <span class="html-italic">p</span> &lt; 0.0001; Ns: not significant.</p> Full article ">
2 pages, 188 KiB  
Editorial
Erasing the Invisible Line to Empower the Pandemic Response
by Nicola Decaro, Alessio Lorusso and Ilaria Capua
Viruses 2021, 13(2), 348; https://doi.org/10.3390/v13020348 - 23 Feb 2021
Cited by 4 | Viewed by 2604
Abstract
A challenging debate has arisen on the role of veterinary expertise in facing the SARS-CoV-2 pandemic. It seems totally unreasonable that in most countries, veterinary diagnostic and tracing forces were not deployed at the start to perform strategic tasks, which could have mitigated [...] Read more.
A challenging debate has arisen on the role of veterinary expertise in facing the SARS-CoV-2 pandemic. It seems totally unreasonable that in most countries, veterinary diagnostic and tracing forces were not deployed at the start to perform strategic tasks, which could have mitigated the outcome of this dramatic health emergency. Erasing the invisible line between human and veterinary virology will empower the response to future pandemics. Full article
(This article belongs to the Special Issue Animal Viruses: State-of-the-Art Research in Italy)
10 pages, 1994 KiB  
Article
Radioligand Assay-Based Detection of Antibodies against SARS-CoV-2 in Hospital Workers Treating Patients with Severe COVID-19 in Japan
by Hidenori Matsunaga, Akiko Makino, Yasuhiro Kato, Teruaki Murakami, Yuta Yamaguchi, Atsushi Kumanogoh, Yuichiro Oba, Satoshi Fujimi, Tomoyuki Honda and Keizo Tomonaga
Viruses 2021, 13(2), 347; https://doi.org/10.3390/v13020347 - 23 Feb 2021
Cited by 3 | Viewed by 3113
Abstract
This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff [...] Read more.
This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff of a general hospital providing treatment to patients with severe COVID-19 were assayed for SARS-CoV-2 nucleocapsid protein (N) IgG using RLA. Nine patients with COVID-19 who had been treated in inpatient settings and had already recovered were used as control subjects, and 186 blood donor samples obtained more than 10 years ago were used as negative controls. Four of the 1000 samples showed apparently positive results, and approximately 10 or more samples showed slightly high counts. Interestingly, a few among the blood donor samples also showed slightly high values. To validate the results, antibody examinations using ELISA and neutralizing antibody tests were performed on 21 samples, and chemiluminescence immunoassay (CLIA) was performed on 201 samples, both resulting in a very high correlation. One blood donor sample showed slightly positive results in both RLA and CLIA, suggesting a cross-reaction. This study showed that five months after the pandemic began in Japan, the staff of a general hospital with a tertiary emergency medical facility had an extremely low seroprevalence of the antibodies against SARS-CoV-2. Further investigation will be needed to determine whether the slightly high results were due to cross-reactions or a low titer of anti-SARS-CoV-2 antibodies. The quantitative RLA was considered sensitive enough to detect low titers of antibodies. Full article
(This article belongs to the Special Issue Antibody Responses to Viral Infections)
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<p>Verification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N protein using electrophoresis. A single band of approximately 50 kDa was recognized for SARS-CoV-2 N.</p> Full article ">Figure 2
<p>Dilution test. Eight samples from patients with coronavirus disease 2019 (COVID-19) were used with two-fold serial dilution.</p> Full article ">Figure 3
<p>Absorption test. Non-radiolabeled (cold) antigens were used to absorb specific antibodies.</p> Full article ">Figure 4
<p>Radioligand assay (RLA) results. Results obtained from all 13 assays were collated, including those from the hospital staff (<span class="html-italic">n</span> = 1000) and blood donors (<span class="html-italic">n</span> = 186). COV3, 4, and 7 were the positive controls.</p> Full article ">Figure 5
<p>Correlation of the results obtained using RLA with those obtained using ELISA and neutralization titers. The 21 selected samples consisted of nine samples from patients with COVID-19 and four apparently positive, six slightly high, and two negative samples from hospital staff. Correlation of the results obtained using RLA with anti-SARS-CoV-2 N determined using ELISA (<b>A</b>), anti-SARS-CoV-2 S determined using ELISA (<b>B</b>), and neutralization antibody titers (<b>C</b>). The correlation coefficients were 0.92, 0.87, 0.93 (Spearman’s rank correlation coefficient), respectively.</p> Full article ">Figure 6
<p>Correlation of the results obtained using RLA and CLIA. The 201 selected samples consisted of nine samples from patients with COVID-19, all 24 samples from the hospital staff having 1.0 or more index values by RLA, 167 negative samples from the hospital staff, and one blood donor sample with slightly high counts. The correlation coefficient was 0.92 among the 52 samples, excluding negative ones whose results were lower than 1.0 in CLIA (Spearman’s rank correlation coefficient). Arrow: Cut-off point recommended by manufacturers.</p> Full article ">
12 pages, 2162 KiB  
Article
Predominance of HBV Genotype B and HDV Genotype 1 in Vietnamese Patients with Chronic Hepatitis
by Nghiem Xuan Hoan, Mirjam Hoechel, Alexandru Tomazatos, Chu Xuan Anh, Srinivas Reddy Pallerla, Le Thi Kieu Linh, Mai Thanh Binh, Bui Tien Sy, Nguyen Linh Toan, Heiner Wedemeyer, C.-Thomas Bock, Peter G. Kremsner, Christian G. Meyer, Le Huu Song and Thirumalaisamy P. Velavan
Viruses 2021, 13(2), 346; https://doi.org/10.3390/v13020346 - 22 Feb 2021
Cited by 10 | Viewed by 3778
Abstract
Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n [...] Read more.
Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n = 115), liver cirrhosis (LC; n = 21), and hepatocellular carcinoma (HCC; n = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the “a” determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (n = 204) and 7% (n = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; n = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; n = 134), followed by HBV-C (31%; n = 64), HBV-D, and HBV-G (3%; n = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; n = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (n = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections. Full article
(This article belongs to the Section Animal Viruses)
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<p>Flow chart of the study design.</p> Full article ">Figure 2
<p>Association of enzyme and platelet levels with clinical diagnoses. Boxplots illustrate medians with 25 and 75 percentiles. (∙) <span class="html-italic">p</span>-values were calculated using the Kruskal–Wallis statistical test. Boxplots were created with SPSS (IBM Corp.). AST, aspartate amino transferase; ALT, alanine amino transferase; PLT, platelets.</p> Full article ">Figure 3
<p>Maximum likelihood phylogenetic tree of HBV S-gene fragment (309 nucleotides). The reference sequences were retrieved from GenBank and labelled with the HBV genotypes and accession numbers. Generated sequences were labelled with the country code, sample ID, and diagnosis (e.g., VN001CHB = Vietnam 001 chronic hepatitis B). Node values indicate statistical support by approximate likelihood ratio test (aLRT) in PhyML 3.0. The scale bar indicates nucleotide substitutions per site.</p> Full article ">Figure 4
<p>Maximum likelihood phylogenetic tree constructed using a fragment of the HDV genome (194 nucleotides). The reference sequences were retrieved from GenBank and labelled with the hepatitis delta virus (HDV) genotypes and the GenBank accession numbers. Sample sequences are labelled with the country code for Vietnam, the sample ID and the diagnosis. Node values indicate statistical support by approximate likelihood ratio test (aLRT) in PhyML 3.0. The scale bar indicates nucleotide substitutions per site.</p> Full article ">Figure 5
<p>Alignment of the HBV sequences containing vaccine escape mutations. AY303915.1 represents an HBV vaccine escape mutant containing the G145A substitution [<a href="#B42-viruses-13-00346" class="html-bibr">42</a>]. Sites of vaccine escape mutations (aa144 and aa145) are highlighted in red.</p> Full article ">
18 pages, 3618 KiB  
Article
Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques
by Petra Mooij, Marieke A. Stammes, Daniella Mortier, Zahra Fagrouch, Nikki van Driel, Ernst J. Verschoor, Ivanela Kondova, Willy M. J. M. Bogers and Gerrit Koopman
Viruses 2021, 13(2), 345; https://doi.org/10.3390/v13020345 - 22 Feb 2021
Cited by 7 | Viewed by 3268
Abstract
Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, [...] Read more.
Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection. Full article
(This article belongs to the Special Issue Non-human Primate Models of Viral and Autoimmune Diseases)
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<p>Virus replication in cynomolgus macaques after H5N1 influenza virus exposure. Virus load measured by RT-PCR in time in throat (<b>A</b>) and nose swabs (<b>B</b>) in animals that received virus by combined-route (left), aerosol (middle), or aerosol delivery with bronchoalveolar lavages (BAL) collection (right). (<b>C</b>) Total viral load in throat and nose calculated for each animal as area under the curve (AUC) divided by the number of days that the animal was in study. Symbols used for individual animals correspond with symbols used in line graphs A and B. (<b>D</b>) Virus in BAL measured at days 2, 4, and 7 after virus inoculation. Statistical analysis of differences in AUC in throat and nose swabs or in BAL was performed by unpaired <span class="html-italic">t</span>-test. NS, not significant.</p> Full article ">Figure 2
<p>Clinical score in cynomolgus macaques after H5N1 influenza virus exposure. The total clinical score is shown for each individual animal in time after infection with H5N1 by combined-route (upper left), aerosol (upper right), or aerosol delivery with BAL collection (lower left). Total clinical score, calculated for each animal as AUC divided by the number of days that the animal was in study is shown in the lower right graph. Symbols used for individual animals in the scatter plots correspond with symbols used in line graphs. NS, not significant.</p> Full article ">Figure 3
<p>Histologic analysis of the lungs. (<b>A</b>) Lung of animal C1 showing broncho-interstitial pneumonia with massive intra-alveolar and intra-bronchiolar edema (hematoxilin–eosin staining (HE) original magnification 200 X). Open arrow, bronchiolar lumen with mixed inflammatory cells and protein-rich edema fluid; asterisk, alveolar lumina with edema and inflammatory cells; closed triangle, alveolar septa expanded by inflammatory infiltrates and edema. (<b>B</b>) Lung of animal A1 showing bronchioles and the surrounding alveoli severely obliterated by the influx of mixed inflammatory cells, fibrin, cell debris, erythrocytes, and protein-rich edema fluid (HE, 200 X). Open arrow, bronchiolar lumen filled with mononuclear inflammatory cells and neutrophils; asterisk, alveolar walls expanded by inflammatory cells; closed triangle, alveolar lumina with fibrin and protein-rich edema; closed arrow, alveolar edema with mixed inflammatory cells. Inset: higher magnification of the inflammatory infiltrates (HE, 400 X). (<b>C</b>) Lung of animal AB2 17–days post infection showing minimal to mild, focal, resolving lesions (HE, 50X). Inset: higher magnification of focal area with residual chronic and regenerative lesions, including type II pneumocyte hyperplasia, mildly expanded alveolar septa by an increase amount of collagen and infiltration by a small number of macrophages and lymphocytes (HE, 200 X). (<b>D</b>) Lung of healthy control macaque (HE, 50 X).</p> Full article ">Figure 4
<p>Computed tomography (CT) score and body temperature. (<b>A</b>) CT score in time depicted for animals that received virus by combined-route (upper left), aerosol delivery (upper right), or aerosol delivery with BAL collection (lower left). Cumulative CT score, calculated for each animal by adding scores measured at day 2, 4, 7, and 14 and dividing these totals by the number of time points, is shown in the lower right graph. (<b>B</b>) Cumulative temperature increase, calculated as the AUC from the actually recorded temperature during 9 days after virus inoculation, minus the circadian temperature pattern recorded before virus exposure, and divided by the number of days that the animal was in study is shown for each individual animal. (<b>C</b>) CT score plotted against temperature. Symbols used for individual animals in the scatter plots correspond with symbols used in line graphs. Statistical analysis of differences in AUC was performed by unpaired <span class="html-italic">t</span>-test. NS, not significant. The correlation was calculated by Pearson correlation test. The black line represents interpolated data, as a linear curve.</p> Full article ">Figure 5
<p>CT images taken on day 4 after virus exposure. Shown are central coronal slices of the lungs on day 4 after virus inoculation of the animals that received virus by combined-route (<b>left row</b>), aerosol (<b>middle row</b>), or aerosol delivery with regular BAL collection (<b>right row</b>).</p> Full article ">Figure 6
<p>Changes in leukocyte subsets and activation in peripheral blood. (<b>A</b>) Peripheral blood lymphocyte and CD3 T-cell count, (<b>B</b>) monocyte count and percentage of monocytes expressing CD16, (<b>C</b>) percentage of CD4 or CD8 T-cells expressing CD69, and (<b>D</b>) percentage of CD4 or CD8 T-cells expressing Ki67 is shown for each individual animal in time for the animals that received combined-route virus inoculation (left side of each graph), aerosol delivery (middle of each graph), or aerosol delivery with BAL collection (right side of each graph). Individual animal numbers are the same for all graphs and only indicated in the upper left graph.</p> Full article ">Figure 7
<p>Cytokine and chemokine expression levels in serum. Shown are IL-6, CXCL10, CCL2, CXCL11, CCL3 and CCL4 in pg/mL in serum, for each individual animal in time for the animals that received combined-route virus inoculation (left side of each graph), aerosol delivery (middle of each graph), or aerosol delivery with BAL collection (right side of each graph). Individual animal numbers are the same for all graphs and only indicated in the upper left graph.</p> Full article ">
21 pages, 3050 KiB  
Review
Role of the Host Genetic Susceptibility to 2009 Pandemic Influenza A H1N1
by Gloria Pérez-Rubio, Marco Antonio Ponce-Gallegos, Bruno André Domínguez-Mazzocco, Jaime Ponce-Gallegos, Román Alejandro García-Ramírez and Ramcés Falfán-Valencia
Viruses 2021, 13(2), 344; https://doi.org/10.3390/v13020344 - 22 Feb 2021
Cited by 12 | Viewed by 5081
Abstract
Influenza A virus (IAV) is the most common infectious agent in humans, and infects approximately 10–20% of the world’s population, resulting in 3–5 million hospitalizations per year. A scientific literature search was performed using the PubMed database and the Medical Subject Headings (MeSH) [...] Read more.
