Viruses

17 pages, 1050 KiB

Open AccessArticle

Association of Increased Programmed Death Ligand 1 Expression and Regulatory T Cells Infiltration with Higher Hepatocellular Carcinoma Recurrence in Patients with Hepatitis B Virus Pre-S2 Mutant after Curative Surgical Resection

by Long-Bin Jeng, Tsai-Chung Li, Shih-Chao Hsu and Chiao-Fang Teng

Viruses 2022, 14(6), 1346; https://doi.org/10.3390/v14061346 - 20 Jun 2022

Cited by 2 | Viewed by 2594

Abstract

Although surgical resection is available as a potentially curative therapy for hepatocellular carcinoma (HCC), high recurrence of HCC after surgery remains a serious obstacle for long-term patient survival. Therefore, the discovery of valuable prognostic biomarkers for HCC recurrence is urgently needed. Pre-S2 mutant [...] Read more.

Although surgical resection is available as a potentially curative therapy for hepatocellular carcinoma (HCC), high recurrence of HCC after surgery remains a serious obstacle for long-term patient survival. Therefore, the discovery of valuable prognostic biomarkers for HCC recurrence is urgently needed. Pre-S2 mutant is a mutant form of hepatitis B virus (HBV) large surface protein which is expressed from the HBV surface gene harboring deletion mutations spanning the pre-S2 gene segment. Pre-S2 mutant-positive HCC patients have been regarded as a high-risk population of HCC recurrence after resection surgery and display increased immune checkpoint programmed death ligand 1 (PD-L1) expression and pro-tumor regulatory T cells (Tregs) infiltration in tumor tissues. In this study, the association of higher levels of PD-L1 expression and Tregs infiltration in tumor tissues with post-operative HCC recurrence in pre-S2 mutant-positive HCC patients was evaluated. We found that patients with pre-S2 mutant in combination with higher levels of PD-L1 expression and Tregs infiltration in tumor tissues were independently associated with a higher risk of HCC recurrence (hazard ratio, 4.109; p value = 0.0011) and poorer recurrence-free survival (median, 8.2 versus 18.0 months; p value = 0.0004) than those of patients with either one or two of these three biomarkers. Furthermore, a combination of pre-S2 mutant, intra-tumoral PD-L1 expression, and tumor-infiltrating Tregs exhibited superior performance in identifying patients at a higher risk of HCC recurrence (area under the receiver operating characteristic curve, 0.8400). Collectively, this study suggests that higher levels of PD-L1 expression and Tregs infiltration in tumor tissues predicted a higher risk of HCC recurrence in pre-S2 mutant-positive HCC patients after curative surgical resection. Full article

(This article belongs to the Special Issue New Frontiers in Small DNA Virus Research)

► Show Figures

Figure 1

13 pages, 1021 KiB

Open AccessReview

Repurposing Molnupiravir for COVID-19: The Mechanisms of Antiviral Activity

by Ashley Jia Wen Yip, Zheng Yao Low, Vincent T. K. Chow and Sunil K. Lal

Viruses 2022, 14(6), 1345; https://doi.org/10.3390/v14061345 - 20 Jun 2022

Cited by 28 | Viewed by 3948

Abstract

Molnupiravir is a β-d-N4-hydroxycytidine-5′-isopropyl ester (NHC) compound that exerts antiviral activity against various RNA viruses such as influenza, SARS, and Ebola viruses. Thus, the repurposing of Molnupiravir has gained significant attention for combatting infection with SARS-CoV-2, the etiological agent of COVID-19. Recently, Molnupiravir [...] Read more.

Molnupiravir is a β-d-N4-hydroxycytidine-5′-isopropyl ester (NHC) compound that exerts antiviral activity against various RNA viruses such as influenza, SARS, and Ebola viruses. Thus, the repurposing of Molnupiravir has gained significant attention for combatting infection with SARS-CoV-2, the etiological agent of COVID-19. Recently, Molnupiravir was granted authorization for the treatment of mild-to-moderate COVID-19 in adults. Findings from in vitro experiments, in vivo studies and clinical trials reveal that Molnupiravir is effective against SARS-CoV-2 by inducing viral RNA mutagenesis, thereby giving rise to mutated complementary RNA strands that generate non-functional viruses. To date, the data collectively suggest that Molnupiravir possesses promising antiviral activity as well as favorable prophylactic efficacy, attributed to its effective mutagenic property of disrupting viral replication. This review discusses the mechanisms of action of Molnupiravir and highlights its clinical utility by disabling SARS-CoV-2 replication, thereby ameliorating COVID-19 severity. Despite relatively few short-term adverse effects thus far, further detailed clinical studies and long-term pharmacovigilance are needed in view of its mutagenic effects. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

► Show Figures

Figure 1

15 pages, 2496 KiB

Open AccessArticle

The Viral Susceptibility of the Haloferax Species

by Zaloa Aguirre Sourrouille, Sabine Schwarzer, Sebastian Lequime, Hanna M. Oksanen and Tessa E. F. Quax

Viruses 2022, 14(6), 1344; https://doi.org/10.3390/v14061344 - 20 Jun 2022

Cited by 5 | Viewed by 3179

Abstract

Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus–host model systems. [...] Read more.

Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus–host model systems. Euryarchaea belonging to the genus Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus–host interactions. Full article

(This article belongs to the Special Issue Archaeal Virology)

► Show Figures

Figure 1

Figure 1
A schematic overview of the virus–host screen. Step 1: Fresh virus stocks made from confluent or semi-confluent plates were prepared on their own host strains and the titers were determined on their own host strains. Step 2: 95 virus stocks (undiluted and 10−2 dilution) were spotted on lawns of 14 Haloferax strains. MGM medium was used as a negative control (CTL). Step 3: After incubation at 37 °C, all virus-Haloferax pairs that resulted in growth inhibition on the spot-on lawn-assay were further tested by plaque assay by making serial dilutions of the virus stock and plating with the Haloferax strains to be tested. Viral plaques observed on Haloferax were counted, the titers were determined, and positive virus-Haloferax pairs were noted in <a href="#viruses-14-01344-t001" class="html-table">Table 1</a>. Full article ">Figure 2
Phylogenetic tree of viruses belonging to the Haloferuviridae and Hafunaviridae families based on average nucleotide identity (ANI) values calculated using VIRIDIC software. Viruses infecting LR2-5 are surrounded by a box. Scale bar represents the number of substitutions per nucleotide position. Place of isolation: pink Senegal, purple Thailand, green Slovenia, orange Israel, and blue Italy. Full article ">Figure 3
(A) Isolation sites of the haloferacalesviruses, mincapviruses, and retbasiphovirus (see also <a href="#app1-viruses-14-01344" class="html-app">Supplementary Table S2</a>). Schemes of the viral morphologies that were observed in each group are also indicated (see also <a href="#viruses-14-01344-t001" class="html-table">Table 1</a>). LR2-5 infecting viruses are circled with a dash line. (B) Schematic representation of the myovirus and siphovirus virion morphologies (not in scale) and a typical genome organization consisting of different functional modules of tailed archaeal viruses. Full article ">Figure 4
Schematic genomic alignment of the (A) mincapviruses, and (B) haloferacalesviruses. Grey bars represent homologous genomic regions. The level of nucleotide identity is reflected by the intensity of grey. Genes encoding major capsid protein (orange) and adhesins (pink) are indicated. The LR2-5 infecting viruses are circled with dashed lines. Identical or very similar genomes are shown as one and virus names are separated by /. Figures are prepared with Easyfig. Full article ">Figure 5
Comparison of the adhesin and tail fiber gene sequence phylogenies to the ability of viruses to infect LR2-5. (a,c) Distribution of Markov jumps (95% HPD) for (i) real states (pink) and (ii) randomized states (grey). (b,d) Bayesian maximum clade credibility tree with discrete trait reconstruction based on adhesin or tail fiber gene sequences, respectively. Asterisk indicates node posterior probabilities higher than 0.8, and viruses in the boxes can infect LR2-5. Color code indicates the genus; blue Mincapvirus, brown Haloferacalesvirus. Full article ">

15 pages, 2426 KiB

Open AccessArticle

Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection

by Yunfei Tian, Yuxin Hao, Maoli Dong, Shuai Li, Dongyue Wang, Fei Jiang, Qingqing Wang, Xiaoli Hao, Yi Yang, Nanhua Chen, Jianzhong Zhu, Junqing Guo, Jiajun Wu, Shaobin Shang and Jiyong Zhou

Viruses 2022, 14(6), 1343; https://doi.org/10.3390/v14061343 - 20 Jun 2022

Cited by 5 | Viewed by 2824

Abstract

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal [...] Read more.

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147–161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination. Full article

(This article belongs to the Topic Veterinary Infectious Diseases)

► Show Figures

Figure 1

Figure 1
mAb 5F12 specifically recognizes recombinant and natural porcine CD69 protein. (A) Recombinant porcine CD69, expressed in pcDNA3.1-poCD69-transfected HEK-293T cells, was detected by mAb 5F12 against porcine CD69. The CD69-positive cells (white arrows) and negative cells were magnified. Scale bars, 40 μm. (B) mAb 5F12 specifically recognizes recombinant porcine CD69 in the cell lysates of pcDNA3.1-poCD69-transfected HEK-293T cells but not in pcDNA3.1-transfected or un-transfected HEK-293T cells (Mock) by Western blotting. (C,D) The expression of CD69 on pig lymphocytes was detected by mAb 5F12 and polyclonal anti-poCD69 (pAb) antibodies, followed by Alexa Fluor 488-labeled goat anti-mouse-IgG antibody, via flow cytometry after stimulation with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h. These CD69+ lymphocytes were gated on lymphocytes within the PBMCs of pigs. *** p < 0.001. Full article ">Figure 2
The identification of the epitope recognized by mAb 5F12 against CD69 by dot-ELISA. (A) Three overlapping peptides covering the CD69 polypeptide (aa 133–161) were synthesized and named Pep 1 (aa 133–147), Pep 2 (aa 140–154), Pep 3 (aa 147–161), an extra cysteine was added at the N terminus of each peptide for the conjugation of KLH. (B) These peptides were conjugated to KLH carrier and their reactivity with anti-poCD69 mAb 5F12 was determined by dot-ELISA. Full article ">Figure 3
Anti-poCD69 mAb 5F12 shows weak or no cross-reactivity with bovine, mouse, and chicken CD69. (A) Homologous comparison of CD69 polypeptide (aa 133–161) in pig samples and human, mouse, and bovine samples. The percentage of homology was indicated. (B) The cross-reactivities of Dylight®755-conjugated anti-poCD69 mAb 5F12 with PBMCs from mice, cows, and chickens were examined by flow cytometry after the in vitro stimulation of PBMCs with or without PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h (Unstim vs. PMA/Iono). The CD69+ lymphocytes were gated on lymphocytes within PBMCs. The data shown are representative of three independent experiments. Full article ">Figure 4
Anti-poCD69 mAb 5F12 is valid for the detection of CD69 expression on different leukocyte subsets of pigs. PBMCs from healthy pigs were stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h, then stained with a cocktail of antibodies containing Dylight®755-CD69, anti-porcine CD3, CD8α, CD4, and CD21 or a cocktail containing Dylight®755-CD69, SLA-II DR, CD163, and CD172a. The expression of CD69 on CD21+ B cells (gated on lymphocytes) and T cell subsets (CD4+, CD8+, CD4+CD8+, gated on CD3+ T lymphocytes), NK cells (gated on CD21−CD3−CD8α+ cells), monocytes/macrophages (MHCII+CD163+), and neutrophils (MHCII−CD163−CD172+) was determined by flow cytometry. Monocytes/macrophages and neutrophils were gated on leukocytes, excluding lymphocytes. Full article ">Figure 5
The detection of CD69 expression on T cells in different organs after PRRSV immunization and infection. PRRSV-free piglets were immunized intranasally and intramuscularly with the rJS-ORF2-6-CON vaccine or were administered RPMI-1640 (as the control), then challenged with a virulent PRRSV NADC30-like SD17-38 isolate on day 42 post-immunization. Peripheral blood, mediastinal lymph nodes (mLN), and spleens were harvested on days 7 and/or 14 post-challenge (dpc) and single-cell suspensions were prepared for the detection of CD69 expression on T cell subsets via flow cytometry. (A) Representative pseudocolor plots (left panel) and the dynamic changes (right panel) of total CD69+ lymphocytes in the PBMCs of unvaccinated (n = 3) and immunized pigs (n = 3) after challenge. (B,C) Representative dot-plots (left panel) and the comparison (right panel) of CD69+CD4+ and CD69+CD8+ T cells in the mLNs and spleens of unvaccinated (n = 3) and immunized pigs (n = 3) at 14 dpc. Data shown are mean ± SEM. ns, no statistical significance. * p < 0.05. Full article ">Figure 6
ASFV infection induces the early activation of different lymphocyte subsets in the blood and lymphoid organs of pigs. Healthy piglets were infected or not infected with the ASFV HLJ/18 strain by intramuscular injection. Peripheral blood, spleens, and submandibular lymph nodes were collected on day 5 after infection and single-cell suspensions were prepared for the detection of CD69 expression on different lymphocyte subsets, using flow cytometry. The percentages of CD69-positive NK cells, B cells, CD8, CD4, and CD4+CD8+ T cells, as well as γδ T cells in the PBMCs (A), spleen (B), and submandibular lymph node cells (C) were compared between mock-infected and ASFV-infected pigs (n = 3). Data shown are mean ± SEM. ns = no statistical significance. ** p < 0.01. Full article ">

14 pages, 1485 KiB

Open AccessArticle

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia

by Nina Kurucz, Jamie Lee McMahon, Allan Warchot, Glen Hewitson, Jean Barcelon, Frederick Moore, Jasmin Moran, Jessica J. Harrison, Agathe M. G. Colmant, Kyran M. Staunton, Scott A. Ritchie, Michael Townsend, Dagmar Meyer Steiger, Roy A. Hall, Sally R. Isberg and Sonja Hall-Mendelin

Viruses 2022, 14(6), 1342; https://doi.org/10.3390/v14061342 - 20 Jun 2022

Cited by 4 | Viewed by 1996

Abstract

The Kunjin strain of West Nile virus (WNV_KUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation [...] Read more.

