Viruses

15 pages, 4275 KiB

Open AccessArticle

Identification and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China

by Jiapeng Li, Xiaoyin Wu, Hui Liu, Xiaomei Wang, Shaokui Yi, Xueting Zhong, Yaqin Wang and Zhanqi Wang

Viruses 2023, 15(4), 1029; https://doi.org/10.3390/v15041029 - 21 Apr 2023

Cited by 4 | Viewed by 2194

Abstract

Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, [...] Read more.

Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5′-untranslated region (UTR) and a 76-nt 3′-UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1. Full article

(This article belongs to the Special Issue Next-Generation Sequencing in Plant Virology 2.0)

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12 pages, 5970 KiB

Open AccessArticle

Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody

by Lie Fu, Xiao-Yu Zhang, Wei-Ping Jin, Chen Wang, Sha-Sha Qian, Meng-Jun Wang, Wen-Hui Wang, Sheng-Li Meng, Jing Guo, Ze-Jun Wang, Xiao-Qi Chen and Shuo Shen

Viruses 2023, 15(4), 1028; https://doi.org/10.3390/v15041028 - 21 Apr 2023

Cited by 1 | Viewed by 1791

Abstract

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the [...] Read more.

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C. By mapping with over-lapped and truncated peptides, a minimal and linear epitope ₂₃PILPGF₂₈ was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A. It may be useful for the development of a cost-effective and pan-Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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Western blotting analysis of mAb 1A11 binding. Proteins of purified CV-A5 full particles (FP) and empty particles (EP) were separated by 4–20% SDS-PAGE. The proteins were blotted to the membrane and were incubated with rabbit anti-CV-A5 serum or mAb 1A11. The lysate of mock-infected Vero cells was used as control. Molecular weight markers in KDa and viral proteins are indicated on the left and right. Full article ">Figure 2
Cross reactivity of mAb 1A11 to 9 serotypes of different enterovirus species. (a) Binding specificity by IFA of the 1A11 mAbs to the Enterovirus species A, B and C was performed. RD cells were infected with 7 serotypes (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16 and EV-A71) of Enterovirus A, 1 serotype (echovirus 11) of Enterovirus B and 1 serotype (poliovirus Sabin 3) of Enterovirus C and were stained with 1A11 in IFA. Mock-infected cells were incubated with mAb as a negative control. (b) Western blotting of proteins of purified viruses of the same 9 serotypes used in (a) and rotavirus G8 was performed using mAb 1A11. Lysate of mock-infected cells was used as a negative control. Full article ">Figure 2 Cont.
Cross reactivity of mAb 1A11 to 9 serotypes of different enterovirus species. (a) Binding specificity by IFA of the 1A11 mAbs to the Enterovirus species A, B and C was performed. RD cells were infected with 7 serotypes (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16 and EV-A71) of Enterovirus A, 1 serotype (echovirus 11) of Enterovirus B and 1 serotype (poliovirus Sabin 3) of Enterovirus C and were stained with 1A11 in IFA. Mock-infected cells were incubated with mAb as a negative control. (b) Western blotting of proteins of purified viruses of the same 9 serotypes used in (a) and rotavirus G8 was performed using mAb 1A11. Lysate of mock-infected cells was used as a negative control. Full article ">Figure 3
Indirect ELISA of the binding specificity and affinity against 9 serotypes of different enterovirus species and a non-enterovirus control rotavirus-G8 (Rota-G8). Data are means ± SDs of the OD450 reading from triplicate wells. ELISA plates were coated with purified viruses including CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, EV-A71, Echo11, Sabin 3, and Rota-G8, at 1 μg/mL. The 10-fold serial dilution of mAb 1A11 was used as detection antibody. Full article ">Figure 4
Epitope mapping of 1A11 mAb binding epitope to CV-A5 VP3. (a) CV-A5 VP3 peptides ELISAs were performed using a panel of 31 peptides spanning the entire VP3 region with 10 overlapped residues to detect reactivity to 1A11. Overlapped residues of peptides 2 to 4 are underlined. Data are means ± SDs of the OD450 reading from triplicate wells. (b) Amino acid sequences of the epitope region among multiple serotypes of different enterovirus species are aligned. Conserved residues are shadowed, and a core conserved sequence is indicated in the box. (c) A panel of 10 truncated peptides in the epitope region, as indicated, was used to detect reactivity to 1A11 in ELISA. The inferred minimal epitope is underlined. Dashes indicate deleted amino acids of peptides b–j from the original sequence of a. Data are means ± SDs of the OD450 reading from triplicate wells. Full article ">Figure 5
Binding ability of 1A11 mAb to single mutation peptides of enterovirus. A panel of 9 single-mutation peptides (mutated residues highlighted) on the epitope region were used to detect reactivity to 1A11 in ELISA. Two amino acids were retained upstream of the N-termini of the mutant epitopes to reduce the effect of the N-terminal amino on the binding reactivity. Data are means ± SDs of the OD450 reading from triplicate wells. Full article ">

19 pages, 3627 KiB

Open AccessArticle

The Synthetic Opioid Fentanyl Increases HIV Replication and Chemokine Co-Receptor Expression in Lymphocyte Cell Lines

by Janani Madhuravasal Krishnan, Ling Kong, Rebekah Karns, Mario Medvedovic, Kenneth E. Sherman and Jason T. Blackard

Viruses 2023, 15(4), 1027; https://doi.org/10.3390/v15041027 - 21 Apr 2023

Cited by 2 | Viewed by 2593

Abstract

Background: In the United States, the illicit use of synthetic opioids such as fentanyl has led to a serious public health crisis. Synthetic opioids are known to enhance viral replication and to suppress immunologic responses, but their effects on HIV pathogenesis remain unclear. [...] Read more.

Background: In the United States, the illicit use of synthetic opioids such as fentanyl has led to a serious public health crisis. Synthetic opioids are known to enhance viral replication and to suppress immunologic responses, but their effects on HIV pathogenesis remain unclear. Thus, we examined the impact of fentanyl on HIV-susceptible and HIV-infected cell types. Methods: TZM-bl and HIV-infected lymphocyte cells were incubated with fentanyl at varying concentrations. Expression levels of the CXCR4 and CCR5 chemokine receptors and HIV p24 antigen were quantified with ELISA. HIV proviral DNA was quantified using SYBR RT-PCR. Cell viability was detected with the MTT assay. RNAseq was performed to characterize cellular gene regulation in the presence of fentanyl. Results: Fentanyl enhanced expression of both chemokine receptor levels in a dose-dependent manner in HIV-susceptible and infected cell lines. Similarly, fentanyl induced viral expression in HIV-exposed TZM-bl cells and in HIV-infected lymphocyte cell lines. Multiple genes associated with apoptosis, antiviral/interferon response, chemokine signaling, and NFκB signaling were differentially regulated. Conclusions: Synthetic opioid fentanyl impacts HIV replication and chemokine co-receptor expression. Increased virus levels suggest that opioid use may increase the likelihood of transmission and accelerate disease progression. Full article

(This article belongs to the Special Issue HIV and Drugs of Abuse 2.0)

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7 pages, 2319 KiB

Open AccessBrief Report

Lipid Accumulation in Host Cells Promotes SARS-CoV-2 Replication

by Tatjana Seitz, Christian Setz, Pia Rauch, Ulrich Schubert and Claus Hellerbrand

Viruses 2023, 15(4), 1026; https://doi.org/10.3390/v15041026 - 21 Apr 2023

Cited by 2 | Viewed by 1909

Abstract

Coronavirus disease-19 (COVID-19) is still affecting the lives of people around the globe and remains a major public health threat. Lipid levels in the host cells have been shown to promote SARS-CoV-2 replication, and since the start of COVID-19 pandemic, several studies have [...] Read more.

Coronavirus disease-19 (COVID-19) is still affecting the lives of people around the globe and remains a major public health threat. Lipid levels in the host cells have been shown to promote SARS-CoV-2 replication, and since the start of COVID-19 pandemic, several studies have linked obesity and other components of the metabolic syndrome with severity of illness, as well as mortality in patients with COVID-19. The aim of this study was to obtain insights into the pathophysiological mechanisms of these associations. First, we established an in vitro model simulating high fatty acid levels and showed that this situation induced the uptake of fatty acids and triglyceride accumulation in human Calu-3 lung cells. Importantly, we found that lipid accumulation significantly enhanced the replication of SARS-CoV-2 Wuhan type or the variant of concern, Delta, in Calu-3 cells. In summary, these findings indicate that hyperlipidemia as found in patients with obesity promotes viral replication and herewith the disease course of COVID-19. Full article

(This article belongs to the Special Issue SARS-CoV-2 and Other Coronaviruses)

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15 pages, 2374 KiB

Open AccessArticle

Effectiveness, Tolerability and Prescribing Choice of Antiviral Molecules Molnupiravir, Remdesivir and Nirmatrelvir/r: A Real-World Comparison in the First Ten Months of Use

by Cosmo Del Borgo, Silvia Garattini, Carolina Bortignon, Anna Carraro, Daniela Di Trento, Andrea Gasperin, Alessandra Grimaldi, Sara Giovanna De Maria, Sara Corazza, Tiziana Tieghi, Valeria Belvisi, Blerta Kertusha, Margherita De Masi, Ombretta D’Onofrio, Gabriele Bagaglini, Gabriella Bonanni, Paola Zuccalà, Paolo Fabietti, Eeva Tortellini, Mariasilvia Guardiani, Alessandra Spagnoli, Raffaella Marocco, Danilo Alunni Fegatelli, Miriam Lichtner and LATINA COVID-group Show full author list Hide full author list

Viruses 2023, 15(4), 1025; https://doi.org/10.3390/v15041025 - 21 Apr 2023

Cited by 7 | Viewed by 3053

Abstract

In 2022, three antiviral drugs—molnupiravir, remdesivir and nirmatrelvir/ritonavir—were introduced for treatment of mild-to-moderate COVID-19 in high-risk patients. The aim of this study is the evaluation of their effectiveness and tolerability in a real-life setting. A single-center observational study was set up, with the [...] Read more.