Influenza A virus (IAV) is the most common infectious agent in humans, and infects approximately 10–20% of the world’s population, resulting in 3–5 million hospitalizations per year. A scientific literature search was performed using the PubMed database and the Medical Subject Headings (MeSH) “Influenza A H1N1” and “Genetic susceptibility”. Due to the amount of information and evidence about genetic susceptibility generated from the studies carried out in the last influenza A H1N1 pandemic, studies published between January 2009 to May 2020 were considered; 119 papers were found. Several pathways are involved in the host defense against IAV infection (innate immune response, pro-inflammatory cytokines, chemokines, complement activation, and HLA molecules participating in viral antigen presentation). On the other hand, single nucleotide polymorphisms (SNPs) are a type of variation involving the change of a single base pair that can mean that encoded proteins do not carry out their functions properly, allowing higher viral replication and abnormal host response to infection, such as a cytokine storm. Some of the most studied SNPs associated with IAV infection genetic susceptibility are located in the FCGR2A, C1QBP, CD55, and RPAIN genes, affecting host immune responses through abnormal complement activation. Also, SNPs in IFITM3 (which participates in endosomes and lysosomes fusion) represent some of the most critical polymorphisms associated with IAV infection, suggesting an ineffective virus clearance. Regarding inflammatory response genes, single nucleotide variants in IL1B, TNF, LTA IL17A, IL8, IL6, IRAK2, PIK3CG, and HLA complex are associated with altered phenotype in pro-inflammatory molecules, participating in IAV infection and the severest form of the disease. Full article
(This article belongs to the Special Issue Host Factors in Viral Infections)
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<p>(<b>a</b>) Publications with the words “influenza A H1N1” and “genetic susceptibility” published between January 2009 to May 2020. (<b>b</b>) Top 25 journals where the 119 articles were published.</p> Full article ">Figure 2
<p>Depicting word cloud showing main keywords in the bibliometric analysis.</p> Full article ">Figure 3
<p>The proposed mechanism to explain <span class="html-italic">IFITM3</span> rs12252 participation in influenza A virus (IAV) infection. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 4
<p>Graphical summary of genetic polymorphisms and their participation in IAV infection. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">
17 pages, 12650 KiB  
Article
Transcriptome Analysis of Responses to Dengue Virus 2 Infection in Aedes albopictus (Skuse) C6/36 Cells
by Manjin Li, Dan Xing, Duo Su, Di Wang, Heting Gao, Cejie Lan, Zhenyu Gu, Tongyan Zhao and Chunxiao Li
Viruses 2021, 13(2), 343; https://doi.org/10.3390/v13020343 - 22 Feb 2021
Cited by 7 | Viewed by 4121
Abstract
Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family, can cause dengue fever (DF) and more serious diseases and thus imposes a heavy burden worldwide. As the main vector of DENV, mosquitoes are a serious hazard. After infection, they [...] Read more.
Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family, can cause dengue fever (DF) and more serious diseases and thus imposes a heavy burden worldwide. As the main vector of DENV, mosquitoes are a serious hazard. After infection, they induce a complex host–pathogen interaction mechanism. Our goal is to further study the interaction mechanism of viruses in homologous, sensitive, and repeatable C6/36 cell vectors. Transcriptome sequencing (RNA-Seq) technology was applied to the host transcript profiles of C6/36 cells infected with DENV2. Then, bioinformatics analysis was used to identify significant differentially expressed genes and the associated biological processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to verify the sequencing data. A total of 1239 DEGs were found by transcriptional analysis of Aedes albopictus C6/36 cells that were infected and uninfected with dengue virus, among which 1133 were upregulated and 106 were downregulated. Further bioinformatics analysis showed that the upregulated DEGs were significantly enriched in signaling pathways such as the MAPK, Hippo, FoxO, Wnt, mTOR, and Notch; metabolic pathways and cellular physiological processes such as autophagy, endocytosis, and apoptosis. Downregulated DEGs were mainly enriched in DNA replication, pyrimidine metabolism, and repair pathways, including BER, NER, and MMR. The qRT-PCR results showed that the concordance between the RNA-Seq and RT-qPCR data was very high (92.3%). The results of this study provide more information about DENV2 infection of C6/36 cells at the transcriptome level, laying a foundation for further research on mosquito vector–virus interactions. These data provide candidate antiviral genes that can be used for further functional verification in the future. Full article
(This article belongs to the Section Invertebrate Viruses)
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<p>Viral growth kinetics of (<b>a</b>) and morphological changes in (<b>b</b>) C6/36 cells infected with DENV2. (<b>a</b>) The <span class="html-italic">Y</span>-axis label represents log10 (DENV2 RNA copies), and the <span class="html-italic">X</span>-axis label represents days post-infection with DENV2. (<b>b</b>) Mock refers to uninfected C6/36 cells (dpi: days post-infection). The magnification is 200 ×.</p> Full article ">Figure 2
<p>Volcano plot of differentially expressed genes. The red dots represent the 1133 upregulated genes; the blue dots represent the 106 downregulated genes; the gray dots represent the unchanged genes.</p> Full article ">Figure 3
<p>Bubble plots comparing GO enrichment and KEGG pathway analysis. (<b>a</b>) The 30 most significantly enriched GO terms for upregulated DEGs. (<b>b</b>) The 30 most significantly enriched GO terms for downregulated DEGs. (<b>c</b>) The 30 most significantly enriched KEGG pathways for upregulated DEGs. (<b>d</b>) The 30 most significantly enriched KEGG pathways for downregulated DEGs. The size of the bubble represents the number of enriched DEGs. The color represents the <span class="html-italic">p</span>-value of the enrichment. The shape of the bubble represents the ontology; circles represent BP, triangles represent CC, and squares represent MF.</p> Full article ">Figure 4
<p>qRT-PCR and transcriptome analysis of 13 differentially expressed genes. Mock means uninfected cells, and the FC values of these cells were set to 1. The relative expression level (FC) of a mRNA transcript refers to the change in expression of DENV2-infected C6/36 cells relative to uninfected cells determined by using the 2<sup>−ΔΔCT</sup> method.</p> Full article ">Figure 5
<p>Overlap analysis of the transcriptomes of C6/36 and Aag2 cells. Blue represents the transcriptome analysis results of Aag2 cells, and green represents the transcriptome analysis results of C6/36 cells. (<b>a</b>) The 16 overlapping DEGs between the Aag2 and C6/36 cell lines. (<b>b</b>) The 7 overlapping GO terms enriched for upregulated DEGs. (<b>c</b>) The 65 overlapping GO terms for downregulated DEGs. (<b>d</b>) The 23 overlapping KEGG pathways enriched for upregulated DEGs. (<b>e</b>) The 8 overlapping KEGG pathways for downregulated DEGs.</p> Full article ">
12 pages, 713 KiB  
Article
Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection
by Snjezana Zidovec-Lepej, Tatjana Vilibic-Cavlek, Ljubo Barbic, Maja Ilic, Vladimir Savic, Irena Tabain, Thomas Ferenc, Ivana Grgic, Lana Gorenec, Maja Bogdanic, Vladimir Stevanovic, Dario Sabadi, Ljiljana Peric, Tanja Potocnik-Hunjadi, Elizabeta Dvorski, Tamara Butigan, Krunoslav Capak, Eddy Listes and Giovanni Savini
Viruses 2021, 13(2), 342; https://doi.org/10.3390/v13020342 - 22 Feb 2021
Cited by 22 | Viewed by 3271
Abstract
Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification [...] Read more.
Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-?/IL-6), Th1 (IL-2/IFN-?), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-? were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-? and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50; serum median 24.48, IQR 11.93–49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-?/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-?) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-? appear to be concentrated mainly in the periphery. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Cytokine expression pattern (pg/mL) in serum (red box plots) and CSF (grey box plots) in patients with WNV infection. Boxes represent median and interquartile range. Error bars indicate the minimum and maximum values.</p> Full article ">
14 pages, 557 KiB  
Article
Maternal Infection and Adverse Pregnancy Outcomes among Pregnant Travellers: Results of the International Zika Virus in Pregnancy Registry
by Manon Vouga, Léo Pomar, Antoni Soriano-Arandes, Carlota Rodó, Anna Goncé, Eduard Gratacos, Audrey Merriam, Isabelle Eperon, Begoña Martinez De Tejada, Béatrice Eggel, Sophie Masmejan, Laurence Rochat, Blaise Genton, Tim Van Mieghem, Véronique Lambert, Denis Malvy, Patrick Gérardin, David Baud and Alice Panchaud
Viruses 2021, 13(2), 341; https://doi.org/10.3390/v13020341 - 22 Feb 2021
Cited by 2 | Viewed by 2998
Abstract
In this multicentre cohort study, we evaluated the risks of maternal ZIKV infections and adverse pregnancy outcomes among exposed travellers compared to women living in areas with ZIKV circulation (residents). The risk of maternal infection was lower among travellers compared to residents: 25.0% [...] Read more.