The Kunjin strain of West Nile virus (WNV_KUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNV_KUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNV_KUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNV_KUN and MVEV transmission by FTA^TM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNV_KUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTA^TM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTA^TM card system as a sensitive and accurate method to monitor the transmission of WNV_KUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a “One Health” issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTA^TM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities. Full article

(This article belongs to the Section Invertebrate Viruses)

► Show Figures

Figure 1

16 pages, 3042 KiB

Open AccessArticle

The Potyviral Protein 6K1 Reduces Plant Proteases Activity during Turnip mosaic virus Infection

by Sayanta Bera, Gabriella D. Arena, Swayamjit Ray, Sydney Flannigan and Clare L. Casteel

Viruses 2022, 14(6), 1341; https://doi.org/10.3390/v14061341 - 20 Jun 2022

Cited by 12 | Viewed by 3052

Abstract

Potyviral genomes encode just 11 major proteins and multifunctionality is associated with most of these proteins at different stages of the virus infection cycle. Some potyviral proteins modulate phytohormones and protein degradation pathways and have either pro- or anti-viral/insect vector functions. Our previous [...] Read more.

Potyviral genomes encode just 11 major proteins and multifunctionality is associated with most of these proteins at different stages of the virus infection cycle. Some potyviral proteins modulate phytohormones and protein degradation pathways and have either pro- or anti-viral/insect vector functions. Our previous work demonstrated that the potyviral protein 6K1 has an antagonistic effect on vectors when expressed transiently in host plants, suggesting plant defenses are regulated. However, to our knowledge the mechanisms of how 6K1 alters plant defenses and how 6K1 functions are regulated are still limited. Here we show that the 6K1 from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and cysteine protease inhibitors when expressed in Nicotiana benthamiana relative to controls. 6K1 stability increased when cysteine protease activity was inhibited chemically, showing a mechanism to the rapid turnover of 6K1 when expressed in trans. Using RNAseq, qRT-PCR, and enzymatic assays, we demonstrate TuMV reprograms plant protein degradation pathways on the transcriptional level and increases 6K1 stability at later stages in the infection process. Moreover, we show 6K1 decreases plant protease activity in infected plants and increases TuMV accumulation in systemic leaves compared to controls. These results suggest 6K1 has a pro-viral function in addition to the anti-insect vector function we observed previously. Although the host targets of 6K1 and the impacts of 6K1-induced changes in protease activity on insect vectors are still unknown, this study enhances our understanding of the complex interactions occurring between plants, potyviruses, and vectors. Full article

(This article belongs to the Special Issue Molecular Plant-Virus Interactions)

► Show Figures

Figure 1

Figure 1
Ectopic expression of 6K1:GFP in Nicotiana benthamiana. The constructs GFP and 6K1:GFP were agroinfiltrated in N. benthamiana leaves. Western blots were performed with proteins extracted from the agroinfiltrated leaves collected over time. Anti-GFP antibodies were used in both western blots and Ponceau staining was performed to check for loading control. The top band in the 6K1:GFP blot represents 6K1 fused GFP. All western blots are representative of at least two replicates which each contained 3 plants per treatment. Full article ">Figure 2
6K1 expression inhibits transcripts related to protease inhibitors and jasmonic acid accumulation. RNA was extracted from N. benthamiana leaves transiently expressing the GFP or 6K1:GFP. Transcript abundance was measured using qRT-PCR of: (A) LOX1 and (B) LOX2, related to jasmonic acid biosynthesis; and of (C) Cystatin, a protease inhibitor. The relative quantification was performed by using actin as reference gene and GFP treatment as the calibrator. Each result is the mean from 5 replicated plants ± SE. The stars (*) denotes if the mean values were significantly different at p < 0.05 as determined from either t-test or Kruskal-Wallis test. (D) Samples were collected from leaves transiently expressing the GFP or 6K1:GFP, and protease activity was quantified. One-way ANOVA was used to determine there was no significant difference between means (n = 10; non-significant; mean ± SE). (E) The GFP or 6K1:GFP constructs were agroinfiltrated in N. benthamiana leaves with and without a cysteine protease inhibitor, E64. Proteins were extracted and SDS-PAGE gels were run with an equal volume of each sample (9 μL). Anti-GFP antibodies were used in both western blots and Ponceau staining was performed to check for loading control. The western blot is representative of at least two replicates which each contained 3 plants per treatment. Full article ">Figure 3
TuMV infection and aphid feeding significantly impacts protein degradation pathways in A. thaliana. RNA-seq was performed with mock-inoculated A. thaliana, A. thaliana one week after TuMV infection (TuMV), A. thaliana 48 h after infestation with the Myzus persicae aphid (Aphid), a vector of TuMV, or from plants with both treatments (Both). Each sample represented a pool of two plants and three samples were taken per treatment (N = 3, 6 plants total). Heatmaps show genes that where at least 1.5 times differentially expressed relative to mock (p-value < 0.1). Differentially expressed genes were grouped according to the following protein degradation pathways: (A) Autophagy, (B) Proteasome, (C) Protease inhibitors, and (D) Proteases. Full article ">Figure 4
TuMV infection decreases plant protease activity and increases 6K1 stability. N. benthamiana leaves were agro-inoculated with TuMV and 6K1:GFP or just 6K1:GFP. (A) Protease activity was quantified and 6K1:GFP quantified with (B) UV light at 120 hpi and (C) western immunoblots over time. For each sample, an equal volume (10 μL) was loaded into each well of an SDS-PAGE gel. Anti-GFP antibodies were used in both western blots and Ponceau staining was performed to check for loading control. Each result is mean from ten biological replicates in (A). All western blots are representative of at least two replicates which each contained three plants per treatment. A one-way ANOVA was used in (A) to check for significance among means (n = 10, mean ± SE, + indicated a p-value of <0.1). Full article ">Figure 5
6K1:GFP expression inhibits plant protease activity in infected plants and increases TuMV accumulation in systemic leaves. (A,B) GFP or, 6K1:GFP constructs were co-infiltrated with TuMV into N. benthamiana leaves. In the control treatment, only TuMV was agro-infiltrated. At 60 h post infiltrations, CP gene-specific primers were used for the quantification of viral RNA relative to the actin in the local (A) and systemic leaves (B). (C,D) In a separate experiment GFP and 6K1:GFP were agroinoculated into local leaves with TuMV (C) or in systemically infected leaves (D) and protease activity measured. Significance was determined using differences at a p-value of * < 0.05 and + < 0.1 as determined from a least significance difference (LSD) test (n = 5, mean ± SE; GLM performed for A and B; n = 10, mean ± SE; one-way ANOVA for C and D). NS indicates non-significant (1 A). Full article ">

11 pages, 2439 KiB

Open AccessArticle

Novel Bacteriophage Specific against Staphylococcus epidermidis and with Antibiofilm Activity

by Rima Fanaei Pirlar, Jeroen Wagemans, Luis Ponce Benavente, Rob Lavigne, Andrej Trampuz and Mercedes Gonzalez Moreno

Viruses 2022, 14(6), 1340; https://doi.org/10.3390/v14061340 - 20 Jun 2022

Cited by 13 | Viewed by 3129

Abstract

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health [...] Read more.

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from −20 °C to 50 °C) and pH values (pH 3–pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis. Full article

(This article belongs to the Section Bacterial Viruses)

► Show Figures

Figure 1

Figure 1
TEM image of the phage CUB-EPI_14 virions. Phage head (59 nm diameter) and tail (238 nm length and 10.7 nm width) measurements were determined with the image processing software ImageJ.JS [<a href="#B16-viruses-14-01340" class="html-bibr">16</a>]. Full article ">Figure 2
Adsorption (upper row) and one-step growth curves (lower row) of CUB-EPI_14 assessed on S. epidermidis strains SE14 (A,B), SE16 (C,D), and SE18 (E,F). Data are expressed as mean ± SD. Full article ">Figure 3
CUB-EPI_14 genome map. Each arrow represents a coding sequence. In red, genes encoding packaging and lysis-associated proteins are displayed; in green, structural proteins; and in blue, DNA- and metabolism-associated proteins (adapted from EasyFig). Full article ">Figure 4
Thermal and pH stability test of CUB-EPI_14. Temperature experiments were performed for 1 h and 24 h at pH 7. pH experiments were performed for 1 h and 24 h at room temperature (25 °C). Error bars represent SD. Full article ">Figure 5
Microcalorimetric heat-flow curves (µW over time) of planktonic (105 CFU/mL) SE14 (A), SE16 (C), and SE18 (E) co-incubated with phage CUB-EPI_14 at different titers (104 to 109 PFU/mL). GC, growth control; NC, negative control. Data of a representative experiment are reported. Time-killing curve of SE14 (B), SE16 (D), and SE18 (F) biofilms treated with CUB-EPI_14 (108 PFU/mL) and untreated monitored at 2 h intervals for the first 10 h and after 24 h. Data are expressed as mean ± SD. Multiple paired Student’s t-test was performed; p-values < 0.005 were considered significant (*). Full article ">

8 pages, 257 KiB

Open AccessArticle

Weight of Clinical and Social Determinants of Metabolic Syndrome in People Living with HIV

by Maria Mazzitelli, Paolo Fusco, Michele Brogna, Alfredo Vallone, Laura D’Argenio, Giuseppina Beradelli, Giuseppe Foti, Carmelo Mangano, Maria Stella Carpentieri, Lucio Cosco, Paolo Scerbo, Armando Priamo, Nicola Serrao, Antonio Mastroianni, Chiara Costa, Maria Teresa Tassone, Vincenzo Scaglione, Francesca Serapide, Enrico Maria Trecarichi and Carlo Torti

Viruses 2022, 14(6), 1339; https://doi.org/10.3390/v14061339 - 20 Jun 2022

Cited by 9 | Viewed by 2105

Abstract

Background. Comorbidities in people living with HIV (PLWH) represent a major clinical challenge today, and metabolic syndrome (MTBS) is one of the most important. Objective. Our objective was to assess the prevalence of MTBS and the role of both clinical/socio-behavioral risk factors for [...] Read more.

Background. Comorbidities in people living with HIV (PLWH) represent a major clinical challenge today, and metabolic syndrome (MTBS) is one of the most important. Objective. Our objective was to assess the prevalence of MTBS and the role of both clinical/socio-behavioral risk factors for MTBS in a cohort of PLWH. Methods. All PLWH, over 18 years of age, attending all Infectious Disease Units in Calabria Region (Southern Italy) for their routine checks from October 2019–January 2020 were enrolled. MTBS was defined by NCEP-ATP III criteria. Logistic regression analysis was performed to assess factors significantly associated with the main outcome (MTBS). Results. We enrolled 356 PLWH, mostly males (68.5%), with a mean age of 49 years (standard deviation: 12), including 98 subjects with and 258 without MTBS. At logistic regression analysis, a statistically significant association was found between MTBS and alcohol use, osteoporosis, polypharmacy, and a history of AIDS. Conclusions. Identifying and addressing risk factors, including those that are socio-behavioral or lifestyle-related, is crucial to prevent and treat MTBS. Our results suggest the importance of implementing educational/multidimensional interventions to prevent MTBS in PLWH, especially for those with particular risk factors (alcohol abuse, osteoporosis, previous AIDS events, and polypharmacy). Moreover, alcohol consumption or abuse should be routinely investigated in clinical practice. Full article

(This article belongs to the Special Issue HIV-1 Infection and Immunometabolism: Relevance to HIV Pathogenesis and Persistence)

16 pages, 1003 KiB

Open AccessReview

Shiftless, a Critical Piece of the Innate Immune Response to Viral Infection

by William Rodriguez and Mandy Muller

Viruses 2022, 14(6), 1338; https://doi.org/10.3390/v14061338 - 20 Jun 2022

Cited by 12 | Viewed by 3389

Abstract

Since its initial characterization in 2016, the interferon stimulated gene Shiftless (SHFL) has proven to be a critical piece of the innate immune response to viral infection. SHFL expression stringently restricts the replication of multiple DNA, RNA, and retroviruses with an extraordinary diversity [...] Read more.