In 2022, three antiviral drugs—molnupiravir, remdesivir and nirmatrelvir/ritonavir—were introduced for treatment of mild-to-moderate COVID-19 in high-risk patients. The aim of this study is the evaluation of their effectiveness and tolerability in a real-life setting. A single-center observational study was set up, with the involvement of 1118 patients, with complete follow-up data, treated between the 5th of January and the 3rd of October 2022 at Santa Maria Goretti’s hospital in Latina, Central Italy. A univariable and a multivariable analysis were performed on clinical and demographic data and composite outcome, the persistence of symptoms at 30 days and time to negativization, respectively. The three antivirals showed a similar effectiveness in containing the progression of the infection to severe COVID-19 and a good tolerability in the absence of serious adverse effects. Persistence of symptoms after 30 days was more common in females than males and less common in patients treated with molnupiravir and nirmatrelvir/r. The availability of different antiviral molecules is a strong tool and, if correctly prescribed, they can have a significant role in changing the natural history of infection for frail persons, in which vaccination could be not sufficient for the prevention of severe COVID-19. Full article

(This article belongs to the Special Issue Efficacy and Safety of Antiviral Therapy)

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Patients’ recruitment algorithm. Full article ">Figure 2
Logistic regression multivariate analysis of composite outcome (pneumonia, ARDS, COVID-19 Death, Non-COVID-19 Death) YRS: years; RDV: remdesivir; MP: molnupiravir; NMV/r: nirmatrelvir/ritonavir; CKD: chronic kidney disease. Full article ">Figure 3
Logistic regression analysis of persistence of symptoms at 30 days. RDV: remdesivir; MP: molnupiravir; NMV/r: nirmatrelvir/ritonavir; CV: cardiovascular disease; Immunodef: immunodeficiency. Full article ">Figure 4
Linear regression analysis of time to negativization. RDV: remdesivir; MP: molnupiravir; NMV/r: nirmatrelvir/ritonavir; CV: cardiovascular disease; Immunodef: immunodeficiency. Full article ">Figure 5
Frequency of self-reported adverse effects and interruptions in the three groups of treatment. MP: molnupiravir, NVM/r: nirmatrelvir/ritonavir, RDV: remdesivir. MP vs. RDV p-Value 0.0001; NVM/r vs. RDV p-Value 0.0001. Full article ">Figure 6
Adverse effects reported by patients GI: gastrointestinal. MP: molnupiravir, NMV/r: nirmatrelvir/r, RDV: remdesivir. Full article ">

10 pages, 981 KiB

Open AccessArticle

Epidemiologic and Clinical Characteristics of Human Bocavirus Infection in Children with or without Acute Gastroenteritis in Acre, Northern Brazil

by Fábyla D’ Tácia Brito Trindade, Endrya Socorro Foro Ramos, Patrícia Santos Lobo, Jedson Ferreira Cardoso, Edvaldo Tavares Penha Júnior, Delana Andreza Melo Bezerra, Mayara Annanda Oliveira Neves, Jorge Alberto Azevedo Andrade, Monica Cristina Moraes Silva, Joana D’Arc Pereira Mascarenhas, Sylvia Fátima Santos Guerra and Luana Silva Soares

Viruses 2023, 15(4), 1024; https://doi.org/10.3390/v15041024 - 21 Apr 2023

Cited by 1 | Viewed by 1858

Abstract

Human bocavirus (HBoV) is an emerging virus detected around the world that may be associated with cases of acute gastroenteritis (AGE). However, its contribution to AGE has not been elucidated. This study aimed to describe the frequency, clinical features, and HBoV species circulation [...] Read more.

Human bocavirus (HBoV) is an emerging virus detected around the world that may be associated with cases of acute gastroenteritis (AGE). However, its contribution to AGE has not been elucidated. This study aimed to describe the frequency, clinical features, and HBoV species circulation in children up to 5 years with or without AGE symptoms in Acre, Northern Brazil. A total of 480 stool samples were collected between January and December 2012. Fecal samples were used for extraction, nested PCR amplification, and sequencing for genotyping. Statistical analysis was applied to verify the association between epidemiological and clinical characteristics. Overall, HBoV-positivity was 10% (48/480), with HBoV-positive rates of 8.4% (19/226) and 11.4% (29/254) recorded in diarrheic and non-diarrheic children, respectively. The most affected children were in the age group ranging between 7 and 24 months (50%). HBoV infection was more frequent in children who live in urban areas (85.4%), use water from public networks (56.2%), and live with adequate sewage facilities (50%). Co-detection with other enteric viruses was 16.7% (8/48) and the most prevalent coinfection was RVA+ HBoV (50%, 4/8). HBoV-1 was the most frequent species detected in diarrheic and non-diarrheic children, responsible for 43.8% (21/48) of cases, followed by HBoV-3 (29.2%, 14/48) and HBoV-2 (25%, 12/48). In this study, HBoV infection was not always associated with AGE, as most HBoV cases belonged to the non-diarrheal group. Future studies are warranted in order to determine the role of HBoV in causing acute diarrhea disease. Full article

(This article belongs to the Special Issue Viral Gastroenteritis 2022)

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13 pages, 3690 KiB

Open AccessArticle

The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus

by Amine Ourahmane, Laura Hertel and Michael A. McVoy

Viruses 2023, 15(4), 1023; https://doi.org/10.3390/v15041023 - 21 Apr 2023

Cited by 1 | Viewed by 1476

Abstract

Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to [...] Read more.

Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact RL13 gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the RL13 gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13’s restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the RL13 gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in RL13 that restored replication and focus size to those of the RL13-frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of RL13 mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13’s temperance functions. Full article

(This article belongs to the Special Issue Molecular Biology of Human Cytomegalovirus)

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The CMV strain Towne genome and genetic modifications made to RL13. (A) Top: map of the strain Towne-varL genome showing the a, b, and c sequences and their inverted a′, b′, and c′ sequence duplications that flank the unique-long (UL) and unique-short (US) regions. A gray dashed line indicates UL/b′ sequences that are present in Towne-varL but deleted in Towne-varS. Bottom: map of the Towne-varL genome as cloned in BAC TL12 showing the location of RL13 and of the BAC origin/GFP cassette. Expanded area: RL13 open reading frames present in the parental virus TL12-RL13FS, which contains a one-bp insertion that frame shifts and prematurely truncates RL13, in the TL12-RL13WT virus, where the one-bp insertion has been removed, and in the TL12-RL13WT-FLAG virus, in which sequences encoding a C-terminal FLAG epitope (DYKDDDDK) have been inserted. (B) ClustalW alignment of RL13 protein sequences as encoded by viruses TL12-RL13FS, TL12-RL13WT, and TL12-RL13WT-FLAG. RL13 sequences encoded by strains KG and Merlin are included to illustrate that RL13 encoded by TL12-RL13WT is similar to RL13 of KG but highly divergent from RL13 of Merlin. Full article ">Figure 2
Repair of RL13 reduces focus size in the CMV Towne-varL genetic background. MRC-5 fibroblast monolayers were infected at low MOI with p0 stocks of viruses TL12-RL13FS or TL12-RL13WT. (A) After eight days, ten GFP+ foci formed by each virus were randomly selected and photographed. (B) Areas of GFP+ foci measured using the ImageJ software (p value from Student’s two-tailed t-test). Original magnification: 100×. Full article ">Figure 3
RL13-FLAG retains the ability to restrict focus size. Replicate p0 stocks of TL12-RL13WT-FLAG were passaged as independent lineages, designated TL12-RL13WT-FLAG–1 and TL12-RL13WT-FLAG–2, then used to infect MRC-5 fibroblast monolayers at low MOI. After eight days, GFP+ foci were photographed (A) and their surface areas measured (B) as described for <a href="#viruses-15-01023-f002" class="html-fig">Figure 2</a>. Data for viruses TL12-RL13FS and TL12-RL13WT from <a href="#viruses-15-01023-f002" class="html-fig">Figure 2</a> are included for comparison (p values from Student’s two-tailed t-test). Original magnification: 100×. Full article ">Figure 4
Detection and localization of RL13-FLAG expressed by virus TL12-RL13WT-FLAG. (A) MRC-5 cultures were infected with a p0 stock of TL12-RL13WT-FLAG or with p10 stocks of TL12-RL13WT-FLAG–1 or TL12-RL13WT-FLAG–2. At day eight post-infection, cultures were stained for FLAG, and images of the FLAG (red) and GFP (green) signal were acquired. The white arrow indicates RL13WT-FLAG accumulation into juxtanuclear structures corresponding to the VAC. Original magnification: 100×. (B) MRC-5 cultures were infected with p0 stocks of TL12-RL13WT-FLAG, TL12-RL13E208K-FLAG, or TL12-RL13FS-FLAG. Cell lysates prepared at day five post-infection were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed using α-FLAG, α-IE1/2, or α-β-actin monoclonal antibodies. The positions of protein molecular weight standards are indicated. Full article ">Figure 5
The E208K substitution in RL13 increases focus size. The indicated virus stocks were used to infect MRC-5 fibroblast monolayers at low MOI. After eight days, GFP+ foci were photographed (A) and their surface areas measured (B) as described for <a href="#viruses-15-01023-f002" class="html-fig">Figure 2</a> (p values from Student’s two-tailed t-test; ns, not significant.). Original magnification: 100×. Full article ">Figure 6
The E208K substitution relieves RL13 restriction of viral replication. Replicate MRC-5 cultures were infected with the indicated viruses at an MOI of 0.3 pfu/cell. On alternate days, post-infection progeny virus yields were determined in culture supernatants (cell-free progeny, A) or in cell sonicates (cell-associated, B) by infecting fresh monolayer cultures and counting the numbers of GFP+ cells per field of view at day three post-infection. Data shown are means of triplicate assays +/− one standard deviation. * TL12-RL13E208K vs. TL12-RL13WT, days 11–13; ** TL12-RL13E208K vs. TL12-RL13WT, days 9–16. Full article ">Figure 7
RL13-FLAG localizes to VAC and is mis-localized by the E208K substitution. MRC-5 monolayers were infected with p0 stocks of TL12-RL13WT-FLAG (A) or TL12-RL13E208K-FLAG (B). After four or 11 days, the cultures were imaged for GFP (green), stained for RL13 with an α-FLAG antibody (red), or stained for pp28 with an α-pp28 antibody (blue). Original magnification: 200×. Full article ">