In this multicentre cohort study, we evaluated the risks of maternal ZIKV infections and adverse pregnancy outcomes among exposed travellers compared to women living in areas with ZIKV circulation (residents). The risk of maternal infection was lower among travellers compared to residents: 25.0% (n = 36/144) versus 42.9% (n = 309/721); aRR 0.6; 95% CI 0.5–0.8. Risk factors associated with maternal infection among travellers were travelling during the epidemic period (i.e., June 2015 to December 2016) (aOR 29.4; 95% CI 3.7–228.1), travelling to the Caribbean Islands (aOR 3.2; 95% CI 1.2–8.7) and stay duration >2 weeks (aOR 8.7; 95% CI 1.1–71.5). Adverse pregnancy outcomes were observed in 8.3% (n = 3/36) of infected travellers and 12.7% (n = 39/309) of infected residents. Overall, the risk of maternal infections is lower among travellers compared to residents and related to the presence of ongoing outbreaks and stay duration, with stays <2 weeks associated with minimal risk in the absence of ongoing outbreaks. Full article
(This article belongs to the Special Issue Emerging Virus Infections in Adverse Pregnancy Outcomes)
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<p>Flow chart. Abbreviations: WG, weeks’ gestation; ZIKV, Zika virus.</p> Full article ">
4 pages, 180 KiB  
Editorial
Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough
by Rami Kantor
Viruses 2021, 13(2), 340; https://doi.org/10.3390/v13020340 - 22 Feb 2021
Cited by 3 | Viewed by 2657
Abstract
Drug resistance remains a global challenge in the fight against the HIV pandemic [...] Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
18 pages, 12416 KiB  
Article
On the Prevalence and Potential Functionality of an Intrinsic Disorder in the MERS-CoV Proteome
by Manal A. Alshehri, Manee M. Manee, Fahad H. Alqahtani, Badr M. Al-Shomrani and Vladimir N. Uversky
Viruses 2021, 13(2), 339; https://doi.org/10.3390/v13020339 - 22 Feb 2021
Cited by 5 | Viewed by 2917
Abstract
Middle East respiratory syndrome is a severe respiratory illness caused by an infectious coronavirus. This virus is associated with a high mortality rate, but there is as of yet no effective vaccine or antibody available for human immunity/treatment. Drug design relies on understanding [...] Read more.
Middle East respiratory syndrome is a severe respiratory illness caused by an infectious coronavirus. This virus is associated with a high mortality rate, but there is as of yet no effective vaccine or antibody available for human immunity/treatment. Drug design relies on understanding the 3D structures of viral proteins; however, arriving at such understanding is difficult for intrinsically disordered proteins, whose disorder-dependent functions are key to the virus’s biology. Disorder is suggested to provide viral proteins with highly flexible structures and diverse functions that are utilized when invading host organisms and adjusting to new habitats. To date, the functional roles of intrinsically disordered proteins in the mechanisms of MERS-CoV pathogenesis, transmission, and treatment remain unclear. In this study, we performed structural analysis to evaluate the abundance of intrinsic disorder in the MERS-CoV proteome and in individual proteins derived from the MERS-CoV genome. Moreover, we detected disordered protein binding regions, namely, molecular recognition features and short linear motifs. Studying disordered proteins/regions in MERS-CoV could contribute to unlocking the complex riddles of viral infection, exploitation strategies, and drug development approaches in the near future by making it possible to target these important (yet challenging) unstructured regions. Full article
(This article belongs to the Section Animal Viruses)
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<p>Schematic representation of the computational analysis applied to the Middle East respiratory syndrome coronavirus (MERS-CoV) proteome to study different aspects of intrinsically disordered viral proteins. Protein sequences were retrieved from NCBI and subjected to several analyses: protein disorder prediction, molecular recognition feature (MoRF) prediction, amino acid composition, identification of protein interaction partners, and short linear motif (SLiM) prediction. In disorder and MoRF predictions, a probability score was given for each amino acid and any residue was considered as disordered/MoRF when the score was above 0.5/0.725, respectively.</p> Full article ">Figure 2
<p>Proportion of MERS-CoV proteins having different degrees of predicted disorder. Proteins were classified according to overall level of intrinsic disorder: highly ordered (PPID &lt; 10%), moderately disordered (10% ≥ PPID &lt; 30%), and highly disordered (PPID ≥ 30%). Predictions were made using seven different tools.</p> Full article ">Figure 3
<p>Percentages of disorder predicted in individual MERS-CoV proteins by seven tools: Espritz, IUPred-L, IUPred-S, PONDR-FIT, VL3, VLXT, and VSL2B. For each predictor, the mean predicted percentage of intrinsic disorder (PPID<sub>mean</sub>) was determined across 20 MERS-CoV genomes.</p> Full article ">Figure 4
<p>Positional distribution of predicted intrinsic disorder for proteins in the MERS-CoV genome. Each line graph represents the distribution of mean disorder probability calculated for a given protein by averaging the per-residue disorder profiles generated by IUPred-short, IUPred-long, PONDRFIT, VL3, VLXT, and VSL2B. Residues with scores above 0.5 are considered disordered.</p> Full article ">Figure 5
<p>Positional distribution of the predicted intrinsic disorder of individual proteins generated by the cleavage of ORF1ab polyproteins in the MERS-CoV genome. Each color represents a per-residue disorder profile generated by IUPred_Long, PONDRFIT, VLXT, IUPred_Short, VL3, or VSL2B. Residues with scores above 0.5 are considered disordered.</p> Full article ">Figure 6
<p>Positional distribution of predicted intrinsic disorder of individual proteins generated by the cleavage of ORF1a polyproteins in MERS-CoV genome. Each color represents a per-residue disorder profile generated by IUPred_Long, PONDRFIT, VLXT, IUPred_Short, VL3, or VSL2B. Residues with scores above 0.5 are considered disordered.</p> Full article ">Figure 7
<p>Disorder propensities of individual proteins generated by the cleavage of ORF1ab protein from MERS-CoV. Plots show the positions of cleavage sites within disorder profiles at the junctions between the cleaved products. (<b>A</b>) Cleavage site between host translation inhibitors nsp1 and nsp2. (<b>B</b>) Cleavage site between nsp2 and papain-like proteinase. (<b>C</b>) Cleavage site between papain-like proteinase and nsp4. (<b>D</b>) Cleavage site between nsp4 and 3C-like proteinase. (<b>E</b>) Cleavage site between 3C-like proteinase and nsp6. (<b>F</b>) Cleavage site between nsp6 and nsp7. (<b>G</b>) Cleavage site between nsp7 and nsp8. (<b>H</b>) Cleavage site between nsp8 and nsp9. (<b>I</b>) Cleavage site between nsp9 and nsp10. (<b>J</b>) Cleavage site between non-structural protein 11 and RNA-directed RNA polymerase. (<b>K</b>) Cleavage site between RNA-directed RNA polymerase and helicase. (<b>L</b>) Cleavage site between helicase and guanine-N7 methyltransferase. (<b>M</b>) Cleavage site between guanine-N7 methyltransferase and uridylate-specific endoribonuclease. (<b>N</b>) Cleavage site between uridylate-specific endoribonuclease and 2’-O-methyltransferase.</p> Full article ">Figure 8
<p>Compositional profiling of MERS-CoV proteins. Positive and negative values respectively correspond to enrichment and depletion of given residues within query proteins. Amino acids are represented as disorder-promoting (red), order-promoting (blue), or neutral (gray) and are ordered from the most depleted to the most enriched.</p> Full article ">Figure 9
<p>Molecular recognition features (MoRFs) predicted in MERS-CoV proteins using MoRFchibi. Positions with scores of 0.752 or greater are considered MoRF residues.</p> Full article ">
12 pages, 530 KiB  
Review
Development of Genome Editing Approaches against Herpes Simplex Virus Infections
by Isadora Zhang, Zoe Hsiao and Fenyong Liu
Viruses 2021, 13(2), 338; https://doi.org/10.3390/v13020338 - 22 Feb 2021
Cited by 10 | Viewed by 4780
Abstract
Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 [...] Read more.
Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases. Full article
(This article belongs to the Special Issue Pathogenesis and Novel Antiviral Targets of Alphaherpesviruses)
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<p>Illustration of the homing endonuclease (HE, meganuclease) and CRISPR/Cas9 System: (<b>a</b>) Homing endonuclease-mediated recognition and binding of a DNA substrate leads to the cleavage of the substrate.; (<b>b</b>) A custom-designed guide RNA (gRNA) hybridizes its target sequence and directs Cas9 endonuclease to cleave a DNA substrate.</p> Full article ">
11 pages, 2113 KiB  
Article
Examination of Staphylococcus aureus Prophages Circulating in Egypt
by Adriana Ene, Taylor Miller-Ensminger, Carine R. Mores, Silvia Giannattasio-Ferraz, Alan J. Wolfe, Alaa Abouelfetouh and Catherine Putonti
Viruses 2021, 13(2), 337; https://doi.org/10.3390/v13020337 - 22 Feb 2021
Cited by 7 | Viewed by 3710
Abstract
Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence [...] Read more.
Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton–Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>Prophage network of shared genes. Each node corresponds with a single predicted prophage sequence. The shape and color of the node represents the identified prophage cluster for the sequence. Two nodes are connected by an edge if they both share a common gene. The weight of the edge represents the number of common genes between two prophages.</p> Full article ">Figure 2
<p>Pangenome of Egyptian <span class="html-italic">S. aureus</span> prophages. Each ring in the graph represents an individual <span class="html-italic">S. aureus</span> prophage sequence, color coded according to the assigned prophage cluster. Each ray in the graph indicates the presence (darker coloration) or absence (lighter coloration) of a given homolog. The number of gene clusters (no. of CDS) and singleton genes (unique genes, i.e., no homologs within other prophage sequences) found within each prophage sequence are shown in the two bar charts.</p> Full article ">Figure 3
<p>Integrase phylogenetic tree. Representative sequences of the 12 <span class="html-italic">S. aureus</span> integrase types (Sa1int–Sa12int) are also included in the tree, shown in bold. Sa1int, Sa2int, Sa3int, and Sa7int branches are colored blue, green, red, and purple, respectively. Virulence factors are indicated for the Egyptian <span class="html-italic">S. aureus</span> prophage sequences and the Sa1int, Sa2int, Sa3int, and Sa7int reference sequences.</p> Full article ">Figure 4
<p>Phylogenetic tree of the terminase large subunit amino acid sequences. Prophage clusters are indicated as are the predicted genera (Biseptimaviruses, Phietaviruses, and Triaviruses).</p> Full article ">
12 pages, 1666 KiB  
Article
An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains
by Esther Shmidov, Itzhak Zander, Ilana Lebenthal-Loinger, Sarit Karako-Lampert, Sivan Shoshani and Ehud Banin
Viruses 2021, 13(2), 336; https://doi.org/10.3390/v13020336 - 21 Feb 2021
Cited by 3 | Viewed by 4573
Abstract
Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were [...] Read more.
Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>The targeted curing method principle. The prophage is bordered by the right attachment site <span class="html-italic">attR</span> (dark green) and the left attachment site <span class="html-italic">attL</span> (orange). The first step is <span class="html-italic">ampR</span> (red) insertion for prophage tagging. The second step is the integration of the plasmid backbone, containing <span class="html-italic">accC1</span> (gray) and <span class="html-italic">sacB</span> (light blue) into the prophage region via homologous recombination. Next, the temporal tagged prophage with the integrated <span class="html-italic">sacB</span> undergoes counter-selection by growing on the sucrose-containing growth medium. The counter-selection outcome can be either (<b>A</b>) unchanged tagged prophage, (<b>B</b>) integration site deletion (marked with an asterisk), (<b>C</b>) <span class="html-italic">sacB</span> mutated variant (asterisk), or (<b>D</b>) prophage curing. Notably, only option (<b>D</b>) would be Crb sensitive.</p> Full article ">Figure 2
<p>Pf4 curing verification. (A-C) Curing verification by PCR, Lanes 1–2 represent the DNA of randomly picked Crb-sensitive colonies, and lane 3 represents the WT PAO1 for control. (<b>A</b>) Amplification of 1800 bp around the Pf4 <span class="html-italic">attB</span> site. (<b>B</b>) Amplification of 1000 bp around the Pf4 <span class="html-italic">attR</span> site that can only be amplified in the integrated form. (<b>C</b>) Amplification of 750 bp around the Pf4 <span class="html-italic">attP</span> site that can only be amplified in the RF form. (<b>D</b>) Curing verification by plaque assay, serial dilutions of Pf4 phage are extracted from WT PAO1 strain used to infect WT PAO1 and ΔPf4 strain.</p> Full article ">Figure 3
<p>The ΔPf4 strain differs from WT in the Pf4 region. Genomic alignment with PAO1 strain is used as a reference, PAO1 WT sequence assembly is indicated in green, and ΔPf4 strain sequence assembly is indicated in blue. Prophage regions are labeled in red; coordinates are taken from PHASTER analysis to the reference PAO1 strain. The cured region is marked with an arrow. We note that there is a difference between our laboratory WT strain and the reference strain around region 2800 bp, but this difference is identical in the Pf4-mutant strain.</p> Full article ">Figure 4
<p>PR2 curing verification by PCR. (<b>A</b>) AmpRin region amplification results either in null, 2000 bp, or 1200 bp in the cured, <span class="html-italic">ampR</span>-containing, or WT strain, respectively; lanes 1–8 represent randomly picked Crb-sensitive colonies, lanes 9–10 represent Crb-resistant colonies, and the WT 39016 is for positive control. (<b>B</b>) Amplification of 2000 bp around the PR2 <span class="html-italic">attB</span> site of ΔPR2 and WT 39016, and positive control (ctrl) amplification of 324 bp PA39016_100004 external gene.</p> Full article ">Figure 5
<p>The ΔPR2 strain differs from WT in the PR2 region solely. Genomic alignment with 39016 strain is used as a reference, 39016 WT sequence assembly is indicated in green, and ΔPR2 strain sequence assembly is indicated in blue. Prophage regions are labeled in red; coordinates are taken from PHASTER analysis to the reference 39016 strain. The cured region is marked with an arrow.</p> Full article ">
15 pages, 719 KiB  
Review
Residual Proviral Reservoirs: A High Risk for HIV Persistence and Driving Forces for Viral Rebound after Analytical Treatment Interruption
by Xiaolei Wang and Huanbin Xu
Viruses 2021, 13(2), 335; https://doi.org/10.3390/v13020335 - 21 Feb 2021
Cited by 6 | Viewed by 3435
Abstract
Antiretroviral therapy (ART) has dramatically suppressed human immunodeficiency virus (HIV) replication and become undetectable viremia. However, a small number of residual replication-competent HIV proviruses can still persist in a latent state even with lifelong ART, fueling viral rebound in HIV-infected patient subjects after [...] Read more.
Antiretroviral therapy (ART) has dramatically suppressed human immunodeficiency virus (HIV) replication and become undetectable viremia. However, a small number of residual replication-competent HIV proviruses can still persist in a latent state even with lifelong ART, fueling viral rebound in HIV-infected patient subjects after treatment interruption. Therefore, the proviral reservoirs distributed in tissues in the body represent a major obstacle to a cure for HIV infection. Given unavailable HIV vaccine and a failure to eradicate HIV proviral reservoirs by current treatment, it is crucial to develop new therapeutic strategies to eliminate proviral reservoirs for ART-free HIV remission (functional cure), including a sterilizing cure (eradication of HIV reservoirs). This review highlights recent advances in the establishment and persistence of HIV proviral reservoirs, their detection, and potential eradication strategies. Full article
(This article belongs to the Special Issue Mechanisms of Viral Persistence)
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<p>Schematic overview of human immunodeficiency virus (HIV)/ simian immunodeficiency virus (SIV) life cycle and measurable viral parameters. Viral particles enter target cells, followed by reverse transcription, integration, transcription, splicing, translation, and virion packaging. The unspliced viral RNAs are transcribed from the integrated provirus. The transported single 5′ capped genomic viral RNAs (~9 Kb) are assembled to the nascent virions. Two or three guanosines 5′ full-length viral RNAs directly translate viral proteins (gag and pol). Rev-dependent export of incompletely spliced RNAs (~4 Kb) to cytoplasm contributes to env, vif, vpr, and vpu expression, whereas rev-independent multiply spliced species (~2 Kb) constitutively express accessory and regulatory proteins (tat, rev, and nef). Cell-associated viral RNA transcripts and viral DNA can be directly measured [<a href="#B18-viruses-13-00335" class="html-bibr">18</a>,<a href="#B19-viruses-13-00335" class="html-bibr">19</a>,<a href="#B20-viruses-13-00335" class="html-bibr">20</a>].</p> Full article ">Figure 2
<p>Representative levels and anatomical tissue distribution of cell-associated SIV RNA/DNA in SIV-infected macaques. Adult Indian-origin rhesus macaques (Macaca mulatta) were intravenously inoculated with 100 TCID50 SIVmac251. After 8 weeks, these animals received three anti-HIV drugs (TFV 20 mg/kg/day; FTC 30 mg/kg/day and DTG 2.5 mg/kg/day) for 20 months. The levels of cell-associated unspliced (US) SIV RNA (<b>A</b>), multiply spliced (MS) SIV tat/rev RNA (<b>B</b>), total SIV DNA (<b>C</b>), circular SIV 2-long terminal repeat (LTR) (<b>D</b>), and integrated proviral DNA (<b>E</b>), in blood, spleen, mesenteric lymph node, axillary lymph node, jejunum, and rectum from SIV-infected animals 3 months after ATI, reaching the levels prior to treatment. (<b>F</b>) Distribution of proviral reservoir in tissues examined. Note that integrated proviral DNA was predominantly distributed in peripheral blood and lymphoid compartments, and rapidly increased to pre-treatment levels after ATI. Cell-associated SIV RNA/DNA are expressed as copies per one-million cells. * <span class="html-italic">p</span> &lt; 0.01, compared with PBMCs.</p> Full article ">
16 pages, 1488 KiB  
Article
Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics
by Bahareh Lashtoo Aghaee, Mohammadali Khan Mirzaei, Mohammad Yousef Alikhani, Ali Mojtahedi and Corinne F. Maurice
Viruses 2021, 13(2), 334; https://doi.org/10.3390/v13020334 - 21 Feb 2021
Cited by 30 | Viewed by 4344
Abstract
Antibiotic resistance causes around 700,000 deaths a year worldwide. Without immediate action, we are fast approaching a post-antibiotic era in which common infections can result in death. Pseudomonas aeruginosa is the leading cause of nosocomial infection and is also one of the three [...] Read more.
Antibiotic resistance causes around 700,000 deaths a year worldwide. Without immediate action, we are fast approaching a post-antibiotic era in which common infections can result in death. Pseudomonas aeruginosa is the leading cause of nosocomial infection and is also one of the three bacterial pathogens in the WHO list of priority bacteria for developing new antibiotics against. A viable alternative to antibiotics is to use phages, which are bacterial viruses. Yet, the isolation of phages that efficiently kill their target bacteria has proven difficult. Using a combination of phages and antibiotics might increase treatment efficacy and prevent the development of resistance against phages and/or antibiotics, as evidenced by previous studies. Here, in vitro populations of a Pseudomonas aeruginosa strain isolated from a burn patient were treated with a single phage, a mixture of two phages (used simultaneously and sequentially), and the combination of phages and antibiotics (at sub-minimum inhibitory concentration (MIC) and MIC levels). In addition, we tested the stability of these phages at different temperatures, pH values, and in two burn ointments. Our results show that the two-phages-one-antibiotic combination had the highest killing efficiency against the P. aeruginosa strain. The phages tested showed low stability at high temperatures, acidic pH values, and in the two ointments. This work provides additional support for the potential of using combinations of phage–antibiotic cocktails at sub-MIC levels for the treatment of multidrug-resistant P. aeruginosa infections. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>Stability of phages in two burn-wound-care products at different temperatures and pHs. (<b>A</b>) Stability of three phages, 6, 32, and 45 in nitrofurazone and silver sulphadiazine over 24 h and (<b>B</b>) stability of phages at different temperatures and pHs. Bars connected by the same letter (a, b, c and d) are not significantly different (<span class="html-italic">p</span> &lt; 0.05, two-way ANOVA, Tukey’s multiple comparisons test); ND, not detected; Int, initial concentration. Error bars represents three biological replicates.</p> Full article ">Figure 2
<p>Genomes and phylogenetic trees of phages 6, 45, and 32. (<b>A</b>) The predicted coding sequences (CDSs) are indicated by arrows. CDSs predicted to encode structural proteins are indicated in blue, hypothetical proteins in grey, and nucleotide metabolism in green. Genes predicted to encode transfer RNA are indicated in red and yellow shows other proteins. (<b>B</b>–<b>D</b>) Phylogenetic tree showing the relationships of the major capsid, of three phages. (<b>E</b>) A single phylogenetic tree of head protein, tail protein, and terminase large subunit combined, of phages 6, 32, and 45. The tree was inferred by using the maximum likelihood (ML) method based on the Whelan and Goldman model [<a href="#B23-viruses-13-00334" class="html-bibr">23</a>]. The analyses were conducted in MEGA.</p> Full article ">Figure 3
<p>Transmission electron microscopy (TEM) of phages 6, 32, and 45. Phages were negatively stained with 2% uranyl acetate. Scale bars represent 100 nm.</p> Full article ">Figure 4
<p>Effect of phage and antibiotic treatment in killing a <span class="html-italic">P</span>. <span class="html-italic">aeruginosa</span> strain from a wound patient. (<b>A</b>) A single-step growth curve of three phages; (<b>B</b>–<b>D</b>) efficacy of phages 6, 32, and 45 against <span class="html-italic">P. aeruginosa</span> isolate #14 at different multiplicities of infection (MOIs); (<b>E</b>) effect of a two-phage cocktail (6 + 32 and 6 + 45) against <span class="html-italic">P. aeruginosa</span> isolate #14; (<b>F</b>) effect of phage 6 in combination with two different antibiotics (gentamicin and ciprofloxacin) on <span class="html-italic">P. aeruginosa</span> isolate #14. PA#14, <span class="html-italic">P. aeruginosa</span> isolate #14. Points connected by the same letter (a or b) are not significantly different; asterisks show significant differences (<span class="html-italic">p</span> &lt; 0.05, two-way ANOVA, Tukey’s multiple comparisons test). Error bars represent three biological replicates. Four different MOIs: 0.01, 0.1, 1, and 10 were used for panels B, C, and D, while a MOI of 1 was used for panels E and F. Bacteria-only controls are in blue; antibiotic + bacteria controls are in brown. Dashed lines in panels B, C, and D represent one common control.</p> Full article ">Figure 5
<p>Combinations of phage and antibiotic treatments against a multi-resistant <span class="html-italic">P</span>. <span class="html-italic">aeruginosa</span> strain. (<b>A</b>,<b>B</b>) Effect of phage + antibiotic combinations on <span class="html-italic">P</span>. <span class="html-italic">aeruginosa</span> isolate #14. (<b>C</b>) Densities of phages at three different time points of the treatment. (<b>D</b>) Bacterial killing effect of phages and gentamicin 1/4 minimum inhibitory concentration (MIC) against <span class="html-italic">P. aeruginosa</span> isolate #14 at lower initial concentrations (10<sup>6</sup> and 10<sup>7</sup> cfu/mL). (<b>E</b>) Efficacy of phages against <span class="html-italic">P. aeruginosa</span> isolate #14 after 12 h of co-incubation and at different stages of the exponential phase (early, middle, and late). (<b>F</b>) Proportion of colonies resistant or susceptible to one or all phages, after 12 and 24 h of co-incubation with phages and antibiotics. PA #14, <span class="html-italic">P. aeruginosa</span> isolate #14; I, phage 6+ phage 32; and ii, phage 6+ phage 45. Points connected by the same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05, two-way ANOVA, Tukey’s multiple comparisons test). Error bars represents three biological replicates. Bacteria-only controls are in blue, while the antibiotics + bacteria controls are in brown.</p> Full article ">
11 pages, 2128 KiB  
Article
Rational Design of a Pan-Coronavirus Vaccine Based on Conserved CTL Epitopes
by Minchao Li, Jinfeng Zeng, Ruiting Li, Ziyu Wen, Yanhui Cai, Jeffrey Wallin, Yuelong Shu, Xiangjun Du and Caijun Sun
Viruses 2021, 13(2), 333; https://doi.org/10.3390/v13020333 - 21 Feb 2021
Cited by 13 | Viewed by 3938
Abstract
With the rapid global spread of the Coronavirus Disease 2019 (COVID-19) pandemic, a safe and effective vaccine against human coronaviruses (HCoVs) is believed to be a top priority in the field of public health. Due to the frequent outbreaks of different HCoVs, the [...] Read more.