Since its initial characterization in 2016, the interferon stimulated gene Shiftless (SHFL) has proven to be a critical piece of the innate immune response to viral infection. SHFL expression stringently restricts the replication of multiple DNA, RNA, and retroviruses with an extraordinary diversity of mechanisms that differ from one virus to the next. These inhibitory strategies include the negative regulation of viral RNA stability, translation, and even the manipulation of RNA granule formation during viral infection. Even more surprisingly, SHFL is the first human protein found to directly inhibit the activity of the -1 programmed ribosomal frameshift, a translation recoding strategy utilized across nearly all domains of life and several human viruses. Recent literature has shown that SHFL expression also significantly impacts viral pathogenesis in mouse models, highlighting its in vivo efficacy. To help reconcile the many mechanisms by which SHFL restricts viral replication, we provide here a comprehensive review of this complex ISG, its influence over viral RNA fate, and the implications of its functions on the virus-host arms race for control of the cell. Full article

(This article belongs to the Special Issue Signaling Pathways in Viral Infection and Antiviral Immunity)

► Show Figures

Figure 1

Figure 1
Shiftless Protein Structure. Shiftless is a 291aa long protein of 33 kDa molecular weight. (A) Highlighted in Red is the PABPC-binding domain (PABPC-BD) which also encompasses the Zinc-Ribbon Domain (112–135) and the Nuclear Localization Signal (121–173). Highlighted in Blue is the -1 Programmed Ribosomal Frameshift (PRF) (169–199). Highlighted in Yellow is the C-terminal domain containing the Glutamic Acid (E)-Rich Domain (270–286) and the Nuclear Export Signal (261–269). (B) Alphafold2 predicted protein structure of Shiftless. The PABPC-BD, -1PRF Domain, and the C-terminal Domain are Red, Blue, and Yellow respectively. Also highlighted in orange is the Nuclear Localization Signal and in pink is the Nuclear Export Signal [<a href="#B26-viruses-14-01338" class="html-bibr">26</a>,<a href="#B27-viruses-14-01338" class="html-bibr">27</a>]. Full article ">Figure 2
Shiftless Mechanisms of Restricting Viral Infection. Shiftless (SHFL) has been demonstrated to be a potent broad-spectrum anti-viral factor that is upregulated in response to viral infection and interferon signaling. SHFL restricts viral infection through several mechanisms summarized here: (1) SHFL directly interfaces with viral genomic RNA and viral mRNA and restricts viral gene expression at various stages between RNA stability and translation. For flaviviruses, SHFL binds to viral genomic RNA at the 3′ end and may relocalize it to Processing bodies to trigger RNA decay or directly interfere with polyprotein translation. (2) SHFL is one of the first human proteins shown to directly restrict the function of the -1 programmed ribosomal frameshift (-1PRF), a cis-RNA element that extends the coding capacity of several retro- and RNA viruses. The -1PRF signal triggers a non-canonical ribosome rotation, signaling the recruitment of SHFL and eukaryotic ribosome release factors (eRF1/eRF3), which synergistically halt and then trigger the premature release of the ribosome from frameshifting viral RNA. (3) For HCV and YFV, SHFL expression decreases the level of PI(4)P in the cell, a lipid precursor that directly contributes to the formation of the viral membranous web, a collection of reorganized lipid membranes that house viral replication compartments. SHFL was also shown to interact with and localize to Stress Granules during HCV infection. (4) Lastly, SHFL has been recently shown to bind to and trigger the degradation of select viral proteins through lysosomal or ubiquitination based pathways. Abbreviations: Shiftless (SHFL), Dengue virus (DENV), Encephalomyocarditis virus (EMCV), Yellow Fever virus (YFV), Hepatitis C virus (HCV), Kaposi’s sarcoma-associated herpesvirus (KSHV), Human Immunodeficiency virus 1 (HIV-1), Japanese Encephalitis virus (JEV), Zika virus (ZIKV), Porcine Epidemic Diarrhea virus (PEDV), Poly-A Binding Protein (PABP), La-associated RNA binding protein (LARP), eukaryotic Initiation Factor 4G (eIF4G), eukaryotic Initiation Factor 4E (eIF4E), Processing bodies (P-bodies). Full article ">

17 pages, 1228 KiB

Open AccessReview

Extracellular Polymeric Substances: Still Promising Antivirals

by Raquel Bello-Morales, Sabina Andreu, Vicente Ruiz-Carpio, Inés Ripa and José Antonio López-Guerrero

Viruses 2022, 14(6), 1337; https://doi.org/10.3390/v14061337 - 19 Jun 2022

Cited by 12 | Viewed by 3254

Abstract

Sulfated polysaccharides and other polyanions have been promising candidates in antiviral research for decades. These substances gained attention as antivirals when they demonstrated a high inhibitory effect in vitro against human immunodeficiency virus (HIV) and other enveloped viruses. However, that initial interest was [...] Read more.

Sulfated polysaccharides and other polyanions have been promising candidates in antiviral research for decades. These substances gained attention as antivirals when they demonstrated a high inhibitory effect in vitro against human immunodeficiency virus (HIV) and other enveloped viruses. However, that initial interest was followed by wide skepticism when in vivo assays refuted the initial results. In this paper we review the use of sulfated polysaccharides, and other polyanions, in antiviral therapy, focusing on extracellular polymeric substances (EPSs). We maintain that, in spite of those early difficulties, the use of polyanions and, specifically, the use of EPSs, in antiviral therapy should be reconsidered. We base our claim in several points. First, early studies showed that the main disadvantage of sulfated polysaccharides and polyanions is their low bioavailability, but this difficulty can be overcome by the use of adequate administration strategies, such as nebulization of aerosols to gain access to respiratory airways. Second, several sulfated polysaccharides and EPSs have demonstrated to be non-toxic in animals. Finally, these macromolecules are non-specific and therefore they might be used against different variants or even different viruses. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

► Show Figures

Figure 1

Figure 1
Mechanisms of antiviral action of microbial exopolymers. EPSs can exert their antiviral activity via different mechanisms. Virucidal agents act by inactivating viruses. Inactivation can occur not only by damaging the virions, but also by blocking them and impeding the adsorption of virions to cells (red cross). Some EPSs can wrap the virions via electrostatic interactions, thus preventing viral adsorption and, therefore, exerting a virucidal effect (1). Other EPSs exert an antiviral effect allowing the viral entry but later impeding the viral replication. Therefore, antivirals inhibit replication of viable viruses in the cells (2). EPSs can also exert antiviral effect by activating immune cells and inducing them to secrete immunomodulators (IMs) and to kill virus-infected cells (3). Full article ">Figure 2
Schematic diagram representing the mechanistic basis of the inhibitory effect of EPSs and other antiviral polyanions on viral adsorption. The figure represents a prototypical polymer interacting with a SARS-CoV-2 virion. The main inhibitory mechanisms include non-covalent and non-specific electrostatic interaction between the negatively charged moieties of the EPSs and the positively charged viral glycoproteins. Viral glycoproteins might also interact with H-bond acceptors of the polymer. Full article ">Figure 3
Alternative routes of administration of EPSs and other polyanions. (A) To overcome the low bioavailability of EPSs, new routes of administration should be tested, especially nebulization of aerosols to access pulmonary alveoli. (B) Aerial virions can be trapped by EPSs before attachment in the respiratory epithelium. To enter cells, viral glycoproteins must first attach to the host cell receptors. EPSs may block the viral glycoproteins and prevent viral entry (red cross). Full article ">

24 pages, 1250 KiB

Open AccessReview

Detection of Ancient Viruses and Long-Term Viral Evolution

by Luca Nishimura, Naoko Fujito, Ryota Sugimoto and Ituro Inoue

Viruses 2022, 14(6), 1336; https://doi.org/10.3390/v14061336 - 18 Jun 2022

Cited by 8 | Viewed by 6083

Abstract

The COVID-19 outbreak has reminded us of the importance of viral evolutionary studies as regards comprehending complex viral evolution and preventing future pandemics. A unique approach to understanding viral evolution is the use of ancient viral genomes. Ancient viruses are detectable in various [...] Read more.

The COVID-19 outbreak has reminded us of the importance of viral evolutionary studies as regards comprehending complex viral evolution and preventing future pandemics. A unique approach to understanding viral evolution is the use of ancient viral genomes. Ancient viruses are detectable in various archaeological remains, including ancient people’s skeletons and mummified tissues. Those specimens have preserved ancient viral DNA and RNA, which have been vigorously analyzed in the last few decades thanks to the development of sequencing technologies. Reconstructed ancient pathogenic viral genomes have been utilized to estimate the past pandemics of pathogenic viruses within the ancient human population and long-term evolutionary events. Recent studies revealed the existence of non-pathogenic viral genomes in ancient people’s bodies. These ancient non-pathogenic viruses might be informative for inferring their relationships with ancient people’s diets and lifestyles. Here, we reviewed the past and ongoing studies on ancient pathogenic and non-pathogenic viruses and the usage of ancient viral genomes to understand their long-term viral evolution. Full article

(This article belongs to the Special Issue Virus Bioinformatics 2022)

► Show Figures

Figure 1

15 pages, 1170 KiB

Open AccessReview

Exploring the Mumps Virus Glycoproteins: A Review

by Jasmine Rae Frost, Saba Shaikh and Alberto Severini

Viruses 2022, 14(6), 1335; https://doi.org/10.3390/v14061335 - 18 Jun 2022

Cited by 6 | Viewed by 2645

Abstract

The resurgence of mumps in vaccinated adult populations has raised concerns about possible waning vaccine immunity or a potential lack of protection to the circulating strain. A number of individual studies have investigated if there are amino acid variations between the circulating wild-type [...] Read more.

The resurgence of mumps in vaccinated adult populations has raised concerns about possible waning vaccine immunity or a potential lack of protection to the circulating strain. A number of individual studies have investigated if there are amino acid variations between the circulating wild-type strains and vaccine strains. In these studies, the HN and F mumps surface glycoproteins have been of interest, because of their role in viral infection, and because the HN protein is the target of neutralizing antibodies. Here, we summarize the single nucleotide variants and their potential effect that have been identified between mumps genotypes in the HN and F proteins. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

► Show Figures

Figure 1

Figure 1
Protein structures generated from Protein Data Base file 5B2D [<a href="#B26-viruses-14-01335" class="html-bibr">26</a>]. (A) Protein binding sites and N-glycosylation sites of mumps HN protein (front view). Illustrated are the HN protein (purple), N-acetyl-D-glucosamine binding sites (pink), and sialic acid receptor binding sites (green). Different N-glycosylation sites are labelled with arrows and boxes indicate the amino acid range. (B) Protein binding sites and N-glycosylation sites of mumps HN protein (back view). Illustrated are the HN protein (purple), N-acetyl-D-glucosamine binding sites (pink), and sialic acid receptor binding sites (green). Different N-glycosylation sites are labelled with arrows and boxes indicate the amino acid range. Full article ">Figure 2
Protein structures generated from Protein Data Base file 5B2D [<a href="#B26-viruses-14-01335" class="html-bibr">26</a>]. (A) Protein binding sites and known epitopes of mumps HN protein (Front view). Protein binding sites and N-glycosylation sites of Mumps HN Protein (back view). Illustrated are the HN protein (purple), N-acetyl-D-glucosamine binding sites (pink), and sialic acid receptor binding sites (green). Different epitope locations are labelled with arrows and boxes indicate the amino acid range. (B) Protein binding sites and known epitopes of mumps HN protein (back view). Protein binding sites and N-glycosylation sites of mumps HN Protein (back view). Illustrated are the HN protein (purple), N-acetyl-D-glucosamine binding sites (pink), and sialic acid receptor binding sites (green). Different epitope locations are labelled with arrows and boxes indicate the amino acid range. Full article ">

10 pages, 2907 KiB

Open AccessArticle

Resistance of SARS-CoV-2 Omicron BA.1 and BA.2 Variants to Vaccine-Elicited Sera and Therapeutic Monoclonal Antibodies

by Hao Zhou, Belinda M. Dcosta, Nathaniel R. Landau and Takuya Tada

Viruses 2022, 14(6), 1334; https://doi.org/10.3390/v14061334 - 18 Jun 2022

Cited by 55 | Viewed by 4147

Abstract

The recent emergence of the Omicron BA.1 and BA.2 variants with heavily mutated spike proteins has posed a challenge to the effectiveness of current vaccines and to monoclonal antibody therapy for severe COVID-19. After two immunizations of individuals with no history of previous [...] Read more.