19 pages, 5033 KiB

Open AccessArticle

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance

by Gregory S. Orf, Ana Olivo, Barbara Harris, Sonja L. Weiss, Asmeeta Achari, Guixia Yu, Scot Federman, Dora Mbanya, Linda James, Samuel Mampunza, Charles Y. Chiu, Mary A. Rodgers, Gavin A. Cloherty and Michael G. Berg

Viruses 2023, 15(4), 1022; https://doi.org/10.3390/v15041022 - 21 Apr 2023

Cited by 6 | Viewed by 3178

Abstract

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from [...] Read more.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species. Full article

(This article belongs to the Special Issue Applications of Next-Generation Sequencing in Virus Discovery 2.0)

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Study flow chart. (a) Testing regime for specimens, and (b) quantification of viral diagnoses and viral identifications via deep sequencing. (Figure created with BioRender.com). Full article ">Figure 2
Summary of non-HxV viruses recovered from specimens U172329 and U172471. (a) Viral families from which a partial or complete genome was recovered from each specimen. Identical sequences of the novel densovirus, novel cyclovirus, and known human gemykibivirus 2 were found in both specimens. (b) Schematic depicting predation/encounter scenarios that may transmit viral genetic material from lower animals to higher animals. (c) Assembled genomes for known and novel DNA viruses described in panel A. (d) Assembled genomes for known and novel RNA viruses described in panel A. (Figure created with BioRender.com). Full article ">Figure 3
Genomic maps and mapping statistics for three viruses found in specimens U172329 and U172471 that have previously been detected in mammals: (a) human blood-associated dicistrovirus, (b) bat cyclovirus, and (c) bastrovirus. In each panel, mismatches represent single-nucleotide polymorphisms detected between the U172329 and U172471 isolates (Figure created using assets from BioRender.com). Full article ">Figure 4
Phylogenetic reconstruction of the Astroviridae, Hepeviridae, and bastroviruses. (a) Amino acid ML phylogeny of the RdRp domain from 133 viral isolates. The amino acid sequences were aligned using the L-INS-i algorithm of MAFFT and the Q.pfam + F + R6 substitution model was selected by IQ-TREE 2 as the most appropriate model to reconstruct the phylogeny. Metadata including sampled host, isolation source, and capsid type are shown as rings outside of the tree. Each clade is presented with a representative genomic schematic with domains of interest from each ORF indicated (domain abbreviations: MT—viral methyltransferase; Hel—helicase; Pol—RNA-dependent RNA polymerase; Pro—serine protease; Cap—capsid). The clade of interest containing the viruses isolated in this study is denoted by a star. (b) An expanded view of the monophyletic group denoted by a star in panel A. Taxa are labeled with isolate name and sampling country. Ultrafast bootstrap support is reported at the nodes. Full article ">Figure 5
Assignment of host class and zoonotic potential. (a) The canonical score plot of a linear discriminant analysis used to classify picorna-like viral sequences into three host groups using all 4 mononucleotide and all 16 dinucleotide frequencies. The plot shows the separation of host groups through the two most statistically significant factors. The training dataset with known host range (n = 945 genomes) was used to establish a scoring profile such that the viral sequences with unknown host could be classified. The ellipses represent the 90% confidence level (i.e., 90% of sequences fitting a host range group fit inside the ellipsis) centered on the centroid of each group. Sequences from the bastroviruses from U172329 and U172471, the dicistrovirus from U172471, and four comparator sequences are labeled separately. (b) Predicted probability of human infectability for all novel viruses identified in this study and closely related comparator sequences. Dots show the mean and bars show the 95% interquartile range of predicted probabilities across the best-performing 10% of iterations. The cut-off for zoonotic potential was set at 0.293 with priority categories assigned as previously described [<a href="#B49-viruses-15-01022" class="html-bibr">49</a>]: low: mean and upper/lower interquartile ranges below cutoff; medium: mean below cutoff but upper interquartile range above cutoff; high: mean above cutoff but lower interquartile range below cutoff; very high—mean and upper/lower interquartile ranges above cutoff. In both panels, the dicistrovirus recovered from specimen U172329 was not analyzed due to its low (45%) total coverage. Full article ">

15 pages, 578 KiB

Open AccessArticle

Human Papillomavirus Infections and Increased Risk of Incident Osteoporosis: A Nationwide Population-Based Cohort Study

by Kevin Sheng-Kai Ma, Ning-Chien Chin, Ting-Yu Tu, Yao-Cheng Wu, Hei-Tung Yip, James Cheng-Chung Wei and Ren-in Chang

Viruses 2023, 15(4), 1021; https://doi.org/10.3390/v15041021 - 21 Apr 2023

Cited by 1 | Viewed by 1811

Abstract

Patients with viral infections are susceptible to osteoporosis. This cohort study investigated the correlation between human papillomavirus (HPV) infections and the risk of osteoporosis via 12,936 patients with new-onset HPV infections and propensity score-matched non-HPV controls enrolled in Taiwan. The primary endpoint was [...] Read more.

Patients with viral infections are susceptible to osteoporosis. This cohort study investigated the correlation between human papillomavirus (HPV) infections and the risk of osteoporosis via 12,936 patients with new-onset HPV infections and propensity score-matched non-HPV controls enrolled in Taiwan. The primary endpoint was incident osteoporosis following HPV infections. Cox proportional hazards regression analysis and the Kaplan-Meier method was used to determine the effect of HPV infections on the risk of osteoporosis. Patients with HPV infections presented with a significantly high risk of osteoporosis (adjusted hazard ratio, aHR = 1.32, 95% CI = 1.06–1.65) after adjusting for sex, age, comorbidities and co-medications. Subgroup analysis provided that populations at risk of HPV-associated osteoporosis were females (aHR = 1.33; 95% CI = 1.04–1.71), those aged between 60 and 80 years (aHR = 1.45, 95% CI = 1.01–2.08 for patients aged 60–70; aHR = 1.51; 95% CI = 1.07–2.12 for patients aged 70–80), and patients with long-term use of glucocorticoids (aHR = 2.17; 95% CI = 1.11–4.22). HPV-infected patients who did not receive treatments for HPV infections were at a greater risk (aHR = 1.40; 95% CI = 1.09–1.80) of osteoporosis, while the risk of osteoporosis in those who received treatments for HPV infections did not reach statistical significance (aHR = 1.14; 95% CI = 0.78–1.66). Patients with HPV infections presented with a high risk of subsequent osteoporosis. Treatments for HPV infections attenuated the risk of HPV-associated osteoporosis. Full article

(This article belongs to the Special Issue Virology Research in Taiwan)

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36 pages, 1943 KiB

Open AccessReview

Current Clinical Landscape and Global Potential of Bacteriophage Therapy

by Nicole Marie Hitchcock, Danielle Devequi Gomes Nunes, Job Shiach, Katharine Valeria Saraiva Hodel, Josiane Dantas Viana Barbosa, Leticia Alencar Pereira Rodrigues, Brahm Seymour Coler, Milena Botelho Pereira Soares and Roberto Badaró

Viruses 2023, 15(4), 1020; https://doi.org/10.3390/v15041020 - 21 Apr 2023

Cited by 34 | Viewed by 7398

Abstract

In response to the global spread of antimicrobial resistance, there is an increased demand for novel and innovative antimicrobials. Bacteriophages have been known for their potential clinical utility in lysing bacteria for almost a century. Social pressures and the concomitant introduction of antibiotics [...] Read more.