With the rapid global spread of the Coronavirus Disease 2019 (COVID-19) pandemic, a safe and effective vaccine against human coronaviruses (HCoVs) is believed to be a top priority in the field of public health. Due to the frequent outbreaks of different HCoVs, the development of a pan-HCoVs vaccine is of great value to biomedical science. The antigen design is a key prerequisite for vaccine efficacy, and we therefore developed a novel antigen with broad coverage based on the genetic algorithm of mosaic strategy. The designed antigen has a potentially broad coverage of conserved cytotoxic T lymphocyte (CTL) epitopes to the greatest extent, including the existing epitopes from all reported HCoV sequences (HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2). This novel antigen is expected to induce strong CTL responses with broad coverage by targeting conserved epitopes against multiple coronaviruses. Full article
(This article belongs to the Section SARS-CoV-2 and COVID-19)
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<p>Phylogenetic analysis of full-length genomes of representative viruses of the existing coronavirus. Lines with different colors represent different coronavirus species, Alpha-coronaviruses (blue lines), Beta-coronaviruses (red lines), Gamma-coronaviruses (green lines), and Delta-coronaviruses (purple lines). Human isolates are highlighted with different colors, whereas strains from other hosts are shown in black. green: HCoV-229E; purple: HCoV-NL63; orange: SARS-CoV; red: SARS-CoV-2; blue: MERS-CoV; yellow: HCoV-OC43; brown: HCoV-HKU1. The scale bar is used to measure the branch length.</p> Full article ">Figure 2
<p>Design of mosaic antigen for a pan-coronavirus vaccine based on conserved CTL epitopes. (<b>A</b>) The schema of the genetic algorithm for the generation of a mosaic vaccine from seven species of HCoVs. (<b>B</b>) The mean epitope-coverage over the circulating viral proteins sequences covered by the CTL epitope in mosaic cocktail. The results indicated coverage (mean per-sequence) of S protein, M protein, N protein, and E protein. ‘Exact’ represents a 100% match, 9 AA out of 9 AA; ‘off-by-1’ indicates a match at 8AA out of 9 AA; ‘off-by-2’ indicates a match at 7AA out of 9 AA. (<b>C</b>) The alignment of the epitopes in mosaic antigens with their counterparts in proteins found in circulating virus using the Positional Epitope Coverage Assessment Tool. Each colored square includes an alignment of amino acid. Each “row” represents an amino acid sequence of the protein in the block. The column represents the relative position of a specific epitope. If a 9-aa in mosaic antigen makes a 100% match to their counterpart found in the vaccine or circulating virus from existing reports, the score is 9 and colored with light yellow; for a 9-aa without any matches, the score is 0 and colored with black. The scores in between are colored codes as the darkness increase. (<b>D</b>) Phylogenetic tree analysis of the mosaic antigens. The trees were midpoint rooted, and these sequences formed four clades, with HCoV-229E and HCoV-NL63 in clade 1; SARS-CoV and SARS-CoV-2 in clade 2; HCoV-HKU1 and HCoV-OC43 in clade 3; and the MERS-CoV in clade 4. Results showed that the mosaic S protein, M protein, N protein, and E protein were evenly distributed in the four clades. The red starts represent the genomic location of each mosaic protein. S: S protein; M: M protein; N: N protein; E: E protein.</p> Full article ">Figure 3
<p>The three-dimensional structure models for these candidate mosaic proteins, based on the computer-guided homology modeling method. Mosaic sequences were submitted to the SWISS-MODEL server to construct the protein structure. We used PyMOL software to visualize the model, and then evaluated its quality with QMEAN tool. <b>A</b>, S protein; <b>B</b>, N protein; <b>C</b>, M protein; <b>D</b>, E protein; <b>1</b>, Merged images of mosaic and nature proteins; <b>2</b>, Proteins found in circulating virus (with PDB ID: 6NZK (from OC43), 6G13 (from MERS-CoV), 5C8S (from SARS-CoV) and 2MM4 (from SARS-CoV) respectively); <b>3</b>, Mosaic protein.</p> Full article ">
22 pages, 4458 KiB  
Review
Single-Molecule FRET Imaging of Virus Spike–Host Interactions
by Maolin Lu
Viruses 2021, 13(2), 332; https://doi.org/10.3390/v13020332 - 21 Feb 2021
Cited by 17 | Viewed by 6427
Abstract
As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate [...] Read more.
As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure–function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure–function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes’ dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions. Full article
(This article belongs to the Special Issue Application of Advanced Imaging to the Study of Virus-Host Interactions)
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<p>Class I viral fusion proteins and proposed model of viral membrane fusion. (<b>A</b>) Schematic drawing of spike precursor and cleaved spike. The spike protein is initially synthesized as a single-chain polypeptide (spike precursor) and later cleaved into a trimer of covalently or non-covalently linked heterodimer. The heterodimer consists of the surface receptor-binding subunit (gray) and the fusion subunit (fusion peptide or fusion loop (FP/FL), dark yellow; N-terminal domain, cyan; C-terminal domain, dark blue). (<b>B</b>) Proposed conformational events of virus spikes during viral membrane fusion. These events are as follows, involving conformational changes in the surface subunit (top row) and changes in the fusion subunit (low row, simplified by only showing the fusion subunit [<a href="#B1-viruses-13-00332" class="html-bibr">1</a>]). (<b>1</b>) Prefusion—conformations of the spike in “closed” and open forms. Spike activation proceeds through an opening of the trimer, usually in response to binding to receptor or due to a cellular cue such as low pH. For non-covalently linked spikes, dissociating/decoupling between the surface/exterior subunit with the fusion subunit has been observed/suggested after the spike opens, such as HIV-1 and SARS-CoV-2 spikes. FP/FL remains sequestered in this process. (<b>2</b>) Exposing, extending, and inserting the FP/FL into the cellular membrane leads to the formation of an extended prehairpin intermediate. (<b>3</b>) Folding back the C-terminal segment of the fusion subunit back on the N-terminal segment core brings viral and cellular membranes into proximity. (<b>4</b>) Further folding and dragging two membranes into contact promotes two membranes’ merging to form a hemifusion stalk. (<b>5</b>) The fusion subunit folds into a stable post-fusion conformation, allowing a fusion pore to form. The intermediate steps from (<b>2</b>) to (<b>4</b>) remain elusive. This proposed model does not specify or speculate the number of spikes required for fusion pore formation.</p> Full article ">Figure 2
<p>Single-molecule Förster resonance energy transfer (smFRET) principle, instrumentation, and imaging of dynamic biomolecules. (<b>A</b>) An example curve depicting energy transfer efficiency (Förster resonance energy transfer (FRET), dashed black line) from an excited donor fluorophore to a neighboring acceptor fluorophore as a function of donor–acceptor distances. FRET values or FRET negatively correlate with the distance within a couple of nanometers between a donor (yellow star) and an acceptor (red star). (<b>B</b>) Widely used smFRET imaging instrumentations: prism- and objective-based total internal reflection fluorescence (TIRF). (<b>C</b>,<b>D</b>) Real-time observations of conformational motions in biomolecules by smFRET. (<b>C</b>) Diagram depicting ideal FRET-derived space-time coordinates of biomolecule conformations. Donor, yellow star; acceptor, red star; relative fluorescence intensity, the star’s size; biomolecule of interest, gray. (<b>D</b>) Example FRET-related traces showing four interconvertible conformations in real time. The host molecule dynamically samples four conformations, reflected by different donor–acceptor energy transfer efficiencies. Donor fluorescence trace, solid yellow line; acceptor fluorescence trace, solid red line; calculated FRET trace, solid blue line; FRET-indicated conformations, dashed black lines.</p> Full article ">Figure 3
<p>Conformational modulations of dynamic SARS-CoV-2 spike (S) proteins by receptors and antibodies. (<b>A</b>) Domain organization of full-length wild-type SARS-CoV-2 S (S1, cyan; S2, dark blue). Green and red arrows indicate the Cy3- and Cy5-labeling sites, respectively. Black arrows indicate protease cleavage sites (S1/S2 and S2′). NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; SD1, subunit domain 1; SD2, subunit domain 2; FP, fusion peptide; HR1/HR2, heptad repeat 1/heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (<b>B</b>,<b>C</b>) smFRET imaging experimental set-up. (<b>B</b>) Virus particles carrying a fluorescently labeled SARS-CoV-2 S protomer among wild-type spikes were immobilized and imaged on a PEG/PEG–biotin-coated PEGylated quartz slide on a prism-based TIRF microscope. The same type of experimental strategy has been used in other virus spike proteins throughout this review. For SARS-CoV-2, two virus particle systems were used to carry S on the surface. HIV-1 lentivirus particles are composed of HIV-1 cores and S proteins on the surface. S-MEN comprises four structural proteins of SARS-CoV-2 (S, spike; M, membrane protein; E, envelope protein; and N, nucleocapsid protein. (<b>C</b>) The binding of the cellular receptor human angiotensin-converting enzyme 2 (hACE2) induces conformational changes of S from the “RBD-down” (based on PDB:6VSB) to the “RBD-up” (PDB: 6VYB/6M0J) conformation. Cy3-labeling site, green ball; Cy5-labeling site, red ball; S1, light cyan; S2, dark blue; hACE2, magenta. (<b>D</b>) Featured findings of S conformations by smFRET imaging. These findings include (1) S dynamically samples four different conformations in real time, and S is in equilibrium exchange between states; (2) the binding of receptor hACE2 shifts S from the ground state (“RBD-down”) to the activated state (“RBD-up”) via an on-path intermediate (existing in the asymmetric S); (3) antibodies can antagonize S either by directly competing with receptor for the binding to S or by stabilizing S in the ground state (“RBD-down”).</p> Full article ">Figure 4
<p>On-path pre-fusion HIV-1 envelope (Env) trimer conformations identified by smFRET imaging. (<b>A</b>) Scheme of wild-type HIV-1 Env, noncovalently associated gp120 and gp41 subunits. (<b>B</b>) smFRET imaged individual HIV-1 viruses carrying a fluorescently labeled protomer within an Env trimer and elsewhere wild-type trimers. The Cy3/Cy5-labeled protomer (Cy3, green; Cy5, red) is in pink, whereas other wild-type protomers are colored gray. Structure is made based on PDB accessions 4ZMJ and 5FUU. (<b>C</b>) Model of Env activation by sequential binding of CD4 receptors. Env dynamically samples three primary conformational states in which State 1 is the predominant one. Upon sequential activation by CD4, Env transits through an asymmetric State 2 to a completely open State 3 (two- or three-CD4-bound trimer). State 2-Env is a single CD4-bound asymmetric trimer, in which the CD4-bound protomer adopts State 3 and the neighboring free protomers adopt State 2. State 2A is an off-path conformation that is highly vulnerable to antibody-dependent cellular cytotoxicity (ADCC). Following CD4 activation, the binding of coreceptors CCR5/CXCR4 lead to virus entry, which smFRET has not informed. (<b>D</b>) Vaccine candidates based upon soluble SOSIP.664 Env trimer resemble State 2, consisting of three State 2 protomers. The design of soluble SOSIP.664 Env is illustrated in the schematic. Results from (<b>D</b>) infer that State 1 Env (<b>C</b>) is structurally unknown.</p> Full article ">Figure 5