The recent emergence of the Omicron BA.1 and BA.2 variants with heavily mutated spike proteins has posed a challenge to the effectiveness of current vaccines and to monoclonal antibody therapy for severe COVID-19. After two immunizations of individuals with no history of previous SARS-CoV-2 infection with BNT162b2 vaccine, neutralizing titer against BA.1 and BA.2 were 20-fold decreased compared to titers against the parental D614G virus. A third immunization boosted overall neutralizing titers by about 5-fold but titers against BA.1 and BA.2 remained about 10-fold below that of D614G. Both Omicron variants were highly resistant to several of the emergency use authorized therapeutic monoclonal antibodies. The variants were highly resistant to Regeneron REGN10933 and REGN10987 and Lilly LY-CoV555 and LY-CoV016 while Vir-7831 and the mixture of AstraZeneca monoclonal antibodies AZD8895 and AZD1061 were significantly decreased in neutralizing titer. Strikingly, a single monoclonal antibody LY-CoV1404 potently neutralized both Omicron variants. Full article

(This article belongs to the Special Issue Basic Sciences for the Conquest of COVID-19)

► Show Figures

Figure 1

Figure 1
Decreased neutralization of Omicron BA.1 and BA.2 pseudotyped viruses by mRNA vaccine-elicited antibodies. (A) The structure of the SARS-CoV-2 Omicron BA.2 spike is indicated. NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; SD1 subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain. Novel mutations found in BA.2 are shown in red. The mutations which are specific to BA.1 are shown in blue. (B) D614G, Omicron BA.1 and BA.2 pseudotyped viruses expressing dual GFP/nanoluciferase reporter genes with codon-optimized spike proteins were described previously [<a href="#B19-viruses-14-01334" class="html-bibr">19</a>]. Virus were incubated with a 2-fold serial dilution of serum for 30 min and applied to target cells. Luciferase activity was measured two days post-infection. Each serum dilution was measured in duplicates and the experiment was done twice with similar results. Statistical significance was calculated by two-sided testing. (**** p ≤ 0.0001). Neutralizing antibody titers of participants without (n = 9) or with (n = 7) SARS-CoV-2 infection were measured with pseudotyped viruses. Sera were collected from participants 1-month post-second vaccination with Pfizer BNT162b2 1-month post-boost. COVID-19 history was determined by symptoms and a PCR+ test or serology. The sera from SARS-CoV-2 experienced were collected prior to February 2021. Thus, the individuals would have been infected with D614G, Alpha or Iota variant. Full article ">Figure 2
Neutralization of BA.2 by therapeutic monoclonal antibodies. (A) Lentiviral pseudotyped viruses were generated as previously described using codon-optimized 19 amino acid deleted spike proteins [<a href="#B24-viruses-14-01334" class="html-bibr">24</a>]. A fixed amount of virus, normalized for reverse transcriptase activity, was treated with 5-fold serially diluted monoclonal antibody, in duplicate, for 30 min and then used to infect ACE2.293T cells. Luciferase activity was measured after 24 h and the data are plotted as curves neutralized to luciferase activity in the absence of antibody. (B) The IC50 was calculated from the neutralization curves by GraphPad Prism 8 software. Values that approached 50% neutralization were estimated, as indicated by an asterisk (*); those that did not approach 50% neutralization were not determined (ND). IC50s with >5-fold decrease in neutralizing titer are shown in orange. Full article ">Figure 3
Neutralization of point mutated virus BA.2 by therapeutic monoclonal antibodies. (A) Individual point mutated viruses were treated with 5-fold serially diluted monoclonal antibodies and then used to infect ACE2.293T cells. Luciferase activity was measured after 24 h. (B) The IC50 was calculated from the neutralization curves using GraphPad Prism 8 software. Mutations found to cause >5-fold decrease in neutralizing titer are shown in orange. Values that did not approach 50% neutralization were not determined (ND). Full article ">Figure 4
The location of mutations that affect monoclonal antibody binding is shown on the antibody:spike protein complex. Complexes were visualized with PyMOL Molecular Graphics System, v2.1.1 (Schrödinger, LLC) software. Mutations in BA.1 that are also in BA.2 previously reported [<a href="#B12-viruses-14-01334" class="html-bibr">12</a>,<a href="#B22-viruses-14-01334" class="html-bibr">22</a>,<a href="#B23-viruses-14-01334" class="html-bibr">23</a>] to have >5-fold effect on neutralizing titer are shown in red. Full article ">

17 pages, 6084 KiB

Open AccessArticle

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years

by Jean-Sébastien Reynard, Justine Brodard, Vivian Zufferey, Markus Rienth, Paul Gugerli, Olivier Schumpp and Arnaud G. Blouin

Viruses 2022, 14(6), 1333; https://doi.org/10.3390/v14061333 - 18 Jun 2022

Cited by 4 | Viewed by 2607

Abstract

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact [...] Read more.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant. Full article

(This article belongs to the Special Issue Closteroviridae)

► Show Figures

Figure 1

10 pages, 1688 KiB

Open AccessArticle

Developing Pseudovirus-Based Neutralization Assay against Omicron-Included SARS-CoV-2 Variants

by Hancong Sun, Jinghan Xu, Guanying Zhang, Jin Han, Meng Hao, Zhengshan Chen, Ting Fang, Xiangyang Chi and Changming Yu

Viruses 2022, 14(6), 1332; https://doi.org/10.3390/v14061332 - 18 Jun 2022

Cited by 16 | Viewed by 4066

Abstract

The global spread of SARS-CoV-2 and its variants poses a serious threat to human health worldwide. Recently, the emergence of Omicron has presented a new challenge to the prevention and control of the COVID-19 pandemic. A convenient and reliable in vitro neutralization assay [...] Read more.

The global spread of SARS-CoV-2 and its variants poses a serious threat to human health worldwide. Recently, the emergence of Omicron has presented a new challenge to the prevention and control of the COVID-19 pandemic. A convenient and reliable in vitro neutralization assay is an important method for validating the efficiency of antibodies, vaccines, and other potential drugs. Here, we established an effective assay based on a pseudovirus carrying a full-length spike (S) protein of SARS-CoV-2 variants in the HIV-1 backbone, with a luciferase reporter gene inserted into the non-replicate pseudovirus genome. The key parameters for packaging the pseudovirus were optimized, including the ratio of the S protein expression plasmids to the HIV backbone plasmids and the collection time for the Alpha, Beta, Gamma, Kappa, and Omicron pseudovirus particles. The pseudovirus neutralization assay was validated using several approved or developed monoclonal antibodies, underscoring that Omicron can escape some neutralizing antibodies, such as REGN10987 and REGN10933, while S309 and ADG-2 still function with reduced neutralization capability. The neutralizing capacity of convalescent plasma from COVID-19 convalescent patients in Wuhan was tested against these pseudoviruses, revealing the immune evasion of Omicron. Our work established a practical pseudovirus-based neutralization assay for SARS-CoV-2 variants, which can be conducted safely under biosafety level-2 (BSL-2) conditions, and this assay will be a promising tool for studying and characterizing vaccines and therapeutic candidates against Omicron-included SARS-CoV-2 variants. Full article

(This article belongs to the Special Issue What SARS-CoV-2 Variants Have Taught Us: Evolutionary Challenges of RNA Viruses)

► Show Figures

Figure 1

15 pages, 2111 KiB

Open AccessArticle

Evaluation of New Polyclonal Antibody Developed for Serological Diagnostics of Tomato Mosaic Virus

by Michaela Mrkvová, Richard Hančinský, Simona Grešíková, Šarlota Kaňuková, Ján Barilla, Miroslav Glasa, Pavol Hauptvogel, Ján Kraic and Daniel Mihálik

Viruses 2022, 14(6), 1331; https://doi.org/10.3390/v14061331 - 18 Jun 2022

Cited by 13 | Viewed by 2830

Abstract

Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to [...] Read more.

Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus’ presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters (sensitivity, specificity, and cross-reactivity) were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential (sequences are revealed) derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant. Full article

(This article belongs to the Special Issue State-of-the-Art Virology Research in Slovakia)

► Show Figures

Figure 1

Figure 1
Regions of the ToMV SL-1 genome targeted by primer pairs (<a href="#viruses-14-01331-t001" class="html-table">Table 1</a>). Full article ">Figure 2
Sensitivity of the compared antibodies in the detection of ToMV by the ELISA assay in differently diluted protein lysates from ToMV-infected tomato plants (mean values and standard deviation error bars). Full article ">Figure 3
Sensitivity of the differently diluted compared antibodies in ToMV-infected tomato plants by the ELISA assay using five antibodies (new-developed AB-1, three commercial against ToMV—Bioreba ToMV, DSMZ ToMV, Loewe ToMV, and one commercial against TMV—Bioreba TMV) (mean values and standard deviation error bars). Full article ">Figure 4
ELISA detection of ToMV at different times after plant inoculation with virus using three antibodies (new-developed AB-1, two commercial—Bioreba, DSMZ) (mean values and standard deviation error bars). Full article ">Figure 5
Reactivity of AB-1 antibody with ToMV isolates, TMV, and PMMoV using Western immunoblotting analysis. Lanes: 1—protein ladder, 2—negative control, 3—ToMV PV-0141, 4—ToMV PV-0143, 5—ToMV PV-0846, 6—ToMV PV-1180, 7—ToMV PV-0135, 8—ToMV SL-1, 9—PMMoV PV-0166, and 10—TMV PV-0107. Full article ">Figure 6
Symptoms of the ToMV SL-1 isolate in tomato, pepper, and eggplant. (A)—symptomatic infected tomato plant (left in pair) and control plant (right in pair), and leaf mosaic symptoms (right), (B)—symptomatic infected pepper plant (left in pair), control plant (right in pair), and leaf shedding from infected plant (right), (C)—symptomatic infected eggplant plant (left in pair), control plant (right in pair), and mild chlorosis on leaf (right), (D)—Western immunoblot detection of ToMV in plants of tomato, pepper, and eggplant. Lanes: 1—protein ladder, 2—positive control (virus), 3—infected tomato, 4—negative control tomato, 5—infected pepper, 6—negative control pepper, 7—infected eggplant, and 8—negative control eggplant. Full article ">Figure 7
Detection of ToMV SL-1 in roots and seeds of infected tomatoes using the AB-1 antibody (mean values and standard deviation error bars). Full article ">Figure 8
Persistence and resilience of ToMV in soil rhizosphere and root residues analyzed by Western immunoblots 24 months after the removal of the infected tomato plants from the soil. (A)—rhizosphere sample, (B)—root residues. Lanes: 1—protein ladder, 2—negative control, 3—positive control (ToMV SL-1), 4—line 706/15, 5—line 730/15, 6—line 756/15, 7—line 762/15, 8—Mobaci variety, and 9—Monalbo variety. (C)—Same rhizosphere samples after sterilization (tomato line 706/15), (D)— same root residues after sterilization (tomato line 706/15). Lanes: 1—protein ladder, 2—negative control, 3—positive control (before sterilization), 4—sterilization for 1 min, 5—sterilization for 2 min, 6—sterilization for 5 min, 7—sterilization for 10 min, and 8—sterilization for 20 min. Full article ">

13 pages, 6507 KiB

Open AccessArticle

SARS-CoV-2, Placental Histopathology, Gravity of Infection and Immunopathology: Is There an Association?

by Leonardo Resta, Antonella Vimercati, Gerardo Cazzato, Margherita Fanelli, Sara Vincenza Scarcella, Giuseppe Ingravallo, Anna Colagrande, Sara Sablone, Mary Stolfa, Francesca Arezzo, Teresa Lettini and Roberta Rossi

Viruses 2022, 14(6), 1330; https://doi.org/10.3390/v14061330 - 18 Jun 2022

Cited by 14 | Viewed by 2472

Abstract

(1) Background: As the pandemic months progress, more and more evidence shows that the placenta acts as a “barrier” to SARS-CoV-2, although rare cases of vertical transmission have been described. (2) Methods: In an attempt to investigate whether the symptoms’ severity was related [...] Read more.

(1) Background: As the pandemic months progress, more and more evidence shows that the placenta acts as a “barrier” to SARS-CoV-2, although rare cases of vertical transmission have been described. (2) Methods: In an attempt to investigate whether the symptoms’ severity was related to different placental histological characteristics and the immune microenvironment, we subdivided 29 placentas from 29 mothers positive for SARS-CoV-2 into two groups, depending on the symptomatology (moderate/severe vs. asymptomatic/mild), performing immunohistochemical investigations for CD4 + and CD8 + T lymphocytes, as well as for CD68 + macrophage. We also evaluated the immuno-expression of the ACE2 receptor at the placental level. These two groups were compared to a control group of 28 placentas from 28 SARS-CoV-2-negative healthy mothers. (3) Results: The symptoms (likely to be related to viremia) were statistically significantly correlated (p < 0.05) with histopathological changes, such as maternal malperfusion, decidual arteriopathy, blood vessel thrombus of fetal relevance. Furthermore, the immuno-expression of ACE2 was significantly lower in SARS-CoV-2-positive groups vs. control group (p = 0.001). (4) Conclusions: There is still much to study and discover regarding the relationship between SARS-CoV-2 and histological changes in placentas and how the latter might contribute to various neonatal clinical outcomes, such as prematurity. Full article

(This article belongs to the Section SARS-CoV-2 and COVID-19)

► Show Figures

Figure 1

13 pages, 1816 KiB

Open AccessArticle

Identification of Phage Receptor-Binding Protein Sequences with Hidden Markov Models and an Extreme Gradient Boosting Classifier

by Dimitri Boeckaerts, Michiel Stock, Bernard De Baets and Yves Briers

Viruses 2022, 14(6), 1329; https://doi.org/10.3390/v14061329 - 17 Jun 2022

Cited by 13 | Viewed by 4900

Abstract

Receptor-binding proteins (RBPs) of bacteriophages initiate the infection of their corresponding bacterial host and act as the primary determinant for host specificity. The ever-increasing amount of sequence data enables the development of predictive models for the automated identification of RBP sequences. However, the [...] Read more.