In response to the global spread of antimicrobial resistance, there is an increased demand for novel and innovative antimicrobials. Bacteriophages have been known for their potential clinical utility in lysing bacteria for almost a century. Social pressures and the concomitant introduction of antibiotics in the mid-1900s hindered the widespread adoption of these naturally occurring bactericides. Recently, however, phage therapy has re-emerged as a promising strategy for combatting antimicrobial resistance. A unique mechanism of action and cost-effective production promotes phages as an ideal solution for addressing antibiotic-resistant bacterial infections, particularly in lower- and middle-income countries. As the number of phage-related research labs worldwide continues to grow, it will be increasingly important to encourage the expansion of well-developed clinical trials, the standardization of the production and storage of phage cocktails, and the advancement of international collaboration. In this review, we discuss the history, benefits, and limitations of bacteriophage research and its current role in the setting of addressing antimicrobial resistance with a specific focus on active clinical trials and case reports of phage therapy administration. Full article

(This article belongs to the Section Bacterial Viruses)

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Bacteriophage adsorption, replication, and release of phage progeny. Attachment of bacteriophages to bacterial cell membranes occurs through a process known as adsorption, whereby phage receptor binding proteins (RBPs) located on the phage tail interact with corresponding, specific receptors on the bacterial cell surface. Receptors can include lipopolysaccharide (the most common receptor on Gram-negative bacteria), polysaccharides, proteins, flagella, and pili. Upon binding, genetic material is transferred from the phage head into the bacterial cell, and host molecular machinery is used to generate new phage progeny in the case of lytic bacteriophages. Phage release occurs via bacterial cell lysis, facilitated by holin–endolysin interaction, resulting in cell membrane breakdown and eventual cell death. Full article ">Figure 2
Process of bacteriophage isolation for therapeutic use. Phage isolation begins with environmental water samples that are filtered to remove bacteria. The resultant filtrate is combined with a bacterial culture of interest in a process known as enrichment, whereby the concentration of phages effective against the specific host bacteria increases. After enrichment, spot assay allows for visualization of phage presence on a solid bacterial lawn. Phages from the resultant phage plaque are combined with a neutral buffer, and serial dilutions are performed before further plating to obtain distinct phage plaques of homogenous morphology. After phage harvest, amplification, sequencing, and characterization are performed to determine phage novelty. Phages are added to bacteriophage banks and international phage directories to be later employed in different applications. Full article ">Figure 3
Bacterial species addressed by clinical trials. Many of the currently registered clinical trials on clinicaltrials.gov address an ESKAPE pathogen (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, or Enterobacter species), predominantly S. aureus, K. pneumoniae, and P. aeruginosa. A. baumannii, E. faecium, and Enterobacter species are less frequently included. Other commonly targeted bacteria which are not considered ESKAPE pathogens include Streptococcus spp., Proteus spp., and E. coli. Bacterial targets denoted “Other” include Burkholderia, Stenotrophomonas, Salmonella, Serratia, Citrobacter, and Morganella. For some clinical trials, specific bacterial species were not defined on clinicaltrials.gov; therefore, some groupings include more than one species. Among the studied Staphylococcus spp., 51.7% are S. aureus, 9.5% S. epidermidis, or 9.5% S. lugdunensis as well as 23.8% undefined species of Staphylococcus. For Enterococcus spp., 25% are E. faecium versus 25% E. faecalis and approximately 50% undefined species of Enterococcus. For Proteus spp., 50% are P. mirabilis and 37.5% P. vulgaris, with 12.5% unknown species of Proteus. For Klebsiella spp., 76.9% are K. pneumoniae and 15.4% K. oxytoca, with the remaining 7.7% being undefined species of Klebsiella. Full article ">Figure 4
Indications for interventional clinical trials by infection site. A large proportion of currently registered bacteriophage clinical trials on clinicaltrials.gov (43.3%) involve the application of phage for either skin and soft tissue infections (SSTI) or gastrointestinal infections (GI). However, other infection sites, such as genitourinary tract infections (GU/UTI) and lung infections (including those in patients with cystic fibrosis) account for an additional 32.4% of the infections evaluated in these trials. The remaining trials involve patients with bacteremia, osteomyelitis, upper respiratory tract infections (URI), and prosthetic joint infections (PJI). A total of 2.7% of current phage clinical trials evaluate their use in treating non-healing wounds or infections of bones, upper respiratory tract, and genitourinary tract for which extensive antibiotic regimens failed or the use of a targeted drug was contraindicated. Full article ">

13 pages, 1187 KiB

Open AccessArticle

Vector Competence of Aedes albopictus for Yellow Fever Virus: Risk of Reemergence of Urban Yellow Fever in Brazil

by Rossela Damasceno-Caldeira, Joaquim Pinto Nunes-Neto, Carine Fortes Aragão, Maria Nazaré Oliveira Freitas, Milene Silveira Ferreira, Paulo Henrique Gomes de Castro, Daniel Damous Dias, Pedro Arthur da Silva Araújo, Roberto Carlos Feitosa Brandão, Bruno Tardelli Diniz Nunes, Eliana Vieira Pinto da Silva, Lívia Carício Martins, Pedro Fernando da Costa Vasconcelos and Ana Cecília Ribeiro Cruz

Viruses 2023, 15(4), 1019; https://doi.org/10.3390/v15041019 - 21 Apr 2023

Cited by 5 | Viewed by 2788

Abstract

The risk of the emergence and reemergence of zoonoses is high in regions that are under the strong influence of anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral diseases in [...] Read more.

The risk of the emergence and reemergence of zoonoses is high in regions that are under the strong influence of anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral diseases in the world, and the Culicidae Aedes albopictus has been proposed as having the potential to transmit the yellow fever virus (YFV). This mosquito inhabits both urban and wild environments, and under experimental conditions, it has been shown to be susceptible to infection by YFV. In this study, the vector competence of the mosquito Ae. albopictus for the YFV was investigated. Female Ae. albopictus were exposed to non-human primates (NHP) of the genus Callithrix infected with YFV via a needle inoculation. Subsequently, on the 14th and 21st days post-infection, the legs, heads, thorax/abdomen and saliva of the arthropods were collected and analyzed by viral isolation and molecular analysis techniques to verify the infection, dissemination and transmission. The presence of YFV was detected in the saliva samples through viral isolation and in the head, thorax/abdomen and legs both by viral isolation and by molecular detection. The susceptibility of Ae. albopictus to YFV confers a potential risk of reemergence of urban YF in Brazil. Full article

(This article belongs to the Special Issue Arboviral Lifecycle)

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Experimental procedure. Legend: (A) Experimental infection of non-human primates (NHP1, NHP2 and NHP3) with YFV; (B) NHP exposure to female Ae. albopictus (AL) on 3 dpi; (C) Exposure of healthy NHPs (NHP4, NHP5 and NHP6) to female Ae. albopictus on 14 dpi and segmentation of females into thorax, abdomen and head and extraction of saliva; (D) Exposure of healthy NHPs (NHP7, NHP8 and NHP9) to females Ae. albopictus on 21 dpi and segmentation of females into thorax, abdomen and head and extraction of saliva; (E) Segmentation of female (in thorax, abdomen, head) and saliva extraction, all samples organized in pools of 10 at 14 and 21 dpi; (F) NHP (negative control) and 50 mosquitoes used as negative controls. Full article ">Figure 2
Indirect immunofluorescence using polyclonal antibodies specific for YFV in C6/36 cell cultures: (A) negative control; (B) positive saliva pool sample (1AALS) on 14 dpi. The magnification is 200×. Full article ">Figure 3
Indirect immunofluorescence using monoclonal antibodies specific for YFV in C6/36 cell cultures: (A) negative control; (B) positive head sample (2AALC) analyzed at 14 dpi; (C) positive head batch sample (3AALC) analyzed at 21 dpi; (D) positive thorax/abdomen batch sample (2AALT) analyzed at 14 dpi; (E) positive thorax/abdomen batch sample (3AALT) analyzed at 21 dpi; (F) positive leg batch sample (2ALP) analyzed at 14 dpi; (G) positive leg batch sample (3AALP) analyzed at 21 dpi. The magnification is 40×. Full article ">

15 pages, 2950 KiB

Open AccessArticle

Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome

by Bárbara Batista Salgado, Maele Ferreira Jordão, Thiago Barros do Nascimento de Morais, Danielle Severino Sena da Silva, Ivanildo Vieira Pereira Filho, Wlademir Braga Salgado Sobrinho, Nani Oliveira Carvalho, Rafaella Oliveira dos Santos, Julia Forato, Priscilla Paschoal Barbosa, Daniel A. Toledo-Teixeira, Kerollen Runa Pinto, Ingrid Silva Correia, Isabelle Bezerra Cordeiro, Júlio Nino de Souza Neto, Enedina Nogueira de Assunção, Fernando Fonseca Almeida Val, Gisely Cardoso Melo, Vanderson de Souza Sampaio, Wuelton Marcelo Monteiro, Fabiana Granja, William M. de Souza, Spartaco Astolfi Filho, Jose Luiz Proenca-Modena, Jaila Dias Borges Lalwani, Marcus Vinícius Guimarães de Lacerda, Paulo Afonso Nogueira and Pritesh Lalwani Show full author list Hide full author list

Viruses 2023, 15(4), 1018; https://doi.org/10.3390/v15041018 - 20 Apr 2023

Cited by 1 | Viewed by 2032

Abstract

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We [...] Read more.