<p>Conformational dynamics of influenza A hemagglutinin (HA) revealed by smFRET imaging. (<b>A</b>) Imaging context of HA trimer on viral particles. Lentiviral particles incorporated with HA trimers are imaged at the single-molecule level. On the virus surface, only one HA protomer (color-coded) within an HA trimer is fluorescently labeled. Two fluorophores (Cy3 in green, Cy5 in red) are attached on HA2 at indicated positions. The structure of the pre-fusion HA, the trimer of HA1/HA2 with labeling sites, is shown (on the basis of PDB 2FK0). (<b>B</b>) The proposed model depicts reversibly and irreversibly conformational dynamics of HA2 during viral membrane fusion (adapted from [<a href="#B29-viruses-13-00332" class="html-bibr">29</a>]). In response to acidic pH and receptors, HA2 shifts conformations from the pre-fusion state to the coiled-coil post-fusion state through multiple fusion-related intermediate states. In the absence of sialic acid receptors, acidic pH triggers HA2 conformational changes in favor of intermediate I and intermediate II, which can be reversed by re-neutralizing the pH. The intermediate I is the conformation in which the fusion peptide is exposed out of the hydrophobic pocket, whereas the fusion peptide in intermediate II is released. The interaction of HA1 to sialic acid-containing endosomal membrane promotes the irreversible process of HA2 adopting coiled-coil post-fusion conformation.</p> Full article ">Figure 6
<p>Ebola virus glycoprotein (GP)-mediated viral membrane fusion revealed by smFRET imaging. (<b>A</b>) Structural organization of Ebola virus envelope glycoprotein (GP). GP is a trimer of GP1/GP2 heterodimer. SP, signal peptide; FL, fusion loop; TM, transmembrane. (<b>B</b>) smFRET imaging of GP in the context of a lentiviral particle. A GP trimer (PDB 5JQ3) carrying a single fluorescently labeled protomer and wild-type GP trimers (gray) were incorporated into a lentiviral particle. Cy3 and Cy5 were attached to the GP2 subunit. Labeled GP protomer: GP1 in magenta; GP2 in blue; Cy3 in green; Cy5 in red. (<b>C</b>) Model of GP-mediated membrane fusion (adapted from [<a href="#B28-viruses-13-00332" class="html-bibr">28</a>]). In this model, acidic pH and Ca<sup>2+</sup> facilitate GP transit from a pre-fusion conformation to an intermediate optimal for NPC1 binding, and this transition is reversible. In the intermediate conformation, the fusion loop moves from the trimer axis towards the host membrane. The binding of NPC1 then triggers at least two irreversible transitions to the post-fusion coiled-coil conformation. NPC1: Niemann-Pick C1.</p> Full article ">
13 pages, 291 KiB  
Article
Risk Factors for Respiratory Syncytial Virus Lower Respiratory Tract Infections: Evidence from an Indonesian Cohort
by Rowena Crow, Kuswandewi Mutyara, Dwi Agustian, Cissy B. Kartasasmita and Eric A. F. Simões
Viruses 2021, 13(2), 331; https://doi.org/10.3390/v13020331 - 21 Feb 2021
Cited by 6 | Viewed by 3230
Abstract
Although risk factors for hospitalization from a respiratory syncytial virus (RSV) are well known, RSV lower respiratory tract infections (LRIs) in the community are much less studied or understood, especially in developing countries. In a prospective, cohort study we studied factors predisposing Indonesian [...] Read more.
Although risk factors for hospitalization from a respiratory syncytial virus (RSV) are well known, RSV lower respiratory tract infections (LRIs) in the community are much less studied or understood, especially in developing countries. In a prospective, cohort study we studied factors predisposing Indonesian infants and children under 5 years of age to developing RSV LRIs. Subjects were enrolled in two cohorts: a birth cohort and a cross-sectional cohort of children <48 months of age. Subjects were visited weekly at home to identify any LRI, using the World Health Organization’s criteria. RSV etiology was determined through analysis of nasal washings by enzyme immunoassay and polymerase chain reaction. Risk factors for the development of the first documented RSV LRI were identified by multivariate analysis using logistic regression and Cox proportional hazard modeling. Of the 2014 children studied, 999 were enrolled within 30 days of birth. There were 149 first episodes of an RSV. Risk factors for an RSV LRI were poverty (p < 0.01), use of kerosene as a cooking fuel (p < 0.05), and household ownership of rabbits and chickens (p < 0.01). Our findings suggested that in a middle-income country such as Indonesia, with a substantial burden of RSV morbidity and mortality, lower socioeconomic status, environmental air quality, and animal exposure are predisposing factors for developing an RSV LRI. Full article
(This article belongs to the Special Issue Respiratory Syncytial Virus)
17 pages, 10089 KiB  
Article
Characterization of the GBoV1 Capsid and Its Antibody Interactions
by Jennifer Chun Yu, Mario Mietzsch, Amriti Singh, Alberto Jimenez Ybargollin, Shweta Kailasan, Paul Chipman, Nilakshee Bhattacharya, Julia Fakhiri, Dirk Grimm, Amit Kapoor, Indrė Kučinskaitė-Kodzė, Aurelija Žvirblienė, Maria Söderlund-Venermo, Robert McKenna and Mavis Agbandje-McKenna
Viruses 2021, 13(2), 330; https://doi.org/10.3390/v13020330 - 20 Feb 2021
Cited by 6 | Viewed by 4421
Abstract
Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better [...] Read more.
Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties. Full article
(This article belongs to the Special Issue Advances in Parvovirus Research 2020)
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<p>The capsid structure of Gorilla bocavirus 1 (GBoV1). (<b>A</b>) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of GBoV1 WT and VP3 only samples confirming the presence of VP1, VP2 and VP3 (~80, 65, 60 kDa) and cryo-electron micrograph showing intact viral particles. (<b>B</b>) Capsid density map of GBoV1 WT contoured at sigma (σ) threshold of 1.0. The radial distance from the center measured in Å is colored as shown. Arrows point to the 5-fold, 3-fold or 2-fold symmetry axis and the 2/5-fold wall. (<b>C</b>) Cross-sectional view of GBoV1 WT density map. (<b>D</b>) Fourier shell correlation (FSC) plot for the cryo-reconstruction with an estimated resolution of 2.76 Å at an FSC threshold of 0.143. Resolution (Å) is presented using a log scale. (<b>E</b>) Atomic model of amino acids 55–61 (βB) represented within their density map contoured at a σ threshold level of 1. C = yellow, O = red and N = blue. Panels B, C and E were made using UCSF-Chimera [<a href="#B41-viruses-13-00330" class="html-bibr">41</a>].</p> Full article ">Figure 2
<p>Structural comparison of GBoV1 to the HBoVs. (<b>A</b>) The VP3 monomer structure of GBoV1 shown as a ribbon diagram, with the secondary structure elements, N- and C-terminus and VRs labeled. The approximate positions of the icosahedral 2-, 3- and 5-fold axes are indicated as filled oval, triangle and pentagon, respectively. (<b>B</b>) VP3 monomer structures of HBoV1 (blue) and GBoV1 (yellow) superposed. The labels are as in panel (<b>A</b>). (<b>C</b>) VP3 monomer structures of HBoV1 (blue), HBoV2 (orange), HBoV3 (green), HBoV4 (red) and GBoV1 (yellow) superposed. The labels are as in panel (<b>A</b>). The color for each model is as given beside panel (<b>C</b>). Images were superposed in the Coot program [<a href="#B42-viruses-13-00330" class="html-bibr">42</a>] and visualized in the PyMol program [<a href="#B46-viruses-13-00330" class="html-bibr">46</a>].</p> Full article ">Figure 3
<p>Structure-based sequence alignment of Human bocavirus 1 (HBoV1)-4 and GBoV1. The structure-based sequence alignment, starting from the first ordered residue (aa33), was generated using distance values from the Coot [<a href="#B42-viruses-13-00330" class="html-bibr">42</a>] superpose tool. Secondary structural elements, β-strands and α-helices, are indicated by blue arrows and red cylinders, respectively. Regions highlighted with orange indicate sequence identity between HBoV1-4 and GBoV1. The locations of the VRs are also indicated based on the previously defined VRs [<a href="#B23-viruses-13-00330" class="html-bibr">23</a>]. Amino acid number, based on HBoV1, is shown above the sequences. Structural variability, defined by amino acids whose Cα atoms are &gt;2 Å apart, are offset low and highlighted in red.</p> Full article ">Figure 4
<p>Cross-reactivity of GBoV1 capsids with HBoV1 antibodies via native dot blot. 10<sup>11</sup>, 10<sup>10</sup> or 10<sup>9</sup> viral capsids were loaded onto a nitrocellulose membrane and tested against H1-H1 (positive control for denatured virus-like particles (VLPs)) and HBoV1 antibodies 15C6, 12C1, 4C2 and 9G12 (detecting conformational epitopes). 10<sup>11</sup> not shown for 15C6 and H1-H1 due to overexposure.</p> Full article ">Figure 5
<p>Antibody epitopes on GBoV1 capsid localized to 5- and 3-fold axes for 15C6 and 12C1. (<b>A</b>) FSC plots for the cryo-reconstruction with an estimated resolution value of 5.3Å, 6.4 Å and 6.2 Å, at an FSC threshold value of 0.143 for GBoV1, GBoV1:15C6 and GBoV1:12C1, respectively. Resolution (Å) is presented using a log<sub>2</sub> scale. (<b>B</b>) Capsid density map of GBoV1 contoured at σ threshold of 1.0. (<b>C</b>) Capsid density map of GBoV1 complexed with 15C6 (GBoV1:15C6) contoured at σ threshold of 1.0. (<b>D</b>) Capsid density map of GBoV1 complexed with 15C6 contoured at σ threshold of 0.5. (<b>E</b>) Cross-sectional view of the GBoV1 complex density map. (<b>F</b>) Cross-sectional view of the GBoV1:15C6 complex density map. (<b>G</b>) Cross-sectional view of the GBoV1:12C1 capsid density map.</p> Full article ">Figure 6
<p>GBoV1-Fab binding interfaces. (<b>A</b>) Close-up view of the GBoV1 WT structure docked to a generic Fab (PDB ID 2FBJ) within the cryo-reconstructed density of GBoV1-15C6 (represented as a gray mesh, contoured at 0.5σ) and (<b>B</b>) GBoV1-12C1. Highlighted VRs are colored as shown in key. Generic Fab (dark brown) consists of a heavy and light chain, each with constant and variable regions. The Fab variable region interacts with the surface of the capsid. The GBoV1 capsid is also colored in tan.</p> Full article ">Figure 7
<p>The GBoV1 15C6 and 12C1 epitopes. (<b>A</b>) Roadmap surface representation of the GBoV1 15C6 epitope. Colored in orange are the modeled contact residues between the GBoV1 capsid and the 15C6 Fab model. Colored in yellow are the residues occluded by the bound 15C6 Fab. (<b>B</b>) Roadmap surface representation of the GBoV1 12C1 epitope. Colored in blue are the modeled contact residues between the GBoV1 capsid and the 12C1 Fab model. Colored in cyan are the residues occluded by the 12C1 Fab. (<b>C</b>) Position of VR-I, VR-II, VR-III and VR-V, VR-VIIIB on the GBoV1 capsid. Amino acid residues that are exposed on the capsid surface are labeled with their 3-letter code and residue number. The 5-fold, 3-fold and 2-fold axis are indicated by a filled pentagon, triangle and ellipse, respectively. The roadmaps were generated with the RIVEM program [<a href="#B44-viruses-13-00330" class="html-bibr">44</a>].</p> Full article ">Figure 8
<p>Dot immunoblot analysis of HBoV1 and GBoV1 against human sera. (<b>A</b>) Representative native dot immunoblots of HBoV1 and GBoV1 against human sera with 10<sup>10</sup> or 10<sup>9</sup> loaded capsid particles. AAV2 and AAV5 are used as controls. Samples tested are as labeled. (<b>B</b>) Bar graph representation of the percentage of positive signal based visual inspection of the 40 dot immunoblots reactivities. <span class="html-italic">n</span> = 3.</p> Full article ">
10 pages, 975 KiB  
Article
Recent Hydroxychloroquine Use Is Not Significantly Associated with Positive PCR Results for SARS-CoV-2: A Nationwide Observational Study in South Korea
by Seongman Bae, Byeongzu Ghang, Ye-Jee Kim, Joon Seo Lim, Sung-Cheol Yun, Yong-Gil Kim, Sang-Oh Lee and Sung-Han Kim
Viruses 2021, 13(2), 329; https://doi.org/10.3390/v13020329 - 20 Feb 2021
Cited by 4 | Viewed by 3961
Abstract
Background: To evaluate the role of hydroxychloroquine (HCQ) as pre-exposure prophylaxis against coronavirus disease 2019 (COVID-19), we investigated the prevalence of positive test results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing according to recent HCQ use in patients who had been [...] Read more.