Receptor-binding proteins (RBPs) of bacteriophages initiate the infection of their corresponding bacterial host and act as the primary determinant for host specificity. The ever-increasing amount of sequence data enables the development of predictive models for the automated identification of RBP sequences. However, the development of such models is challenged by the inconsistent or missing annotation of many phage proteins. Recently developed tools have started to bridge this gap but are not specifically focused on RBP sequences, for which many different annotations are available. We have developed two parallel approaches to alleviate the complex identification of RBP sequences in phage genomic data. The first combines known RBP-related hidden Markov models (HMMs) from the Pfam database with custom-built HMMs to identify phage RBPs based on protein domains. The second approach consists of training an extreme gradient boosting classifier that can accurately discriminate between RBPs and other phage proteins. We explained how these complementary approaches can reinforce each other in identifying RBP sequences. In addition, we benchmarked our methods against the recently developed PhANNs tool. Our best performing model reached a precision-recall area-under-the-curve of 93.8% and outperformed PhANNs on an independent test set, reaching an F1-score of 84.0% compared to 69.8%. Full article

(This article belongs to the Special Issue Virus Bioinformatics 2022)

► Show Figures

Figure 1

Figure 1
A schematic overview of data processing steps. Collected phage genomes from INPHARED were processed and coding sequences were extracted from each genome. Coding sequences were divided into two groups based on a regular expression that covered the various annotations of RBPs. Both groups were further processed to exclude sequences that had unknown amino acids, unwanted keywords, or extreme lengths. Full article ">Figure 2
Visualization of the iterative manual search of RBP-related protein domains in the Pfam database, starting from the Phage_T7_tail domain. The iterative procedure entailed alternately identifying domains at the N- or C-terminus that were linked to the domains identified in the previous iteration of the manual search. Full article ">Figure 3
Count plot of the identified RBP-related HMMs from the Pfam database, grouped into N-terminal domains (top) and C-terminal domains (bottom). At the N-terminus, two domains occur substantially more than all the others (namely Phage_T7_tail and Tail_spike_N). At the C-terminus, Peptidase_S74 is the most occurring domain. Full article ">Figure 4
Architectures of the RBPs detected with RBP-related HMMs from the Pfam database, together with their number of occurrences and divided into three categories (N-terminal domains, cleaving domains, and C-terminal binding domain or chaperone). Examples of N-terminal domains are Phage_T7_tail and Tail_spike_N. Examples of cleaving domains are Lipase_GDSL_2 and Pectate_lyase_3. Examples of binding domains and chaperones are Phage_fiber_C and CBM_4_9. Only the architectures that occurred more than five times were visualized. The top-five occurring architectures are single-domain architectures, indicating that there are many unknown RBP-related protein domains in the Pfam database. Full article ">Figure 5
Venn diagram of the concordance between correct positive predictions made by the three benchmarked methods: our domain-based approach, our XGBoost classifier, and PhANNs. Full article ">

11 pages, 1443 KiB

Open AccessArticle

Human Intramuscular Hyperimmune Gamma Globulin (hIHGG) Anti-SARS-CoV-2—Characteristics of Intermediates and Final Product

by Elzbieta Lachert, Joanna Lasocka, Artur Bielawski, Ewa Sulkowska, Katarzyna Guz, Krzysztof Pyrc, Agnieszka Dabrowska, Agata Wawryniuk-Malmon, Magdalena Letowska, Krzysztof Tomasiewicz and Piotr Grabarczyk

Viruses 2022, 14(6), 1328; https://doi.org/10.3390/v14061328 - 17 Jun 2022

Cited by 2 | Viewed by 2640

Abstract

This study aims to characterize the intermediates, and the final product (FP) obtained during the production of human intramuscular hyperimmune gamma globulin anti-SARS-CoV-2 (hIHGG anti-SARS-CoV-2) and to determine its stability. Material and methods: hIHGG anti-SARS-CoV-2 was fractionated from 270 convalescent plasma donations [...] Read more.

This study aims to characterize the intermediates, and the final product (FP) obtained during the production of human intramuscular hyperimmune gamma globulin anti-SARS-CoV-2 (hIHGG anti-SARS-CoV-2) and to determine its stability. Material and methods: hIHGG anti-SARS-CoV-2 was fractionated from 270 convalescent plasma donations with the Cohn method. Prior to fractionation, the plasma was inactivated (Theraflex MB Plasma). Samples were defined using enzyme immunoassays (EIA) for anti-S1, anti-RBD S1, and anti-N antibodies, and neutralization assays with SARS-CoV-2 (VN) and pseudoviruses (PVN, decorated with SARS-CoV-2 S protein). Results were expressed as a titer (EIA) or 50% of the neutralization titer (IC₅₀) estimated in a four-parameter nonlinear regression model. Results: Concentration of anti-S1 antibodies in plasma was similar before and after inactivation. Following fractionation, the anti-S1, anti-RBD, and anti-N (total tests) titers in FP were concentrated approximately 15-fold from 1:4 to 1:63 (1800 BAU/mL), 7-fold from 1:111 to 1:802 and from 1:13 to 1:88, respectively. During production, the IgA (anti-S1) antibody titer was reduced to an undetectable level and the IgM (anti-RBD) titer from 1:115 to 1:24. The neutralizing antibodies (nAb) titer increased in both VN (from 1:40 to 1:160) and PVN (IC₅₀ from 63 to 313). The concentration of specific IgG in the FP did not change significantly for 14 months. Conclusions: The hIHGG anti-SARS-CoV-2 was stable, with concentration up to approximately 15-fold nAb compared to the source plasma pool. Full article

(This article belongs to the Special Issue Transfusion Transmitted Viral Infections)

► Show Figures

Figure 1

Figure 1
hIHGG anti-SARS-CoV-2 production. Full article ">Figure 2
Histogram of anti-SARS-CoV-2 antibodies reactivity in EIA assays in convalescent plasma units used for the production of hIHGG anti-SARS-CoV-2: (a) Wantai anti-S1 RBD Ab Total—IgM, IgA and IgG (n = 270, S/Co < 1 negative result, S/Co > 19.9 over result); (b) iFlash anti-S1 IgG (n = 206, <10 AU/mL—negative); (c) Euroimmun anti-S1 IgG (n = 265, S/Co < 0.8 negative, 0.8–1.1 gray zone, >1.1 positive); (d) Euroimmun IgA (n = 177, S/Co < 0.8 negative, 0.8–1.1 gray zone, >1.1 positive). Above bars % of tested samples is presented. Full article ">Figure 2 Cont.
Histogram of anti-SARS-CoV-2 antibodies reactivity in EIA assays in convalescent plasma units used for the production of hIHGG anti-SARS-CoV-2: (a) Wantai anti-S1 RBD Ab Total—IgM, IgA and IgG (n = 270, S/Co < 1 negative result, S/Co > 19.9 over result); (b) iFlash anti-S1 IgG (n = 206, <10 AU/mL—negative); (c) Euroimmun anti-S1 IgG (n = 265, S/Co < 0.8 negative, 0.8–1.1 gray zone, >1.1 positive); (d) Euroimmun IgA (n = 177, S/Co < 0.8 negative, 0.8–1.1 gray zone, >1.1 positive). Above bars % of tested samples is presented. Full article ">Figure 3
Comparison of iFlash test reactivity (anti-S1 SARS-CoV-2 IgG antibody assay) in 206 CP donations before and after inactivation—no statistically significant differences in the pairwise Wilcoxon test were observed, p = 0.502. Full article ">

20 pages, 3353 KiB

Open AccessArticle

Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner

by Liubov Cherkashchenko, Kai Rausalu, Sanjay Basu, Luke Alphey and Andres Merits

Viruses 2022, 14(6), 1327; https://doi.org/10.3390/v14061327 - 17 Jun 2022

Cited by 8 | Viewed by 3000

Abstract

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the [...] Read more.

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Figure 1

Figure 1
Schematic overview of used plasmids and verification of nsP2 expression in transfected C6/36 cells. (A). Trans-replicase plasmid for expression of alphavirus ns-polyprotein. Ubi—full-length Aedes aegypti polyubiquitin promoter; UL—transcribed leader of polyubiquitin gene containing naturally occurring intron; SV40Ter—SV40 late polyadenylation region. (B) Constructs expressing template RNAs for trans-replicases. AlbPolI—truncated (−250 to −1) promoter for Aedes albopictus RNA polymerase I; AlbTer—tentative terminator for Aedes albopictus RNA polymerase I. The 5′ and 3′ UTRs and SG promoter are from CHIKV, SINV, SFV, RRV, MAYV, EILV, VEEV or EEEV; nsP1N—region encoding for the N-terminal region of nsP1; HDV RZ—antisense strand ribozyme of hepatitis delta virus. (C). Constructs expressing nsP2 of CHIKV or SINV. nsP1*—region encoding for 10 C-terminal amino acid residues of nsP1; DmHSP70Ter—transcription terminator of Drosophila melanogaster hsp70 gene. (A–C). The vector backbones are not shown; drawings are not in scale. (D). C6/36 cells were transfected with pPubi-CHIKV-nsP2 (left) or pPubi-SINV-nsP2 (right). Cells were harvested at 12, 18, 24, 36 or 48 hpt and lysed in 1× Laemmli buffer. Proteins were separated using SDS-PAGE in 10% gels and transferred to PVDF membranes. nsP2 proteins were detected using anti-CHIKV and anti-SINV nsP2 antibodies, and β-actin was detected as the loading control. (E) C6/36 cells were transfected with (left panel) pPubi-CHIKV-nsP2 (WT), pPubi-CHIKV-nsP2CA, pPubi-CHIKV-nsP2EV, pPubi-CHIKV-nsP2YA+EV, pPubi-CHIKV-nsP2ALT/ERR, pPubi-CHIKV-nsP2KR/DD; (right panel) pPubi-SINV-nsP2 (WT), pPubi-SINV-nsP2CA, pPubi-SINV-nsP2ND, pPubi-SINV-nsP2ND+PQ, pPubi-SINV-nsP2PQ or pPubi-SINV-nsP2KR/DD. Cells were harvested at 48 hpt and analyzed as described for (D). Full article ">Figure 2
Co-expression of CHIKV and SINV nsP2 inhibits activity of trans–replicase of homologous virus. (A). Mutations introduced to ns-polyprotein of CHIKV. Mutation G534V leads to expression of P1GV234 polyprotein with uncleavable 1/2 processing site, while mutation G1332V leads to expression of P12GV34 polyprotein with uncleavable 2/3 processing site. (B). C6/36 cells grown at 96-well plate were co-transfected with Alb-FG-CHIKV, wt or mutant ns-polyprotein expression plasmid (Ubi-P1234-CHIKV, Ubi-P1GV234-CHIKV, Ubi-P12GV34-CHIKV or Ubi-P1234GAA-CHIKV) and pPubi-CHIKV-nsP2CA, pPubi-CHIKV-nsP2, pPubi-CHIKV-nsP2EV, pPubi-CHIKV-nsP2YA+EV, pPubi-CHIKV-nsP2ALT/ERR, pPubi-CHIKV-nsP2KR/DD or with dummy plasmid (no-nsP2 control). Cells were incubated at 28 °C and lysed 48 hpt. Data represent the luciferase activity (Fluc and Gluc) from Ubi-P1234-CHIKV (left), Ubi-P1GV234-CHIKV (middle) and Ubi-P12GV34-CHIKV (right) transfected cells normalized to the Ubi-P1234GAA control cells. Value obtained for P1234GAA control was taken as 1. (C). C6/36 cells grown at 96-well plate were co-transfected with Alb-FG-SINV, Ubi-P1234-SINV or Ubi-P1234GAA-SINV and pPubi-SINV-nsP2CA, pPubi-SINV-nsP2, pPubi-SINV-nsP2ND, pPubi-SINV-nsP2ND+PQ, pPubi-SINV-nsP2PQ, pPubi-SINV-nsP2KR/DD or with dummy plasmid (no-nsP2 control). The experiment was performed and data analyzed as described for panel B. (B,C). Means ± SD from three biological replicates are shown. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA test). Full article ">Figure 3
Efficiency of infection of C6/36 cells by CHIKV VRPs is reduced by expression of CHIKV or SINV nsP2 proteins. C6/36 cells grown in 12-well plates were transfected with pB-IE1.dsR (no nsP2), pPubi-CHIKV-nsP2CA, pPubi-CHIKV-nsP2, pPubi-SINV-nsP2CA or pPubi-SINV-nsP2. At 48 hpt, cells were infected with VRPs containing CHIKVRepl-ZsGreen replicon at a multiplicity of infection of approximately 0.4. Cells were harvested at 16 h post-infection and fixed and analyzed with an Attune NxT acoustic focusing cytometer. Y-axes: percentage of ZsGreen-positive cells (i.e., harboring replicating CHIKV replicon) from DsRed-positive cells (i.e., cells successfully transfected with nsP2 expression or control plasmid). Means ± SD from two independent experiments performed in triplicate are shown. ns, not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA test). Full article ">Figure 4
Co-expression of nsP2 of CHIKV or SINV inhibits activity of trans-replicases of heterologous alphaviruses. (A) C6/36 cells grown on 96-well plates were co-transfected with matching pairs of AlbPolI-FG and Ubi-P1234 or Ubi-P1234GAA plasmids of alphaviruses shown at X-axes and with pPubi-CHIKV-nsP2CA, pPubi-CHIKV-nsP2 or dummy plasmid (no-nsP2 control). Cells were incubated at 28 °C and lysed 48 hpt. Data represent the Gluc activity from Ubi-P1234-CHIKV transfected cells normalized to the Ubi-P1234GAA control cells. Value obtained for P1234GAA control was taken as 1. (B). Experiment was performed as described for panel A except that pPubi-SINV-nsP2CA or pPubi-SINV-nsP2 plasmids were used. (A,B). Means ± SD are shown for three biological replicates. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA test). Full article ">