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We observed that the SARS-CoV-2 infection elicits a robust IgA and IgG response against the N-terminal (N1) and C-terminal (N3) region of the N protein, whereas we failed to detect IgA antibodies and observed a weak IgG response against the disordered linker region (N2) in COVID-19 patients. N and S protein-specific IgG1, IgG2 and IgG3 response was significantly elevated in hospitalized patients with severe disease compared to outpatients with non-severe disease. IgA and total IgG antibody reactivity gradually increased after the first week of symptoms. Magnitude of RBD-ACE2 blocking antibodies identified in a competitive assay and neutralizing antibodies detected by PRNT assay correlated with disease severity. Generally, the IgA and total IgG response between the discharged and deceased COVID-19 patients was similar. However, significant differences in the ratio of IgG subclass antibodies were observed between discharged and deceased patients, especially towards the disordered linker region of the N protein. Overall, SARS-CoV-2 infection is linked to an elevated blood antibody response in severe patients compared to non-severe patients. Monitoring of antigen-specific serological response could be an important tool to accompany disease progression and improve outcomes. Full article

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

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SARS-CoV-2 nucleocapsid and spike proteins elicit a robust IgA, total IgG and IgG-subclass response in COVID-19 patients. (A) Sketch depicts SARS-CoV-2 full-length and truncated nucleocapsid (N) and spike (S) proteins. (B) RT-PCR confirmed COVID-19 patients (n = 128) with non-severe disease (outpatient n = 80) and hospitalized patients with severe disease (inpatient n = 48) included in the study. (C–E) 100 ng of purified SARS-CoV-2 proteins were coated on ELISA plates and 1:100 patient serum dilution was used in an indirect ELISA to estimate serum IgA, IgG and IgG-subclass response. Antigen-specific antibody response among (C) all COVID-19 patients, (D) outpatients and inpatients, and (E) discharged (n = 24, samples n = 59) and deceased (n = 24, samples n = 72) inpatients was compared. Reactivity index (RI) values were calculated as a ratio between sample optical density and pre-pandemic negative samples. Horizontal red or black lines denote the median antibody range. Each curve in the graph represents reactivity towards each antigen, Splines were plotted using the Fit Spline program in GraphPad Prism software. ANOVA test with Tukey’s post hoc test was used to compare differences in the RI between antigens. Two-way ANOVA with Šidák post hoc correction for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 2
Representative COVID-19 patients and their humoral response: 100 ng of purified SARS-CoV-2 protein and 1:100 patient serum dilution was used in an indirect ELISA to estimate serum IgA, total IgG, IgG-subclass response and RBD-ACE inhibitory antibodies. Each column represents one patient, and each row represents specific response to one antibody isotype or RBD-ACE2 inhibitory antibodies. Each graph has reactivity index for SARS-CoV-2 full-length or truncated antigens tested. Outpatient samples were collected only at recruitment, whereas inpatients were followed longitudinally after hospitalization. Vertical dotted blank and red lines indicate hospitalized and intubation period, respectively (samples n = 3). Full article ">Figure 3
RBD-ACE2 interactions blocking antibodies and neutralizing antibody increase with symptomatic days and correlates with disease severity. (A) Commercial RBD-ACE2 competitive assay was used to assess inhibitory antibodies among COVID-19 patients (n = 111, samples n = 190) versus healthy individuals (n = 13). (B–D) Percentage RBD-ACE2 inhibition was evaluated post-onset of symptoms (outpatients n = 70; discharged inpatients n = 20, samples n = 54; deceased inpatients n = 21, samples n = 66). (E–H) Plasma samples were tested by PRNT in Vero cells after incubation with 300 plaque forming units (PFU) (outpatients n = 40; discharged inpatients n = 6, samples n = 18; deceased inpatients n = 8, samples n = 24). (E) Correlation between RBD-ACE2 competitive assay and live-virus PRNT assay (samples n = 82). (F) Outpatient and inpatient patient samples were serially diluted for the PRNT assay. Each data point represents the mean of all plasma samples for each group at each dilution level and error bars represent SD. (G,H) Percentage neutralization values were compared to days after onset of symptoms to evaluate neutralizing antibody kinetics. Solid lines representing the tendency for the neutralizing antibodies were created by the Fit Spline program in GraphPad Prism software. Two-way ANOVA with Šidák post hoc correction for multiple comparisons was applied. **** p < 0.0001. Full article ">Figure 4
Elevated antigen-specific IgG3 response correlated with severe disease outcome. IgG subclass antibody ratio correlates with disease outcome. Ratio of full-length and truncated N and S protein-specific IgG subclass antibody response was compared between (A) outpatients (n = 45) and inpatients (n = 38, samples n = 121) or (B) hospitalized patients with discharged (n = 20, samples n = 55) or deceased (n = 18, samples n = 66) disease outcome. Samples with more than seven days after the start of symptoms were included in this analysis. T-test was performed to compare patient groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">

15 pages, 3258 KiB

Open AccessArticle

Epidemiological Surveillance Reveals the Rise and Establishment of the Omicron SARS-CoV-2 Variant in Brazil

by Joice do Prado Silva, Aline Brito de Lima, Luige Biciati Alvim, Frederico Scott Varella Malta, Cristiane Pinheiro Toscano Brito Mendonça, André Henrique Barbosa de Carvalho, Jéssica Silqueira Hickson Rios, Paula Luize Camargos Fonseca, Daniel Costa Queiroz, Luíza Campos Guerra de Araújo e Santos, Alessandro Clayton de Souza Ferreira, Renan Pedra de Souza, Renato Santana de Aguiar and Danielle Alves Gomes Zauli

Viruses 2023, 15(4), 1017; https://doi.org/10.3390/v15041017 - 20 Apr 2023

Cited by 4 | Viewed by 1999

Abstract

The introduction of SARS-CoV-2 variants of concern (VOCs) in Brazil has been associated with major impacts on the epidemiological and public health scenario. In this study, 291,571 samples were investigated for SARS-CoV-2 variants from August 2021 to March 2022 (the highest peak of [...] Read more.

The introduction of SARS-CoV-2 variants of concern (VOCs) in Brazil has been associated with major impacts on the epidemiological and public health scenario. In this study, 291,571 samples were investigated for SARS-CoV-2 variants from August 2021 to March 2022 (the highest peak of positive cases) in four geographical regions of Brazil. To identify the frequency, introduction, and dispersion of SARS-CoV-2 variants in 12 Brazilian capitals, VOCs defining spike mutations were identified in 35,735 samples through genotyping and viral genome sequencing. Omicron VOC was detected in late November 2021 and replaced the Delta VOC in approximately 3.5 weeks. We estimated viral load differences between SARS-CoV-2 Delta and Omicron through the evaluation of the RT-qPCR cycle threshold (Ct) score in 77,262 samples. The analysis demonstrated that the Omicron VOC has a lower viral load in infected patients than the Delta VOC. Analyses of clinical outcomes in 17,586 patients across the country indicated that individuals infected with Omicron were less likely to need ventilatory support. The results of our study reinforce the importance of surveillance programs at the national level and showed the introduction and faster dispersion of Omicron over Delta VOC in Brazil without increasing the numbers of severe cases of COVID-19. Full article

(This article belongs to the Collection Mathematical Modeling of Viral Infection)