Background: To evaluate the role of hydroxychloroquine (HCQ) as pre-exposure prophylaxis against coronavirus disease 2019 (COVID-19), we investigated the prevalence of positive test results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing according to recent HCQ use in patients who had been tested using nationwide health-insurance data of South Korea. Methods: All adults tested for SARS-CoV-2 from 20 January 2020 to 15 May 2020 were identified. HCQ users were defined as patients who had been pretreated with HCQ for at least 30 days until the date of SARS-CoV-2 testing. The prevalence of positive PCR results for SARS-CoV-2 was compared between HCQ users and nonusers. Results: Of a total of 216,686 individuals who had been tested for SARS-CoV-2, 743 (0.3%) were pretreated with HCQ. The prevalence of positive results was not significantly different between HCQ users (2.2%) and nonusers (2.7%; P = 0.35), with an odds ratio of 0.79 (95% confidence interval (CI), 0.48–1.30). Propensity score-matched-cohort analysis showed similar results in terms of the prevalence of positive results (2.2% in HCQ users vs. 3.1% in nonusers; P = 0.18), with an odds ratio of 0.69 (95% CI, 0.40–1.19). The rate of positive PCR was not significantly different in long-term HCQ users (more than 3 or 6 months) compared with nonusers. Conclusions: In this population-based study, recent exposure to HCQ was not significantly associated with a lower risk of SARS-CoV-2 infection. Our data do not support the use of HCQ as pre-exposure prophylaxis against COVID-19. Full article
(This article belongs to the Special Issue Vaccines and Therapeutics against Coronaviruses)
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<p>Flowchart of population selection and propensity-score-matched analysis.</p> Full article ">
16 pages, 2848 KiB  
Article
Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity
by Amanda Carroll-Portillo, Cristina N. Coffman, Matthew G. Varga, Joe Alcock, Sudha B. Singh and Henry C. Lin
Viruses 2021, 13(2), 328; https://doi.org/10.3390/v13020328 - 20 Feb 2021
Cited by 38 | Viewed by 7296
Abstract
For decades, bacteriophage purification has followed structured protocols focused on generating high concentrations of phage in manageable volumes. As research moves toward understanding complex phage populations, purification needs have shifted to maximize the amount of phage while maintaining diversity and activity. The effects [...] Read more.
For decades, bacteriophage purification has followed structured protocols focused on generating high concentrations of phage in manageable volumes. As research moves toward understanding complex phage populations, purification needs have shifted to maximize the amount of phage while maintaining diversity and activity. The effects of standard phage purification procedures such as polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) density gradients on both diversity and activity of a phage population are not known. We have examined the effects of PEG precipitation and CsCl density gradients on a number of known phage (M13, T4, and ?X 174) of varying structure and size, individually and as mixed sample. Measurement of phage numbers and activity throughout the purification process was performed. We demonstrate that these methods, used routinely to generate “pure” phage samples, are in fact detrimental to retention of phage number and activity; even more so in mixed phage samples. As such, minimal amounts of processing are recommended to introduce less bias and maintain more of a phage population. Full article
(This article belongs to the Section Bacterial Viruses)
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<p>Characteristics of bacteriophage used. Arrowhead = T4, * = ΦX 174, <b>+</b> = M13.</p> Full article ">Figure 2
<p>Work flow for phage preparation. Outline of phage preparation procedure showing where collection points occurred, purification steps taken (polyethylene glycol (PEG) precipitation followed by cesium chloride (CsCl) gradient purification) and types of analyses used (measures).</p> Full article ">Figure 3
<p>TEM of phage samples. Focused regions of selected images from each set of phage samples to demonstrate enhancement of phage populations after PEG concentration and CsCl gradient purification. An example ΦX phage particle is denoted with an arrowhead in each sample frame where they are found. Scale bars are 1 μm.</p> Full article ">Figure 4
<p>Phage activity decreases after PEG and/or CsCl purification. Graphical representation of the percent difference between the detected number of plaque forming units (active phage) and the expected number calculated from stock phage solutions for each individual phage preparation: (<b>A</b>) M13, (<b>B</b>) T4, and (<b>C</b>) ΦX. Tables accompanying each graph list numerical values for the respective bar and the stock phage activity value from which calculations were based.</p> Full article ">Figure 5
<p>Greater loss of phage nucleic acid after CsCl purification than PEG precipitation. Graphical representation (left panels) of the mean percent change in nucleic acid fluorescent intensity (FI) relative to expected levels after treatments for (<b>A</b>) M13, (<b>B</b>) T4, (<b>C</b>) ΦX, and (<b>D</b>) mixed phages. The fluorescent intensity averages for each sample as well as the expected levels (stock) are listed (middle panels) and an overlay of representative histograms for each phage from all samples tested (right panels) show shifts in fluorescent intensities from expected (black lines, stock) of nucleic acid labeling due to treatments.</p> Full article ">Figure 6
<p>Selected phage numbers are significantly decreased after PEG precipitation and/or CsCl purification. Mean percent change in phage numbers as determined through qPCR quantitation of DNA is shown for the individual (left column) and mixed (right column) phage samples for (<b>A</b>) M13, (<b>B</b>) T4, and (<b>C</b>) ΦX. (<b>D</b>) The average numbers of phage/mL calculated for each sample set show consistent and significant decrease in phage after PEG precipitation (T4, ΦX but not M13) and CsCl purification when compared to stock. UD = undetectable.</p> Full article ">
19 pages, 4252 KiB  
Article
First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523
by Kristin Köppen, Grisna I. Prensa, Kerstin Rydzewski, Hana Tlapák, Gudrun Holland and Klaus Heuner
Viruses 2021, 13(2), 327; https://doi.org/10.3390/v13020327 - 20 Feb 2021
Cited by 3 | Viewed by 2962
Abstract
Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to [...] Read more.
Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation. Full article
(This article belongs to the Special Issue Viruses of Microbes 2020: The Latest Conquests on Viruses of Microbes)
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<p>Organization of <span class="html-italic">Francisella</span> bacteriophage vB_<span class="html-italic">Fhi</span>M_KIRK as prophage (<b>A</b>) or its episomal form (<b>B</b>)<b>.</b> (<b>A</b>)<b>:</b> The prophage is integrated within the tRNA-Val gene (pink arrow). The <span class="html-italic">att</span> sites (<span class="html-italic">attL</span> and <span class="html-italic">attR</span>) are indicated by a pink trapezium. The <span class="html-italic">attR</span> site corresponds to the 3′ end of the tRNA-Val. Chromosomal genes are given in black and genes of the bacteriophage are given in different colours according to their respective putative function based on BLASTp analysis (see also <a href="#viruses-13-00327-t001" class="html-table">Table 1</a>). A putative origin of replication (OriR) is indicated. Gene numbers are indicated below the genes as published for <span class="html-italic">Fhi</span> 3523 (FN3523; CP002558) or as determined in this work for the bacteriophage KIRK (FhV_0001 to FhV_0051). Location of the spacer DNAs identified in the CRISPR-Cas systems of different <span class="html-italic">Francisella</span> strains are indicated above of the genes by red arrows (Schunder et al., 2013; modified). Primer used in this study are indicated as brown arrows (for details see text). (<b>B</b>)<b>:</b> Gene organization of the episomal form of bacteriophage KIRK are given in different colours (see (<b>A</b>)) due to their putative function and are clustered in “replication and regulation” and “phage particle production”. The site-specific DNA region of KIRK (<span class="html-italic">attP</span>) responsible for the integration into the genome of host cells (<span class="html-italic">attB</span>, not shown) is indicated.</p> Full article ">Figure 2
<p>The putative origin of replication of vB_<span class="html-italic">FhiM</span>_KIRK. The origin is composed of the putative inceptor signal for DNA replication (dotted lines, three times), found within a direct repeat sequence (arrows), and four misc-binding sites (underlined sequence). The region is localized within gene <span class="html-italic">fhv_0043</span> (see <a href="#viruses-13-00327-f001" class="html-fig">Figure 1</a>).</p> Full article ">Figure 3
<p>Phylogenetic tree analysis of the bacteriophage KIRK. 7 loci concatenated protein sequence of genes <span class="html-italic">fhv_0018, 0024, 0012, 0008, 0025, 0028, 0023</span>) and available homologous proteins from bacteriophages <span class="html-italic">Escherichia</span> T4 (<span class="html-italic">Tevenvirinae</span>, AF158101), <span class="html-italic">Escherichia</span> 186 (<span class="html-italic">Peduovirinae</span>, NC_001317), <span class="html-italic">Escherichia</span> P2 (<span class="html-italic">Peduovirinae</span>, KC618326), <span class="html-italic">Vibrio</span> VHML (<span class="html-italic">Vhmlvirus</span>, NC_004456), <span class="html-italic">Vibrio</span> VP585 (<span class="html-italic">Vhmlvirus</span>, NC_027981), <span class="html-italic">Wolbachia</span> WO2 (MK976036), <span class="html-italic">Wolbachia</span> WO (MN180249), <span class="html-italic">Ralstonia</span> phiRSP (<span class="html-italic">Jilinvirus</span>, MH252365), <span class="html-italic">Pseudomonas</span> PPpW3 (<span class="html-italic">Jilinvirus</span>, NC_023006), <span class="html-italic">Enterobacter</span> Arya (<span class="html-italic">Jilinvirus</span>, NC_031048), <span class="html-italic">Escherichia</span> ECO-1230-10 (<span class="html-italic">Jilinvirus</span>, GU903191) and <span class="html-italic">Escherichia</span> EcoM-ep3 (<span class="html-italic">Jilinvirus</span>, NC_025430) were used for amino acid sequences alignment using the ClustalO program in Geneious. The phylogenetic tree was generated by using Geneious Tree Builder, Neighbor-Joining method and <span class="html-italic">Escherichia</span> T4 phage as outgroup.</p> Full article ">Figure 4
<p>Bacteriophage induction. (<b>A</b>): Semi-quantitative PCR analyses. Supernatants of <span class="html-italic">Fhi</span> 3523 lysates (S) and control DNA of <span class="html-italic">Fhi</span> 3523 (C) were analyzed using primer Fha-2/Fha-3 (product size: 538 bp) with 5, 10, 15 and 20 PCR amplification cycles. (<b>B</b>–<b>D</b>): <span class="html-italic">Fhi</span> 3523 was exposed to UV radiation at 254 nm for 0, 60 and 90 s (<b>B</b>); treated with 0, 0.5, 1 and 5 µg/mL mitomycin C (<b>C</b>); or incubated at 37, 40, 42 and 44 °C (<b>D</b>). Samples were collected after various time points post-induction and used for PCR analyses with Fha-2/Fha-3 and 10 amplification PCR cycles. (<b>E</b>): PCR analyses were performed targeting the phage KIRK using primers Fha-2/Fha-3 (left, 538 bp) and bacterial genome of <span class="html-italic">Fhi</span> 3523 with primers Fhis_R13/Fhis_U13A (right, 1289 bp) after 0 and 90 s of UV radiation (254 nm) at different time points. (<b>F</b>): Supernatants (1, 2, 3, three replicates) of <span class="html-italic">Fhi</span> 3523 cultures treated with (+) or without (−) UV for 90 s at 254 nm were analyzed by PCR detecting the phage (Fha-2/Fha-3, 10 PCR cycles, upper row) and the bacterial <span class="html-italic">Fhi</span> 3523 chromosome (Fhis_U13A/Fhis_R13, lower row). All supernatants were treated with DNase and RNase and sterile filtered prior using in PCR analyses, except for sample marked by asterisk (control) which was not treated with DNase and RNase. (<b>G</b>): Purified, UV-induced phage samples (P) and control DNA of <span class="html-italic">Fhi</span> 3523 (C) were analyzed targeting FhaGI-1 (PCR 1–4) and chromosomal regions of <span class="html-italic">Fhi</span> 3523 (PCR 5, 6), respectively. 1: Fha-2/Fha-3 (538 bp); 2: Fha996_U/Fha997_R (2024 bp); 3: F1_out_U/F2_out_R (554 bp); 4: F2_out_U/F3_out_R (530 bp); 5: Fha-1/Fha-4 (617 bp); 6: Fhis_R13/Fhis_U13A (1289 bp). C = control DNA of <span class="html-italic">Fhi</span> 3523 (whole DNA of bacterial cell lysates, see <a href="#sec2dot2-viruses-13-00327" class="html-sec">Section 2.2</a>); NC = no template control; DNA ladder 1 kb GeneRuler was used.</p> Full article ">Figure 5
<p>Electron micrographs of <span class="html-italic">Francisella</span> phage vB_<span class="html-italic">FhiM</span>_KIRK. (<b>A</b>–<b>C</b>): Transmission EM of purified KIRK samples (see <a href="#sec2dot5-viruses-13-00327" class="html-sec">Section 2.5</a>) stained with uranyl acetate. Phage particles are composed of an icosahedral head ~52 nm in diameter and a tail structure of ~82 nm in length and ~9 nm in width. (<b>D</b>,<b>E</b>): Thin section EM of UV treated <span class="html-italic">Fhi</span> 3523 cells, showing multiple phage particles inside of one bacterial cell. Bars = 50 nm.</p> Full article ">Figure 6
<p>The recombinant bacteriophage KIRK<sub>rec</sub>. (<b>A</b>): Transmission EM of purified KIRK<sub>rec</sub> stained with uranyl acetate. Phage particles are composed of an icosahedral head ~57 nm. (<b>B</b>): Infection of <span class="html-italic">Francisella</span> with KIRK<sub>rec</sub>. <span class="html-italic">Fth</span> LVS and <span class="html-italic">F-</span>W12 were transformed with the episomal form of KIRK<sub>rec</sub> by electroporation. Obtained clones were tested regarding the circular form of KIRK<sub>rec</sub> using primer Fha-2/Fha-3 and the chromosomal integration using Fha-1/Fha-2 and Fha-3/Fha-4, here Fha-1 and Fha-4 are species-specific primers binding in the genome of <span class="html-italic">F</span>-W12 (Fha-1<sup>W12</sup>, Fha-4<sup>W12</sup>) and <span class="html-italic">Fth</span> LVS (Fha-1, Fha-4*), respectively, see <a href="#app1-viruses-13-00327" class="html-app">Supplemental Table S1</a>. 1, 2: <span class="html-italic">F</span>-W12 and <span class="html-italic">Fth</span> LVS KIKR<sub>rec</sub> clones, respectively; W: chromosomal DNA of <span class="html-italic">F</span>-W12, rec: <span class="html-italic">F</span>-W12 KIRK<sub>rec</sub> clone which was obtained by in vitro synthesis and cloning (see <a href="#app1-viruses-13-00327" class="html-app">Supplemental Materials</a>), and was used for extraction of circular form of KIRK<sub>rec</sub>; NC: no template control; L: chromosomal DNA of <span class="html-italic">Fth</span> LVS.</p> Full article ">
23 pages, 3799 KiB  
Article
Design and Synthesis of HCV-E2 Glycoprotein Epitope Mimics in Molecular Construction of Potential Synthetic Vaccines
by Theodorus J. Meuleman, Vanessa M. Cowton, Arvind H. Patel and Rob M. J. Liskamp
Viruses 2021, 13(2), 326; https://doi.org/10.3390/v13020326 - 20 Feb 2021
Cited by 4 | Viewed by 3026
Abstract
Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so [...] Read more.
Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design. Full article
(This article belongs to the Special Issue Novel Advances in Vaccines against HCV)
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<p>Illustration of cyclized synthetic peptides as described earlier by Meuleman et al. [<a href="#B33-viruses-13-00326" class="html-bibr">33</a>] based on epitope I (411–424) denoted in <span style="color:green">green</span> (<b>4</b>); epitope II (436–448) denoted in <span style="color:red">red</span> (<b>5</b>); epitope III (521–537) denoted in <span style="color:blue">blue</span> (<b>6</b>); and, their spatial orientation within the crystal structure of the E2 core glycoprotein, as obtained by Kong et al. [PDB 4MWF] [<a href="#B38-viruses-13-00326" class="html-bibr">38</a>]. Epitope IV (611–617) denoted in <span style="color:#B4A923">yellow</span> was not considered for mimicry, as it is not recognized as a (continuous) epitope by antibodies.</p> Full article ">Figure 2
<p>Continuous epitope mimics <b>20</b>, <b>21</b>, and <b>22</b>, as described by Meuleman et al [<a href="#B33-viruses-13-00326" class="html-bibr">33</a>].</p> Full article ">Figure 3
<p>Continuous epitope mimics <b>20</b>, <b>21</b>, and <b>22</b> are separately presented on mcKLH using thiol-maleimide conjugation.</p> Full article ">Figure 4
<p>Discontinuous epitope mimic <b>27</b> is presented on mcKLH using ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) amide bond formation.</p> Full article ">Figure 5
<p>ELISA of immobilized sE2 against sera obtained from the final bleed (FB). Including: group O &amp; W – <b>20</b>-mcKLH; group P &amp; X – <b>21</b>-mcKLH; group Q &amp; Y – <b>22</b>-mcKLH; group R &amp; Z – <b>27</b>-mcKLH. Sera obtained from the pre-immunization bleed (PB) were included as a negative control. Monoclonal antibody AP33 was included as positive control. (<b>A</b>) Three-fold dilutions of the immunization of groups O, P, Q, and R, including: FB (starting dilution of 1:50); PB (starting dilution of 1:50); AP33 (starting concentrations of 2.0 μg/mL and 0.1 μg/mL). O1, P1, Q1, R1, etc. refer to an individual animal in the corresponding group. (<b>B</b>) Three-fold dilutions of the immunization of groups W, X, Y, and Z, including: FB (starting dilution of 1:50); PB (starting dilution of 1:50); AP33 (starting concentrations of 2.0 μg/mL and 0.1 μg/mL). W1, X1, Y1, Z1, etc. refer to an individual animal in the corresponding group. (<b>C</b>) The absorbance of FB and PB at a 1:50 dilution, each dot indicates serum per animal. The ELISA was performed in triplicate, with the exception of group O – epitope I (<b>20</b>-mcKLH) that was done in duplicate. The background signal (no mAb) was subtracted. Error bars represent the standard error of the mean. *: <span class="html-italic">p</span> &lt; 0.05, ****: <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 6
<p>Levels of neutralization of HCVpp of strain H77 incubated with IgG(100 µg/mL) obtained from immunization experiments. Monoclonal antibody AP33 (50 µg/mL) and 1:7 (50 µg/mL) were included as the positive control.</p> Full article ">Figure 7
<p>Illustrating the compatibility of conjugate cyclic peptides <b>4</b>, <b>5</b>, and <b>6</b> on TAC-scaffold <b>19</b> to obtain various variations of discontinuous epitopes.</p> Full article ">Scheme 1
<p>Synthesis of triethylene glycol monotrityl thioether ethylamine 10 and subsequent instalment on triazacyclophane (TAC)-scaffold 11 [<a href="#B20-viruses-13-00326" class="html-bibr">20</a>,<a href="#B31-viruses-13-00326" class="html-bibr">31</a>].</p> Full article ">Scheme 2
<p>Synthesis of triethylene glycol Boc-amide ethylamine 18 and subsequent instalment onto TAC-scaffold 11 [<a href="#B20-viruses-13-00326" class="html-bibr">20</a>,<a href="#B31-viruses-13-00326" class="html-bibr">31</a>] to obtain the improved TAC-scaffold 19.</p> Full article ">Scheme 3
<p>The assembly of discontinuous epitope mimic 27 based on the HCV E2 glycoprotein by sequential selective incorporation of continuous epitopes 4–6.</p> Full article ">
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