13 pages, 2437 KiB

Open AccessArticle

ORFeome Phage Display Reveals a Major Immunogenic Epitope on the S2 Subdomain of SARS-CoV-2 Spike Protein

by Rico Ballmann, Sven-Kevin Hotop, Federico Bertoglio, Stephan Steinke, Philip Alexander Heine, M. Zeeshan Chaudhry, Dieter Jahn, Boas Pucker, Fausto Baldanti, Antonio Piralla, Maren Schubert, Luka Čičin-Šain, Mark Brönstrup, Michael Hust and Stefan Dübel

Viruses 2022, 14(6), 1326; https://doi.org/10.3390/v14061326 - 17 Jun 2022

Cited by 7 | Viewed by 3107

Abstract

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate [...] Read more.

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants. Full article

(This article belongs to the Section Bacterial Viruses)

► Show Figures

Figure 1

Figure 1
NGS data analysis. (A) NGS reads per nucleotide position plotted for each nucleotide position of the SARS-CoV-2 genome (Wuhan variant; Genbank No.: MT326090.1). A higher signal referred to a better coverage of the corresponding nucleotide position. (B) average reads per nucleotide position (as indicated in (A)) plotted against the length of the corresponding ORF, indicating the quality of coverage for each ORF. Structural ORFs are indicated in blue and ORFs encoding accessory proteins are indicated in red. Full article ">Figure 2
(A) alignment results of ORFeome phage display clones selected on STE73-6C10 scFv-hFc. Top sequence: spike of Wuhan variant, Genbank No.: MT326090.1. (B) Peptide microarray results of STE73-6C10 on the spike protein. The antibody recognizes the peptides 207-209 which correspond to the amino acid sequence 619–639 of the Wuhan variant, Genbank No.: MT326090.1. Full article ">Figure 3
(A) Titration of serum samples from Italian patients on the identified mimotope synthetic peptide DPSKPSKRSFIEDLLFNKVTLADA. The serum samples used in ORFeome phage display panning are indicated with colors (red: patient 1, blue: patient 17 and green: patient 18). Patient samples from northern Italy that were not subject of ORFeome phage display are indicated in grey. Fitted curves were obtained by the Logistic5 function in OriginPro2018. (B) Peptide microarray analysis of the IgG response of patient sera 1, 17 and 18 on the four structural SARS-CoV-2 proteins. Left: Spike; Upper right: Nucleocapsid; Middle right: Membrane; Lower right: Envelope. Areas marked in red correspond to identified hits for linear epitopes detectable. Hit identification was carried out by visual inspection. Full article ">

12 pages, 1284 KiB

Open AccessReview

Direct-Acting Antiviral Agents for Hepatitis C Virus Infection—From Drug Discovery to Successful Implementation in Clinical Practice

by Christopher Dietz and Benjamin Maasoumy

Viruses 2022, 14(6), 1325; https://doi.org/10.3390/v14061325 - 17 Jun 2022

Cited by 20 | Viewed by 3666

Abstract

Today, hepatitis C virus infection affects up to 1.5 million people per year and is responsible for 29 thousand deaths per year. In the 1970s, the clinical observation of unclear, transfusion-related cases of hepatitis ignited scientific curiosity, and after years of intensive, basic [...] Read more.

Today, hepatitis C virus infection affects up to 1.5 million people per year and is responsible for 29 thousand deaths per year. In the 1970s, the clinical observation of unclear, transfusion-related cases of hepatitis ignited scientific curiosity, and after years of intensive, basic research, the hepatitis C virus was discovered and described as the causative agent for these cases of unclear hepatitis in 1989. Even before the description of the hepatitis C virus, clinicians had started treating infected individuals with interferon. However, intense side effects and limited antiviral efficacy have been major challenges, shaping the aim for the development of more suitable and specific treatments. Before direct-acting antiviral agents could be developed, a detailed understanding of viral properties was necessary. In the years after the discovery of the new virus, several research groups had been working on the hepatitis C virus biology and finally revealed the replication cycle. This knowledge was the basis for the later development of specific antiviral drugs referred to as direct-acting antiviral agents. In 2011, roughly 22 years after the discovery of the hepatitis C virus, the first two drugs became available and paved the way for a revolution in hepatitis C therapy. Today, the treatment of chronic hepatitis C virus infection does not rely on interferon anymore, and the treatment response rate is above 90% in most cases, including those with unsuccessful pretreatments. Regardless of the clinical and scientific success story, some challenges remain until the HCV elimination goals announced by the World Health Organization are met. Full article

(This article belongs to the Special Issue Antiviral Molecular Mechanisms)

► Show Figures

Figure 1

Figure 1
Timeline of HCV discovery and advances in clinical and basic virologic research. Adapted from [<a href="#B2-viruses-14-01325" class="html-bibr">2</a>]. Full article ">Figure 2
Clinical presentation and natural course of HCV infection. In addition to the acute and chronic clinical picture, selected extrahepatic manifestations are shown. Phenomena with the strongest evidence for association with chronic HCV infection are printed in bold font. The other phenomena were observed with higher prevalence in HCV-infected individuals than in controls, but evidence was less strong [<a href="#B9-viruses-14-01325" class="html-bibr">9</a>]. Additional references [<a href="#B7-viruses-14-01325" class="html-bibr">7</a>,<a href="#B8-viruses-14-01325" class="html-bibr">8</a>]. Created with BioRender.com. Full article ">Figure 3
Viral replication and points of attack of direct-acting antiviral agents. Shown are key steps of viral replication and the modes of action of different DAAs. After binding and cellular entry of the viral particle, the ssRNA is released and translated into the HCV polyprotein. DAAs interfere with the viral replication at different stages. NS3/4A protease inhibitors block the processing of the HCV polyprotein. NS5B inhibitors interfere with the viral RNA polymerase. For NS5A inhibitors, an interaction with viral and host proteins is assumed, while the exact mode of action is unknown. Adapted from [<a href="#B44-viruses-14-01325" class="html-bibr">44</a>]. Created with BioRender.com. Full article ">

15 pages, 2944 KiB

Open AccessArticle

Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana

by Tingting Ma, Shuai Fu, Kun Wang, Yaqin Wang, Jianxiang Wu and Xueping Zhou

Viruses 2022, 14(6), 1324; https://doi.org/10.3390/v14061324 - 17 Jun 2022

Cited by 3 | Viewed by 2316

Abstract

Remorin (REM) is a plant-specific plasma membrane-associated protein regulating plasmodesmata plasticity and restricting viral cell-to-cell movement. Here, we show that palmitoylation is broadly present in group 1 remorin proteins in Nicotiana benthamiana and is crucial for plasma membrane localization and accumulation. By screening [...] Read more.

Remorin (REM) is a plant-specific plasma membrane-associated protein regulating plasmodesmata plasticity and restricting viral cell-to-cell movement. Here, we show that palmitoylation is broadly present in group 1 remorin proteins in Nicotiana benthamiana and is crucial for plasma membrane localization and accumulation. By screening the four members of N. benthamiana group 1 remorin proteins, we found that only NbREM1.5 could significantly hamper tobacco mosaic virus (TMV) cell-to-cell movement. We further showed that NbREM1.5 interacts with the movement protein of TMV in vivo and interferes with its function of expanding the plasmodesmata size exclusion limit. We also demonstrated that palmitoylation is indispensable for NbREM1.5 to hamper plasmodesmata permeability and inhibit TMV cell-to-cell movement. Full article

(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)

► Show Figures

Figure 1

Figure 1
Acyl-resin assisted capture (Acyl-RAC) to detect palmitoylation of NbREMs and their respective palmitoylation-defective mutants in N. benthamiana leaves. (a) NbREM1.1, (b) NbREM1.3, (c) NbREM1.5, (d) NbREM1.8, (e) NbREM1.1-C206A, (f) NbREM1.3-C177A, (g) NbREM1.5-C172A, (h) NbREM1.5-C172/175A, (i) NbREM1.8-C195A. (j) The relative palmitoylation levels of NbREMs and their respective palmitoylation-defective mutants. Data are mean ± SD (n = 3). Immunoblotting was performed using anti-flag antibody. NH2OH indicates presence (+) or absence (−) of hydroxylamine required for acyl group cleavage during the thiopropyl Sepharose 6b capture step. Thiopropyl Sepharose 6b enriched proteins were eluted and represent palmitoylated proteins (palmitoylation). Prior to thiopropyl Sepharose 6b capture the samples were removed as an input loading control (input). Full article ">Figure 2
Protein accumulation assay comparison of NbREM1.1 and NbREM1.1-C206A (a), NbREM1.3 and NbREM1.3-C177A (b), NbREM1.8 and NbREM1.8-C195A (c), NbREM1.5 and NbREM1.5-C172A (d), NbREM1.5 and NbREM1.5-C172/175A (e) in N. benthamiana leaves. Data are mean ± SD (n = 4). Asterisks mark significant differences according to two-tailed Student’s t-test; * p < 0.05; ** p < 0.01; ns, no significant difference. The opposite halves of N. benthamiana leaves separately expressed flag-tagged NbREMs or their palmitoylation-defective mutants by agro-infiltration. Immunoblotting with anti-actin antibody was used as a loading control. Immunoblotting with anti-flag antibody was used to detect protein accumulation of NbREMs and its palmitoylation-defective mutants. Full article ">Figure 3
Subcellular localization of GFP-NbREMs and their respective palmitoylation-defective mutants in RFP-H2B transgenic N. benthamiana protoplast cells. (a) GFP-NbREM1.1 and GFP-NbREM1.1-C206A, (b) GFP-NbREM1.3 and GFP-NbREM1.3-C177A, (c) GFP-NbREM1.8 and GFP-NbREM1.8-C195A, (d) GFP-NbREM1.5, GFP-NbREM1.5-C172A, and GFP-NbREM1.5-C172/175A. The Z-stacks of optical sections were constructed to view the localization in protoplast using ZEN Black software. GFP-NbREMs and their palmitoylation-defective mutants were transiently expressed in transgenic N. benthamiana expressing RFP-H2B, of which the nuclei was marked by red fluorescence. The released protoplasts were collected and examined by a Zeiss LSM 880 or 980 AiryscanTM upright laser scanning confocal microscope at 48 hpi. Bar, 20 μm. Full article ">Figure 4
NbREM1.5 negatively regulates tobacco mosaic virus (TMV) cell-to-cell movement. (a) The effect of four NbREMs on TMV-GFP cell-to-cell movement at 4 days post-inoculation (dpi) and 8 dpi. TMV-GFP was co-agroinfiltrated with flag-NbREMs or scarlet-flag (a negative control) on the opposite halves of N. benthamiana leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 dpi for upper row and 8 dpi for lower row. Leaves were captured under a portable UV lamp. Bar, 1 cm. (b) Statistical analyses of the effects of the four NbREMs on TMV-GFP cell-to-cell movement in the lower row of (a). Data are mean ± SEM (n = 50). Asterisks mark significant differences according to two-tailed Student’s t-test; ** p < 0.01; ns, no significant difference. (c) Detection of the infection foci of TMV-GFP in TRV-mCherry (control) and NbREM1.5 knock-down (TRV-NbREM1.5) N. benthamiana plant leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 dpi under the portable UV lamp. (d) Statistical analyses of TMV-GFP infection foci area in TRV-mCherry (as a control) and NbREM1.5 knock-down (TRV-NbREM1.5) N. benthamiana plant leaves. Data are mean ± SEM (n = 133). Asterisks mark significant differences according to two-tailed Student’s t-test; *** p < 0.001. Full article ">Figure 5
Palmitoylation is indispensable for NbREM1.5 to negatively regulate TMV cell-to-cell movement. (a) NbREM1.5 interacts with TMV MP in vivo by BiFC. Reconstituted YFP signals were observed at 48 h post-inoculation (hpi) by fluorescence confocal microscope in N. benthamiana leaf epidermal cells. Bar, 20 μm. (b) Effect of NbREM1.5 and its palmitoylation-defective mutants NbREM1.5-C172/175A on cell-to-cell diffusion of GFP under co-expression with TMV-MP. GUS-flag was used as the negative control. Confocal images were taken at 48 hpi. Bar, 20 μm. (c) Statistical analyses of the effect of NbREM1.5 and NbREM1.5-C172/175A on cell-to-cell diffusion of GFP under co-expression with TMV-MP. Data are mean ± SD (n = 65). Asterisks mark significant differences and “ns” marks no significant difference according to two-tailed Student’s t-test; *** p < 0.001. (d) Effect of NbREM1.5-C172/175A on TMV-GFP cell-to-cell movement. TMV-GFP was co-agroinfiltrated with flag-NbREM1.5-C172/175A or scarlet-flag (as a negative control) on the opposite halves of N. benthamiana leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 days post-inoculation under a portable UV lamp. Bar, 1 cm. (e) Statistical analyses of the effect of NbREM1.5-C172/175A on TMV-GFP cell-to-cell movement. Data are mean ± SEM (n = 45). Asterisks mark significant differences according to two-tailed Student’s t-test; ** p < 0.01. Full article ">