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Descriptive analysis of the study design. (A) Twelve capitals distributed along four macro-regions of Brazil were included in this study. A total of 77,262 samples with confirmed positive COVID-19 diagnoses were evaluated. The blue circle size represents the proportion of positive samples in each Brazilian capital; (B) the cartogram of sample representativeness according to the population of each Brazilian region; (C) the sex and age profile of the study sample (data were grouped by sex and age clusters); and (D) the sex profile by Brazilian capital of the study sample. The gray and magenta bars correspond to men’s and women’s records, respectively. Full article ">Figure 2
Monitoring of Delta and Omicron VOCs in Brazil. Delta and Omicron variants were represented by red and dark purple colors, respectively. (A) Percentage of non-SGTF and SGTF samples per epidemiological week in 35,735 samples across Brazil. (B) Absolute numbers of non-SGTF and SGTF profiles in the positive RT-PCR samples per epidemiological week and positive rate; (C) Transition period between non-SGTF and SGTF samples by capital and epidemiological week. Blank spaces indicate the absence of samples in a particular capital within the specified epidemiological week. Full article ">Figure 3
Phylogenetic reconstruction of SARS-CoV-2 during the fourth wave of SARS-CoV-2 in Brazil (epidemiological weeks 35/2021 to 09/2022). The dataset was constructed based on the genomes used by Nextclade as references for Omicron classification (n = 1531). Maximum likelihood phylogenetic tree inferred from our dataset to confirm lineage classification. Purple-tipped shapes indicate genomes generated in our study (n = 109). Node values correspond to bootstrap values. The clades corresponding to the variants BA.1, BA.2, BA.4, and BA.5 are highlighted in gray, lilac, pink, and purple, respectively. Full article ">Figure 4
Comparative analysis of RT-qPCR Ct values between Delta and Omicron SARS-CoV-2 VOC dominance periods. Colors represent the dominant variant, with a frequency above 90% in the analyzed period, being Delta (red) or Omicron (dark purple). Violin plots displaying the distribution of Ct data according to the predominant lineage for the viral target genes N (A) and ORF1ab (C), and the MS2 internal process control (E). Boxplots display Ct value variation along the epidemiological weeks comprised in the study period (August 29, 2021–March 5, 2022) for the viral target genes N (B), ORF1ab (D), and MS2 internal control (F). A statistical comparison between periods denotes that Omicron VOC might be associated with lower viral loads in the upper respiratory tract than Delta VOC infection. White bars represent the first quartile, the median and the third quartile. Black circles represent the outliers * p < 0.001. Full article ">Figure 5
Forest plot of effect sizes measured between Omicron (case) and Delta (control) groups in COVID-19 patients for: (A) need for ventilatory support: 9828 cases and 6488 controls; (B) intensive care unit admission: 9914 cases and 6467 controls; and (C) death: 9815 cases and 6443 controls, in a meta-analysis of 12 different Brazilian capitals according to the periods in which Omicron (case) and Delta (control) VOCs had a minimum prevalence of 90% in each capital city. Full article ">

14 pages, 244 KiB

Open AccessArticle

How do German General Practitioners Manage Long-/Post-COVID? A Qualitative Study in Primary Care

by Beatrice E. Bachmeier, Salome Hölzle, Mohamed Gasser and Marjan van den Akker

Viruses 2023, 15(4), 1016; https://doi.org/10.3390/v15041016 - 20 Apr 2023

Cited by 8 | Viewed by 2060

Abstract

Background: Many patients with ongoing complaints after a SARS-CoV-2 infection are treated in primary care. Existing medical guidelines on how to diagnose and treat Long-/Post-COVID are far from being comprehensive. This study aims to describe how German general practitioners (GPs) deal with this [...] Read more.

Background: Many patients with ongoing complaints after a SARS-CoV-2 infection are treated in primary care. Existing medical guidelines on how to diagnose and treat Long-/Post-COVID are far from being comprehensive. This study aims to describe how German general practitioners (GPs) deal with this situation, what problems they experience when managing such patients, and how they solve problems associated with the diagnosis and treatment of Long-/Post-COVID. Methods and Findings: We conducted a qualitative study and interviewed 11 GPs. The most commonly described symptoms were ongoing fatigue, dyspnea, chest tightness and a decrease in physical capacity. The most common way to identify Long-/Post-COVID was by exclusion. Patients suffering from Long-/Post-COVID were generally treated by their GPs and rarely referred. A very common non-pharmacological intervention was to take a wait-and-see approach and grant sick leave. Other non-pharmacological interventions included lifestyle advices, physical exercise, acupuncture and exercises with intense aromas. Pharmacological treatments focused on symptoms, like respiratory symptoms or headaches. Our study’s main limitations are the small sample size and therefore limited generalizability of results. Conclusions: Further research is required to develop and test pharmaceutical and non-pharmaceutical interventions for patients with Long-/Post-COVID. In addition, strategies to prevent the occurrence of Long-/Post-COVID after an acute infection with SARS-CoV-2 have to be developed. The routine collection of data on the diagnosis and management of Long-/Post-COVID may help in the formulation of best practices. It is up to policymakers to facilitate the necessary implementation of effective interventions in order to limit the huge societal consequences of large groups of patients suffering from Long-/Post-COVID. Full article

(This article belongs to the Special Issue COVID-19: Prognosis and Long-Term Sequelae)

14 pages, 4682 KiB

Open AccessReview

Asfarviruses and Closely Related Giant Viruses

by Sihem Hannat, Bernard La Scola, Julien Andreani and Sarah Aherfi

Viruses 2023, 15(4), 1015; https://doi.org/10.3390/v15041015 - 20 Apr 2023

Cited by 2 | Viewed by 2334

Abstract

Acanthamoeba polyphaga mimivirus, so called because of its “mimicking microbe”, was discovered in 2003 and was the founding member of the first family of giant viruses isolated from amoeba. These giant viruses, present in various environments, have opened up a previously unexplored [...] Read more.

Acanthamoeba polyphaga mimivirus, so called because of its “mimicking microbe”, was discovered in 2003 and was the founding member of the first family of giant viruses isolated from amoeba. These giant viruses, present in various environments, have opened up a previously unexplored field of virology. Since 2003, many other giant viruses have been isolated, founding new families and taxonomical groups. These include a new giant virus which was isolated in 2015, the result of the first co-culture on Vermamoeba vermiformis. This new giant virus was named “Faustovirus”. Its closest known relative at that time was African Swine Fever Virus. Pacmanvirus and Kaumoebavirus were subsequently discovered, exhibiting phylogenetic clustering with the two previous viruses and forming a new group with a putative common ancestor. In this study, we aimed to summarise the main features of the members of this group of giant viruses, including Abalone Asfarvirus, African Swine Fever Virus, Faustovirus, Pacmanvirus, and Kaumoebavirus. Full article

(This article belongs to the Special Issue Research Progresses of Giant Viruses: A Themed Issue Dedicated to Professor Jean-Michel Claverie)

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Genome synteny between Abalone Asfarvirus genome and the ASFV-BA71V genome. Schematic genome alignment obtained using the Mauve software [<a href="#B102-viruses-15-01015" class="html-bibr">102</a>]. The analysis was performed using the genome of Abalone Asfarvirus (LC637659.1), draft genome and ASFV strain BA71V (NC_001659.2). The blocks illustrated above the x axis are in the positive strand (forward sense), while blocks below the x axis are in the negative strand (reverse sense). The names of each virus are indicated below the sequence. The connected lines represent the relatively similar blocks between the genomes. Full article ">Figure 2
Phylogenetic tree based on the DNA polymerase homologs of Asfarviridae and relative viruses. ASFV contains 38 DNA polymerase sequences. Protein alignment was performed using Mafft software (v7.471) with standard parameters. The tree was built using IQ-TREE 1.6.12 with LG + F + I + G4 as best-fit model and 10,000 ultrafast bootstrap replication. Poxviridae sequences were used as an outgroup. Full article ">Figure 3
Mauve alignment genome of some Asfarviridae and other related viruses. Schematic genome alignment obtained using the Mauve software [<a href="#B102-viruses-15-01015" class="html-bibr">102</a>]. The analysis was performed using the genome of ASFV strain BA71V (NC_001659.2), Abalone Asfarvirus (LC637659.1), Faustovirus E12 (KJ614390.1), Pacmanvirus (LT706986.1) and Kaumoebavirus (KX552040.1). The blocks illustrated above the x axis are in the positive strand (forward sense), while blocks below the x axis are in the negative strand (reverse sense). Names of each virus are indicated below the sequence. The connected lines represent the relatively similar blocks between the genomes. Full article ">

14 pages, 2730 KiB

Open AccessArticle

Human Cytomegalovirus UL23 Antagonizes the Antiviral Effect of Interferon-γ by Restraining the Expression of Specific IFN-Stimulated Genes

by Hankun Wang, Weijian Peng, Jialin Wang, Chunling Zhang, Wangchun Zhao, Yanhong Ran, Xiaoping Yang, Jun Chen and Hongjian Li

Viruses 2023, 15(4), 1014; https://doi.org/10.3390/v15041014 - 20 Apr 2023

Cited by 3 | Viewed by 1771

Abstract

Interferon-γ (IFN-γ) is a critical component of innate immune responses in humans to combat infection by many viruses, including human cytomegalovirus (HCMV). IFN-γ exerts its biological effects by inducing hundreds of IFN-stimulated genes (ISGs). In this study, RNA-seq analyses revealed that HCMV tegument [...] Read more.

Interferon-γ (IFN-γ) is a critical component of innate immune responses in humans to combat infection by many viruses, including human cytomegalovirus (HCMV). IFN-γ exerts its biological effects by inducing hundreds of IFN-stimulated genes (ISGs). In this study, RNA-seq analyses revealed that HCMV tegument protein UL23 could regulate the expression of many ISGs under IFN-γ treatment or HCMV infection. We further confirmed that among these IFN-γ stimulated genes, individual APOL1 (Apolipoprotein-L1), CMPK2 (Cytidine/uridine monophosphate kinase 2), and LGALS9 (Galectin-9) could inhibit HCMV replication. Moreover, these three proteins exhibited a synergistic effect on HCMV replication. UL23-deficient HCMV mutants induced higher expression of APOL1, CMPK2, and LGALS9, and exhibited lower viral titers in IFN-γ treated cells compared with parental viruses expressing full functional UL23. Thus, UL23 appears to resist the antiviral effect of IFN-γ by downregulating the expression of APOL1, CMPK2, and LGALS9. This study highlights the roles of HCMV UL23 in facilitating viral immune escape from IFN-γ responses by specifically downregulating these ISGs. Full article

(This article belongs to the Special Issue 65-Year Anniversary of the Discovery of Cytomegalovirus)

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14 pages, 3558 KiB

Open AccessArticle

Topical Protease Inhibitor Decreases Anal Carcinogenesis in a Transgenic Mouse Model of HPV Anal Disease

by Laura C. Gunder, Hillary R. Johnson, Evan Yao, Tyra H. Moyer, Heather A. Green, Nathan Sherer, Wei Zhang and Evie H. Carchman

Viruses 2023, 15(4), 1013; https://doi.org/10.3390/v15041013 - 20 Apr 2023

Cited by 1 | Viewed by 1632

Abstract

Anal cancer is a major health problem. This study seeks to determine if the topical protease inhibitor Saquinavir (SQV), is effective at the prevention of anal cancer in transgenic mice with established anal dysplasia. K14E6/E7 mice were entered into the study when the [...] Read more.