23 pages, 982 KiB

Open AccessReview

Influenza B: Prospects for the Development of Cross-Protective Vaccines

by Liudmila M. Tsybalova, Liudmila A. Stepanova, Edward S. Ramsay and Andrey V. Vasin

Viruses 2022, 14(6), 1323; https://doi.org/10.3390/v14061323 - 17 Jun 2022

Cited by 11 | Viewed by 3413

Abstract

In this review, we analyze the epidemiological and ecological features of influenza B, one of the most common and severe respiratory infections. The review presents various strategies for cross-protective influenza B vaccine development, including recombinant viruses, virus-like particles, and recombinant proteins. We provide [...] Read more.

In this review, we analyze the epidemiological and ecological features of influenza B, one of the most common and severe respiratory infections. The review presents various strategies for cross-protective influenza B vaccine development, including recombinant viruses, virus-like particles, and recombinant proteins. We provide an overview of viral proteins as cross-protective vaccine targets, along with other updated broadly protective vaccine strategies. The importance of developing such vaccines lies not only in influenza B prevention, but also in the very attractive prospect of eradicating the influenza B virus in the human population. Full article

(This article belongs to the Special Issue Viruses Research in Russia 2022)

► Show Figures

Figure 1

12 pages, 575 KiB

Open AccessReview

Cardiac Complications of COVID-19 in Low-Risk Patients

by Akash Srinivasan, Felyx Wong, Liam S. Couch and Brian X. Wang

Viruses 2022, 14(6), 1322; https://doi.org/10.3390/v14061322 - 17 Jun 2022

Cited by 11 | Viewed by 5243

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has resulted in over 6 million deaths and significant morbidity across the globe. Alongside common respiratory symptoms, COVID-19 is associated with a variety of cardiovascular complications in the acute and post-acute phases of infection. The suggested pathophysiological [...] Read more.

The coronavirus disease 2019 (COVID-19) pandemic has resulted in over 6 million deaths and significant morbidity across the globe. Alongside common respiratory symptoms, COVID-19 is associated with a variety of cardiovascular complications in the acute and post-acute phases of infection. The suggested pathophysiological mechanisms that underlie these complications include direct viral infection of the myocardium via the angiotensin-converting enzyme 2 (ACE2) protein and a cytokine release syndrome that results in indirect inflammatory damage to the heart. Patients with pre-existing cardiovascular disease and co-morbidities are generally more susceptible to the cardiac manifestations of COVID-19. However, studies have identified a variety of complications in low-risk individuals, including young adults and children. Myocarditis and paediatric inflammatory multisystem syndrome temporally associated with COVID-19 (PIMS) are among the adverse events reported in the acute phase of infection. Furthermore, patients have reported cardiac symptoms persisting beyond the acute phase in post-COVID syndrome. This review summarises the acute and chronic cardiac consequences of COVID-19 in low-risk patients, explores the pathophysiology behind them, and discusses new predictive factors for poor outcomes. Full article

(This article belongs to the Special Issue Post-COVID Syndrome)

► Show Figures

Figure 1

16 pages, 1799 KiB

Open AccessArticle

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening

by Hirotaka Sato, Tomoyuki Murakami, Ryosuke Matsuura, Masako Abe, Seiji Matsuoka, Yoko Yashiroda, Minoru Yoshida, Hirofumi Akari, Yosuke Nagasawa, Masami Takei and Yoko Aida

Viruses 2022, 14(6), 1321; https://doi.org/10.3390/v14061321 - 16 Jun 2022

Cited by 2 | Viewed by 2986

Abstract

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by [...] Read more.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 μM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy. Full article

(This article belongs to the Special Issue Viral Accessory Proteins)

► Show Figures

Figure 1

Figure 1
The first screening of the HIV-1 Vpr inhibitor using yeast-based high-throughput screening. (a) The growth defect of fission yeast strains with the HIV Vpr overexpression. The fission yeast cells harboring the Vpr-wild type (portion 1) or Vpr mutant R80A (portion 2), or the empty control (portion 3), were streaked on an MM medium for overexpression (upper plate) and an SD medium for repression (lower plate), and they were incubated at 30 °C for 3 days. (b) Twenty µM of 140,000 candidate compounds was added to the Vpr-expressing yeast cultured in an MM medium and cultured for 20 h. Yeast cell growth was assessed by WST-1 assay, and the proliferation recovery rate was calculated. The red line represents the hit criteria of the proliferation recovery rate of 15%, which was set as the threshold by over three-fold the standard deviation of all compounds. Full article ">Figure 2
Secondary screening of Vpr inhibitor using HeLa cell-based high-throughput screening. (a) HeLa cells were treated with 10 µM of 268 candidate compounds for 48 h, and the effect on the cell viability of each compound was assessed by WST-1 assay. The red line represents the threshold for high toxicity when the treated cells showed viability under 70% compared to the DMSO-treated control. (b) The HeLa cells were transfected with pME/Flag-Vpr-IRES-ZsGreenI and incubated at 37 °C. At 4 h post-transfection, 224 candidate compounds were added at 10 µM and incubated for an additional 44 h. The cells were fixed, stained with Hoechst 33342, and analyzed using CELAVIEW RS-100. The red lines represent the threshold for inhibition ranging from over 30% and not exceeding 100% of the G2/M arrest recovery rate. (c) The inhibition of the Vpr-induced G2/M cell cycle arrest was confirmed by the flow cytometry. The HeLa cells were transfected with either pME/Flag-Vpr-IRES-ZsGreenI (FVpr-ZsG) or the control pME/Flag-IRES-ZsGreenI (ZsG) and incubated at 37 °C. At 4 h post-transfection, transfected HeLa cells were treated either with 10 µM of 17 candidate compounds or vehicle (DMSO only). After 48 h, the cells were collected and stained with propidium iodide, and the DNA content of ZsGreenI-positive cells was measured by flow cytometry. The cell cycle was analyzed by Modfit software, and the G2/G1 ratio was calculated. Results of the G2/G1 ratio of 17 candidate compounds-treated cells (upper panel) and the typical model of the cell cycle analysis of a representative drug, VTD227 (bottom panel), are shown. Arrowheads indicate peaks of cells at the G1 and G2/M phases. The G2/M:G1 ratio is indicated in the upper right of each graph. Full article ">Figure 3
Third screening of Vpr inhibitor using human monocyte-derived macrophages (MDM). (a) Human peripheral blood mononuclear cells (PBMCs) were collected from healthy donor 1, and MDM were differentiated from monocytes isolated from human PBMCs. MDM cells were treated with 10 µM of 7 candidate compounds for 10 days, and the effect on the cell viability of each compound was assessed by a WST-1 assay. (b) MDM were differentiated from monocytes isolated from healthy donors 1 and 2, infected with HIV-1 NF462, and then treated with 10 µM of 4 candidate compounds. After 8 days, we collected the supernatant and assessed the inhibitory effect of the compounds on virus replication by p24 ELISA. Full article ">Figure 4
The inhibitory activity of selected compounds on the virus replication in HIV-1 NF462-infected human monocyte-derived macrophages (MDM). (a) Structure of the final selected compounds, VTD227, VTD232, and VTD 263. (b) Dose-dependent cell cytotoxicity for MDM of the final selected compounds. Differentiated MDM from healthy donors 1 and 3 were treated with 0, 0.4, 2, 10, 25, or 50 µM of 4 candidate compounds for 48 h, and cell viability was assessed by WST-1 assay. (c) Inhibition on the replication of HIV-1 NF462 by the final selected compounds. Differentiated MDM from healthy donors 1 and 4 were infected either with NF462 HIV-1 or NF462delVpr and replaced with a new medium containing 0, 0.001, 0.01, 0.1, 1, or 10 µM of 4 candidate compounds. The cells were maintained for 8 days, and the levels of virus production in the culture supernatants were measured by the p24 antigen, ELISA. A calculated 50% replication inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) are shown in <a href="#viruses-14-01321-t002" class="html-table">Table 2</a>. Each column and error bar represent the mean SD of the results from the duplicate samples from two donors. Full article ">Figure 5
The binding affinity to the Vpr protein of the final selected compounds. To analyze whether the compounds directly bind to the HIV Vpr protein, the binding affinity of the compounds with a purified GST-Vpr protein was assessed by Biacore. GST-Vpr immobilized on the sensor CM5 chips and flew analyte as 50 µM of the compounds, VTD227, VTD232, and VTD263 ((Left) panel), and as a dose from 3.13 to 100 µM of the compound, VTD227 ((Center) panel). This dissociation constant (Kd) value was calculated by two independent experiments using equilibration analysis ((Right) panel), as shown in <a href="#viruses-14-01321-t002" class="html-table">Table 2</a>. N.D. means as not determined. Full article ">

16 pages, 1688 KiB

Open AccessArticle

A Novel Flavi-like Virus in Alfalfa (Medicago sativa L.) Crops along the Snake River Valley

by Jennifer Dahan, Yuri I. Wolf, Gardenia E. Orellana, Erik J. Wenninger, Eugene V. Koonin and Alexander V. Karasev

Viruses 2022, 14(6), 1320; https://doi.org/10.3390/v14061320 - 16 Jun 2022

Cited by 8 | Viewed by 3579

Abstract

Alfalfa is an important perennial forage crop in Idaho supporting dairy and cattle industries that is typically grown in the same field for as many as 4 years. Alfalfa stands of different ages were subjected to screening for viruses using high-throughput sequencing and [...] Read more.

Alfalfa is an important perennial forage crop in Idaho supporting dairy and cattle industries that is typically grown in the same field for as many as 4 years. Alfalfa stands of different ages were subjected to screening for viruses using high-throughput sequencing and RT-PCR. The two most common viruses found were alfalfa mosaic virus and bean leafroll virus, along with Medicago sativa amalgavirus, two alphapartitiviruses, and one deltapartitivirus. Additionally, a new flavi-like virus with an unusual genome organization was discovered, dubbed Snake River alfalfa virus (SRAV). The 11,745 nt, positive-sense (+) RNA genome of SRAV encodes a single 3835 aa polyprotein with only two identifiable conserved domains, an RNA-dependent RNA polymerase (RdRP) and a predicted serine protease. Notably, unlike all +RNA virus genomes in the similar size range, the SRAV polyprotein contained no predicted helicase domain. In the RdRP phylogeny, SRAV was placed inside the flavi-like lineage as a sister clade to a branch consisting of hepaci-, and pegiviruses. To the best of our knowledge, SRAV is the first flavi-like virus identified in a plant host. Although commonly detected in alfalfa crops in southern Idaho, SRAV sequences were also amplified from thrips feeding in alfalfa stands in the area, suggesting a possible role of Frankliniella occidentalis in virus transmission. Full article

(This article belongs to the Special Issue A Tribute to Giovanni P. Martelli)

► Show Figures

Figure 1

Figure 1
Mosaic, vein clearing, and flecking on the foliage of alfalfa samples ALF1060 (a) and ALF1061 (b) collected from location 1 in July 2020. Full article ">Figure 2
Schematic representation of the Snake River alfalfa virus (SRAV) genome in comparison to the genome organization of yellow fever virus (YFV, flavivirus) and bovine viral diarrhea virus (BVDV, pestivirus). Homologous protein domains in all three polyproteins, Tryp_SPc (trypsin-like serine protease) and RdRP (RNA-dependent RNA polymerase), are indicated by arrows. Full article ">Figure 3
Phylogenetic analysis of the RdRP domains of flavi-like viruses and phylogenetic placement of the newly described Snake River alfalfa virus (SRAV). Numbered nodes indicate aBayes support values; large clades are collapsed and depicted as triangles. Virus genera of Flaviviridae currently approved by the International Committee on Taxonomy of Viruses (ICTV) are shown in bold italic; two clades of unclassified arthropod flavi-like viruses are provisionally denoted as “Jingmen” and “Hermitage”. See <a href="#app1-viruses-14-01320" class="html-app">Supplementary Figure S3</a> for the expanded tree. Full article ">Figure 4
RT-PCR testing of the August 2021 alfalfa and insect samples for the presence of the Snake River alfalfa virus (SRAV); PCR products were analyzed in agarose gel and visualized under UV light. A: composite sample of the aphids collected from field 4B; T1 and T2: composite samples of thrips collected from fields 14B and 4B, respectively; the next ten samples are alfalfa samples from fields 4B and F14, respectively, representing five individual plants per field; (-): negative control (water); C: positive control (sample ALF1060); M1 and M2: DNA size markers. The red arrow indicates the position of the SRAV-specific, 843 bp band amplified with the ANPV_3 primers (<a href="#app1-viruses-14-01320" class="html-app">Supplemental Table S3</a>). Full article ">

20 pages, 4540 KiB

Open AccessArticle

Pathogenesis of West Nile Virus Lineage 2 in Domestic Geese after Experimental Infection

by Hannah Reemtsma, Cora M. Holicki, Christine Fast, Felicitas Bergmann, Martin Eiden, Martin H. Groschup and Ute Ziegler

Viruses 2022, 14(6), 1319; https://doi.org/10.3390/v14061319 - 16 Jun 2022

Cited by 5 | Viewed by 2668

Abstract

West Nile virus (WNV) is an emerging infectious pathogen circulating between mosquitoes and birds but also infecting mammals. WNV has become autochthonous in Germany, causing striking mortality rates in avifauna and occasional diseases in humans and horses. We therefore wanted to assess the [...] Read more.

West Nile virus (WNV) is an emerging infectious pathogen circulating between mosquitoes and birds but also infecting mammals. WNV has become autochthonous in Germany, causing striking mortality rates in avifauna and occasional diseases in humans and horses. We therefore wanted to assess the possible role of free-ranging poultry in the WNV transmission cycle and infected 15 goslings with WNV lineage 2 (German isolate). The geese were monitored daily and sampled regularly to determine viremia, viral shedding, and antibody development by molecular and serological methods. Geese were euthanized at various time points post-infection (pi). All infected geese developed variable degrees of viremia from day 1 to day 10 (maximum) and actively shed virus from days 2 to 7 post-infection. Depending on the time of death, the WN viral genome was detected in all examined tissue samples in at least one individual by RT-qPCR and viable virus was even re-isolated, except for in the liver. Pathomorphological lesions as well as immunohistochemically detectable viral antigens were found mainly in the brain. Furthermore, all of the geese seroconverted 6 days pi at the latest. In conclusion, geese are presumably not functioning as important amplifying hosts but are suitable sentinel animals for WNV surveillance. Full article

(This article belongs to the Special Issue Flaviviruses and Flavivirus Vaccines)

► Show Figures

Figure 1

Figure 1
Planned time schedule of the animal experiment, including the sampling procedure over 21 dpi. Full article ">Figure 2
Weight gain of all geese throughout the experiment. Infected geese shown in blue, controls in green, and G 11 (severely diseased) in red. Full article ">Figure 3
Viremia of the geese. The negative results of the controls (G 16–G 18) are not shown. (a) Results of RT-qPCR without G 11 (left) and for all WNV-inoculated geese (top right), using different y-scales. (b) Results of virus titration for RT-qPCR-positive samples. Full article ">Figure 4
Viral shedding of the geese, without G 11 (left), and of all WNV-inoculated geese (top right), as estimated by RT-qPCR and using different y-scales. The negative results for the controls (G 16–G 18) are not shown. (a) Oropharyngeal shedding. (b) Cloacal shedding. Full article ">Figure 5
Seroconversion of all WNV-inoculated geese. Data are presented in a box-and-whisker plot, with the box including 50% of the values for each group and the dark-blue line in the middle of each group representing the median value. (a) Antibodies examined by a conventional competition IgG ELISA dyed in orange. (b) Antibodies examined by VNT dyed in pink. Full article ">Figure 6
WN viral load in different organs of all WNV-inoculated geese as estimated by RT-qPCR (negative values are not shown) in: (a) brain and feather pulp; (b) spleen and heart; (c) bursa fabricii and kidney; and (d) liver and lung samples. Full article ">Figure 7
Viral distribution in different organs presented as the means of three WNV-inoculated geese each at 3 dpi and 6 dpi in copies/µL total RNA, as estimated by RT-qPCR. Full article ">Figure 8
Histomorphological lesions induced by WNV infection in geese. (a) Small glial nodule with central neuronal necrosis in brain/cerebrum (arrow), G 5, 10 dpi. (b) Multifocal perivascular cuffs (arrowheads) in the brain/cerebrum. Clearly visible are the mild foci of encephalomalacia with gemistocytes clearing the cellular debris (arrow), G 11, 7 dpi. (c) Gut with distinct fibrinoid-necrotizing arteritis (arrow) in a medium size vessel of the tunica muscularis, G 11, 7 dpi. H&E, bars = 20 µm. Full article ">Figure 9
Immunohistochemical detection of WNV antigens in geese. (a) Plexus submucosus in the gut of G 1 (6 dpi) with viral antigen (arrow). (b) Plexus myentericus in the gut of G 11 (7 dpi). Viral antigens are distinctly seen in neurons and glial cells (arrows). Additionally, slight lymphohistiocytic infiltration can be seen (star). (c) Cerebellum/molecular layer of G 11 with a foci of altered neurons and glial cells accumulating viral antigens. Immunohistochemistry, pab (in-house) OM8; bars = 20 µm. Full article ">

15 pages, 1264 KiB

Open AccessArticle

Genomic Epidemiology of SARS-CoV-2 in Seychelles, 2020–2021

by John Mwita Morobe, Brigitte Pool, Lina Marie, Dwayne Didon, Arnold W. Lambisia, Timothy Makori, Khadija Said Mohammed, Zaydah R. de Laurent, Leonard Ndwiga, Maureen W. Mburu, Edidah Moraa, Nickson Murunga, Jennifer Musyoki, Jedida Mwacharo, Lydia Nyamako, Debra Riako, Pariken Ephnatus, Faith Gambo, Josephine Naimani, Joyce Namulondo, Susan Zimba Tembo, Edwin Ogendi, Thierno Balde, Fred Athanasius Dratibi, Ali Ahmed Yahaya, Nicksy Gumede, Rachel A. Achilla, Peter K. Borus, Dorcas W. Wanjohi, Sofonias K. Tessema, Joseph Mwangangi, Philip Bejon, David J. Nokes, Lynette Isabella Ochola-Oyier, George Githinji, Leon Biscornet and Charles N. Agoti Show full author list Hide full author list

Viruses 2022, 14(6), 1318; https://doi.org/10.3390/v14061318 - 16 Jun 2022

Cited by 4 | Viewed by 3128

Abstract

Seychelles, an archipelago of 155 islands in the Indian Ocean, had confirmed 24,788 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 31st of December 2021. The first SARS-CoV-2 cases in Seychelles were reported on the 14th of March 2020, but [...] Read more.

Seychelles, an archipelago of 155 islands in the Indian Ocean, had confirmed 24,788 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 31st of December 2021. The first SARS-CoV-2 cases in Seychelles were reported on the 14th of March 2020, but cases remained low until January 2021, when a surge was observed. Here, we investigated the potential drivers of the surge by genomic analysis of 1056 SARS-CoV-2 positive samples collected in Seychelles between 14 March 2020 and 31 December 2021. The Seychelles genomes were classified into 32 Pango lineages, 1042 of which fell within four variants of concern, i.e., Alpha, Beta, Delta and Omicron. Sporadic cases of SARS-CoV-2 detected in Seychelles in 2020 were mainly of lineage B.1 (lineage predominantly observed in Europe) but this lineage was rapidly replaced by Beta variant starting January 2021, and which was also subsequently replaced by the Delta variant in May 2021 that dominated till November 2021 when Omicron cases were identified. Using the ancestral state reconstruction approach, we estimated that at least 78 independent SARS-CoV-2 introduction events occurred in Seychelles during the study period. The majority of viral introductions into Seychelles occurred in 2021, despite substantial COVID-19 restrictions in place during this period. We conclude that the surge of SARS-CoV-2 cases in Seychelles in January 2021 was primarily due to the introduction of more transmissible SARS-CoV-2 variants into the islands. Full article

(This article belongs to the Topic Acute Respiratory Viruses Molecular Epidemiology)

► Show Figures

Figure 1

Figure 1
(A) Seychelles government intervention levels as measured by the Oxford stringency index [<a href="#B15-viruses-14-01318" class="html-bibr">15</a>]. (B) An epidemic curve for Seychelles derived from the daily positive case numbers obtained from <a href="https://ourworldindata.org/coronavirus/country/seychelles" target="_blank">https://ourworldindata.org/coronavirus/country/seychelles</a> (accessed on 5 May 2022). (C) Percentage of the population administered with vaccine; data obtained from <a href="https://ourworldindata.org/coronavirus/country/seychelles" target="_blank">https://ourworldindata.org/coronavirus/country/seychelles</a> (accessed on 5 May 2022). (D) Monthly temporal pattern of SARS-CoV-2 lineages and variants in Seychelles among the 1056 samples sequenced from COVID-19 positive cases from the Seychelles (25 June 2020, to 31 December 2021). (E) Monthly temporal distribution of Delta VOC lineages among samples sequenced from COVID-19 positive cases from the Seychelles (25 June 2020, to 31 December 2021). (F) Monthly temporal distribution of Omicron VOC lineages among samples sequenced from COVID-19 positive cases from the Seychelles (25 June 2020, to 31 December 2021). Full article ">Figure 2
SARS-CoV-2 Pango lineages in the sequenced 1056 Seychelles samples and timing of detections (circle size scaled by number of daily detections). Full article ">Figure 3
Genetic distance-resolved lineage-specific phylogenetic trees for Omicron, Alpha, Beta, Delta VOC, and Non-VOC. Seychelles genomes are indicated with colored tip labels. (A) Phylogeny of Non-VOC that combined 14 Seychelles sequences and 875 global sequences. (B) Phylogeny of Alpha VOC that combined 5 Seychelles sequences and 246 global sequences. (C) Phylogeny of Beta VOC that combined 29 Seychelles sequences and 187 global sequences. (D) Phylogeny of Delta VOC that combined 863 Seychelles sequences and 2676 global sequences. (E) Phylogeny of Omicron VOC that combined 145 Seychelles sequences and 1195 global sequences. Full article ">Figure 4
(A) Time-resolved global phylogeny that combined 1056 Seychelles sequences (coloured tip labels) and 5179 global reference sequences. (B) The number of viral imports and exports into and out of Seychelles. (C) Cumulative number of viral imports and export over time into Seychelles. Full article ">

15 pages, 1198 KiB

Open AccessReview

Prevention and Control of Porcine Epidemic Diarrhea: The Development of Recombination-Resistant Live Attenuated Vaccines

by Xiaoyu Niu and Qiuhong Wang

Viruses 2022, 14(6), 1317; https://doi.org/10.3390/v14061317 - 16 Jun 2022

Cited by 19 | Viewed by 3418

Abstract

Porcine epidemic diarrhea (PED), causing up to 100% mortality in neonatal pigs, is a highly contagious enteric disease caused by PED virus (PEDV). The highly virulent genogroup 2 (G2) PEDV emerged in 2010 and has caused huge economic losses to the pork industry [...] Read more.

Porcine epidemic diarrhea (PED), causing up to 100% mortality in neonatal pigs, is a highly contagious enteric disease caused by PED virus (PEDV). The highly virulent genogroup 2 (G2) PEDV emerged in 2010 and has caused huge economic losses to the pork industry globally. It was first reported in the US in 2013, caused country-wide outbreaks, and posed tremendous hardship for many pork producers in 2013–2014. Vaccination of pregnant sows/gilts with live attenuated vaccines (LAVs) is the most effective strategy to induce lactogenic immunity in the sows/gilts and provide a passive protection via the colostrum and milk to suckling piglets against PED. However, there are still no safe and effective vaccines available after about one decade of endeavor. One of the biggest concerns is the potential reversion to virulence of an LAV in the field. In this review, we summarize the status and the major obstacles in PEDV LAV development. We also discuss the function of the transcriptional regulatory sequences in PEDV transcription, contributing to recombination, and possible strategies to prevent the reversion of LAVs. This article provides insights into the rational design of a promising LAV without safety issues. Full article

(This article belongs to the Special Issue Animal Coronavirus Pathogenesis and Immunity)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Viruses, Volume 14, Issue 6 (June 2022) – 229 articles

Further Information

Guidelines

MDPI Initiatives

Follow MDPI