Anal cancer is a major health problem. This study seeks to determine if the topical protease inhibitor Saquinavir (SQV), is effective at the prevention of anal cancer in transgenic mice with established anal dysplasia. K14E6/E7 mice were entered into the study when the majority spontaneously developed high-grade anal dysplasia. To ensure carcinoma development, a subset of the mice was treated with a topical carcinogen: 7,12-Dimethylbenz[a]anthracene (DMBA). Treatment groups included: no treatment, DMBA only, and topical SQV with/without DMBA. After 20 weeks of treatment, anal tissue was harvested and evaluated histologically. SQV was quantified in the blood and anal tissue, and tissue samples underwent analysis for E6, E7, p53, and pRb. There was minimal systemic absorption of SQV in the sera despite high tissue concentrations. There were no differences in tumor-free survival between SQV-treated and respective control groups but there was a lower grade of histological disease in the mice treated with SQV compared to those untreated. Changes in E6 and E7 levels with SQV treatment suggest that SQV may function independently of E6 and E7. Topical SQV decreased histological disease progression in HPV transgenic mice with or without DMBA treatment without local side effects or significant systemic absorption. Full article

(This article belongs to the Special Issue Efficacy and Safety of Antiviral Therapy)

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16 pages, 1540 KiB

Open AccessArticle

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia

by Khalil Dachraoui, Ifhem Chelbi, Imen Labidi, Raja Ben Osman, Aida Sayadi, Mourad Ben Said, Saifedine Cherni, Mohammed Abdo Saghir Abbas, Rémi Charrel and Elyes Zhioua

Viruses 2023, 15(4), 1012; https://doi.org/10.3390/v15041012 - 20 Apr 2023

Cited by 1 | Viewed by 1806

Abstract

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of [...] Read more.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum. Full article

(This article belongs to the Special Issue Sand Fly-Borne Phleboviruses, Volume II)

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8 pages, 3662 KiB

Open AccessBrief Report

Role of Histamine and Related Signaling in Kaposi’s Sarcoma-Associated Herpesvirus Pathogenesis and Oncogenesis

by Jungang Chen, Jiao Song, Karlie Plaisance-Bonstaff, Shengyu Mu, Steven R. Post, Lu Dai and Zhiqiang Qin

Viruses 2023, 15(4), 1011; https://doi.org/10.3390/v15041011 - 20 Apr 2023

Viewed by 1631

Abstract

Although Kaposi’s sarcoma-associated herpesvirus (KSHV) has been reported to cause several human cancers including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), the mechanisms of KSHV-induced tumorigenesis, especially virus–host interaction network, are still not completely understood, which therefore hinders the development of effective [...] Read more.

Although Kaposi’s sarcoma-associated herpesvirus (KSHV) has been reported to cause several human cancers including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), the mechanisms of KSHV-induced tumorigenesis, especially virus–host interaction network, are still not completely understood, which therefore hinders the development of effective therapies. Histamine, together with its receptors, plays an important role in various allergic diseases by regulating different inflammation and immune responses. Our previous data showed that antagonists targeting histamine receptors effectively repressed KSHV lytic replication. In the current study, we determined that histamine treatment increased cell proliferation and anchorage-independent growth abilities of KSHV-infected cells. Furthermore, histamine treatment affected the expression of some inflammatory factors from KSHV-infected cells. For clinical relevance, several histamine receptors were highly expressed in AIDS-KS tissues when compared to normal skin tissues. We determined that histamine treatment promoted KSHV-infected lymphoma progression in immunocompromised mice models. Therefore, besides viral replication, our data indicate that the histamine and related signaling are also involved in other functions of KSHV pathogenesis and oncogenesis. Full article

(This article belongs to the Special Issue Opportunistic Viral Infections)

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Figure 1
Histamine treatment promotes the growth of KSHV-infected cells. (A,B) HUVEC with or without KSHV infection ((A), MOI~3) or TIVE and KSHV long-term-infected TIVE-LTC (B) were treated with indicated concentrations of histamine for 48 h; then, the cell proliferation was measured using the WST-1 assays. (C,D) The anchorage-independent growth abilities of iSLK.219 and TIVE-LTC were determined using the soft agar assays. Error bars represent the S.D. for three independent experiments. * = p < 0.05; ** = p < 0.01 (vs. the vehicle groups). Full article ">Figure 2
The impacts of inflammatory factors by histamine treatment. (A) TIVE-LTC were treated with indicated concentrations of histamine for 48 h; then, gene transcripts were quantified by using RT-qPCR. Error bars represent the S.D. for three independent experiments. * = p < 0.05; ** = p < 0.01 (vs. the vehicle groups). (B) Protein expression was measured using Western blot. Full article ">Figure 3
The upregulation of histamine receptor expression within AIDS-KS tissues. Expression of viral latent protein LANA and histamine receptors H1-H4 (HRH1-HRH4) in AIDS-KS tissues and normal skin tissues were determined and compared by using immunohistochemical (IHC) staining as described in the Methods section (the magnification at ×40). The arrows indicate representative HRHs-positive KS tumor cells. Bars: 50 μm. Full article ">Figure 4
Histamine treatment promotes KSHV-infected lymphoma progression in vivo. (A–C) NOD/SCID mice were injected i.p. with BCBL-1 cells. Then, 72 h later, histamine (1.0 mg/kg) or vehicle were administered i.p. as described in the Methods section, and weights were recorded weekly. At the end of the treatment period, the ascites and spleens were collected from the histamine- or vehicle-treated mice for comparison. (D) The gene transcripts in ascites were quantified by using RT-qPCR. * = p < 0.05; ** = p < 0.01 (vs. the vehicle groups). Full article ">

14 pages, 3287 KiB

Open AccessArticle

High-Risk Regions of African Swine Fever Infection in Mozambique

by Azido Ribeiro Mataca, Francisco Alyson Silva Oliveira, Ângelo André Lampeão, José Pereira Mendonça, Maria Aparecida Scatamburlo Moreira, Rinaldo Aparecido Mota, Wagnner José Nascimento Porto, David Germano Gonçalves Schwarz and Abelardo Silva-Júnior

Viruses 2023, 15(4), 1010; https://doi.org/10.3390/v15041010 - 20 Apr 2023

Cited by 1 | Viewed by 1888

Abstract

African swine fever (ASF) is a transboundary infectious disease that can infect wild and domestic swine and requires enhanced surveillance between countries. In Mozambique, ASF has been reported across the country, spreading between provinces, mainly through the movement of pigs and their by-products. [...] Read more.

African swine fever (ASF) is a transboundary infectious disease that can infect wild and domestic swine and requires enhanced surveillance between countries. In Mozambique, ASF has been reported across the country, spreading between provinces, mainly through the movement of pigs and their by-products. Subsequently, pigs from bordering countries were at risk of exposure. This study evaluated the spatiotemporal distribution and temporal trends of ASF in swine in Mozambique between 2000 and 2020. During this period, 28,624 cases of ASF were reported across three regions of the country. In total, the northern, central, and southern regions presented 64.9, 17.8, and 17.3% of the total cases, respectively. When analyzing the incidence risk (IR) of ASF per 100,000 pigs, the Cabo Delgado province had the highest IR (17,301.1), followed by the Maputo province (8868.6). In the space-time analysis, three clusters were formed in each region: (i) Cluster A involved the provinces of Cabo Delgado and Nampula (north), (ii) Cluster B involved the province of Maputo and the city of Maputo (south), and (iii) Cluster C consisted of the provinces of Manica and Sofala (central) in 2006. However, when analyzing the temporal trend in the provinces, most were found to be decreasing, except for Sofala, Inhambane, and Maputo, which had a stationary trend. To the best of our knowledge, this is the first study to evaluate the spatial distribution of ASF in Mozambique. These findings will contribute to increasing official ASF control programs by identifying high-risk areas and raising awareness of the importance of controlling the borders between provinces and countries to prevent their spread to other regions of the world. Full article

(This article belongs to the Special Issue Endemic and Emerging Swine Viruses 2023)

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14 pages, 1278 KiB

Open AccessArticle

Intact Proviral DNA Analysis of the Brain Viral Reservoir and Relationship to Neuroinflammation in People with HIV on Suppressive Antiretroviral Therapy

by Dana Gabuzda, Jun Yin, Vikas Misra, Sukrutha Chettimada and Benjamin B. Gelman

Viruses 2023, 15(4), 1009; https://doi.org/10.3390/v15041009 - 20 Apr 2023

Cited by 13 | Viewed by 2427

Abstract

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes [...] Read more.

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1–92); 3′ defective, 509 (225–858); 5′ defective, 519 (273–906); and total proviruses, 1063 (501–2074) copies/10⁶ cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3′ and 5′ defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/10⁶ cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/10⁶ cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure. Full article

(This article belongs to the Special Issue HIV Neurological Disorders)

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Figure 1
IPDA analysis of HIV proviruses in brain tissue from 28 HIV+ individuals on ART. Log10 copy number (standardized to 106 cells) of proviral genomes defined as intact (blue), 3′ defective (green), 5′ defective (yellow), or total (sum of intact, 3′ defective, and 5′ defective; red) was measured in autopsy brain tissue (frontal white matter) from HIV+ individuals on ART (n = 28) (top left). Log10 copy number of intact and total proviruses in groups by neurocognitive impairment (NCI, n = 15) vs. no neurocognitive impairment (No NCI, n = 13), last CD4 count <350 (n = 18) vs. last CD4 count ≥350 (n = 10), or HIVE score 0 (n = 19) vs. HIVE score 1 or 2 (n = 9) (top right and bottom panels). Horizontal bars represent medians, boxes span the interquartile range (IQR), and whiskers extend to extreme data points within 1.5 times the IQR. Full article ">Figure 2
Correlation analysis of intact versus total HIV provirus or gag DNA levels in brain tissue from HIV+ individuals on ART. (A) Correlation between log10 intact provirus copy number and log10 total provirus copy number or log10 gag DNA copy number (standardized to 106 cells) in brain tissue (frontal lobe white matter) among 28 HIV+ individuals. (B) Correlation analysis as described in sensitivity analysis (A) restricted to 18 HIV+ individuals with detectable intact proviruses (>1 intact copy per 106 cells). Spearman’s rho and p-values are shown. Full article ">Figure 3
Differential expression of genes related to neuroinflammation and white matter integrity in groups stratified by level of intact proviruses in brain tissue from 28 HIV+ individuals on ART. Differential gene expression in brain tissue (frontal white matter) from 28 HIV+ individuals stratified by frequency of intact proviruses per 106 cells in brain tissue >5 copies vs. ≤5 copies per 106 cells (n = 12 and n = 16, respectively). Horizontal bars represent medians, boxes span the interquartile range (IQR), and whiskers extend to extreme data points within 1.5 times the IQR. p-values calculated using Welch’s t-test. Full article ">

16 pages, 3392 KiB

Open AccessArticle

Virulence Profiles of Wild-Type, P.1 and Delta SARS-CoV-2 Variants in K18-hACE2 Transgenic Mice

by Yasmin da Silva Santos, Thais Helena Martins Gamon, Marcela Santiago Pacheco de Azevedo, Bruna Larotonda Telezynski, Edmarcia Elisa de Souza, Danielle Bruna Leal de Oliveira, Jamille Gregório Dombrowski, Livia Rosa-Fernandes, Giuseppe Palmisano, Leonardo José de Moura Carvalho, Maria Cecília Rui Luvizotto, Carsten Wrenger, Dimas Tadeu Covas, Rui Curi, Claudio Romero Farias Marinho, Edison Luiz Durigon and Sabrina Epiphanio

Viruses 2023, 15(4), 999; https://doi.org/10.3390/v15040999 - 19 Apr 2023

Cited by 5 | Viewed by 2273

Abstract

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and [...] Read more.

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice. Full article

(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)

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15 pages, 1798 KiB

Open AccessArticle

Evaluating Novel Quantification Methods for Infectious Baculoviruses

by Keven Lothert, Elena Bagrin and Michael W. Wolff

Viruses 2023, 15(4), 998; https://doi.org/10.3390/v15040998 - 19 Apr 2023

Viewed by 2523

Abstract

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, [...] Read more.

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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Figure 1
Amplification plots for the qPCR evaluation of different baculovirus concentrations in the range of 0 (i.e., blank)−1.1 × 107 IU/mL (A) as well as the linear regression over the concentration range (B). The number of replicate measurements was n = 39 for 1.1 × 102 IU/mL and 1.1 × 107 IU/mL and n = 18 for all other concentrations and the blank. Full article ">Figure 2
Amplification plots of the baculovirus DNA by qPCR measurements. The DNA was extracted from samples taken either from the supernatant or from the cell pellet of infected cells after 5 min (i.e., 0 h) of incubation and compared to a negative control (A). The amount of viral DNA detectable in the cell pellet was monitored over the course of 7 h (B) and compared to values after 18 h of infection (C). All samples were prepared in triplicates, with error bars only being displayed in (A) to allow a clearer view of the data. Full article ">Figure 3
Development of the flow cytometric quantification protocol for baculoviruses (either wild-type or virus expressing the green fluorescent protein, BV-wt and BV-GFP, respectively). In a first approach, (A) the detection capability for both viruses was determined without further method optimization after 18 h of cell infection and subsequent flow cytometric detection of fluorescent cells. Depending on the staining procedure, green (no staining, left column), yellow (PE staining, middle column), or red fluorescence (APC staining, right column) was evaluated. The infection kinetics of the three approaches (B) indicate the earliest possible time of quantification at which the linear calibration range was subsequently determined for each individually optimized strategy (C). Error bars depict standard deviation of technical triplicates (A,B) and 18 replicates with n = 6 on three different days for (C). Full article ">Figure 4
Quantification of the percentage of fluorescing cells after BV infection and fluorescent labeling of the mRNA of the viral gp64 protein within the first four hours of infection. A multiplicity of infection (MOI) of 1 was used and compared to a negative control without virus infection (error bars indicate the standard deviation of technical triplicates). Full article ">

11 pages, 3302 KiB

Open AccessArticle

Structural Consequences of Antigenic Variants of Human A/H3N2 Influenza Viruses

by David Francis Burke

Viruses 2023, 15(4), 1008; https://doi.org/10.3390/v15041008 - 19 Apr 2023

Cited by 1 | Viewed by 1495

Abstract

The genetic basis of antigenic drift of human A/H3N2 influenza virus is crucial to understanding the constraints of influenza evolution and determinants of vaccine escape. Amino acid changes at only seven positions near the receptor binding site of the surface hemagglutinin protein have [...] Read more.

The genetic basis of antigenic drift of human A/H3N2 influenza virus is crucial to understanding the constraints of influenza evolution and determinants of vaccine escape. Amino acid changes at only seven positions near the receptor binding site of the surface hemagglutinin protein have been shown to be responsible for the major antigenic changes for over forty years. Experimental structures of HA are now available for the majority of the observed antigenic clusters of A/H3N2. An analysis of the HA structures of these viruses reveals the likely consequences of these mutations on the structure of HA and thus, provides a structural basis for the antigenic changes seen in human influenza viruses. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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Figure 1
Structure of the trimer of the haemagglutinin protein of A/H3N2 influenza virus. (a) The five antigenic sites A–E are coloured cyan, red, green, orange and blue, respectively. The receptor binding site is coloured in gold. (b) Close-up of the receptor binding site of HA (coloured in gold). The seven amino acid positions at which mutations have been shown to be sufficient for human H3 antigenic evolution are highlighted. Full article ">Figure 2
(a) The substitution Glu156Lys which is responsible for the antigenic change between viruses in the SI87(blue) and BE92(orange) clusters. Compared to the structure of the SI87 representative (A/Sichuan/2/1987; PDB entry 6pyp), the model of the BE92 representative (A/Netherlands/179/1993) is predicted to induce an additional structural rearrangement involving the sidechain of Tyr159. (b) The same substitution occurs between the earlier clusters BK79(blue) and TX77(orange). The structures of A/Texas/1/1977(PDB entry 6mxu) and A/Philippines/2/1982(PDB entry 6mym) show that, due to the presence of a smaller amino acid at position 159 (Ser) the mutation Glu156Lys does not give rise to additional structural changes thus resulting in a much smaller antigenic change. Full article ">Figure 3
(a) Structures are available for viruses from the HK68(A/Hong-Kong/1/1968 PDB entry 4fnk) and EN72(A/Port-Chalmers/1/1973 PDB 4we5) clusters which contain Gln189 and show little conformational variability. (b) Many experimental structures are available for viruses which contain Lys189. Structures from the clusters VI75 (A/Victoria/3/1975; PDB entry 4gms), TX77 (A/Texas/1/1977; PDB entry 6mxu), BK79 (A/Netherlands/209/1980; PDB entry 6n08; A/Philippines/2/1982; PDB entry 6mym),VI09 (A/Victoria/361/2011; PDB entry 4we9), 3C.1 (A/Texas/50/2012; PDB entry 5w08; A/Singapore/H2011.447/2011; PDB entry 4o5n; A/Alberta/26/2012; PDB entry 4we7), 3C.2A (A/Michigan/15/2014; PDB entry 6bkp), and 3C.3A (A/Switzerland/9715293/2013; PDB entry 6pdx) show a large difference in conformational flexibility. Full article ">Figure 4
Interactions with neighbouring amino acids are shown to alter the allowed conformations sampled by the side-chains at position 189. (a) Experimental structures of viruses from the clusters VI75 (A/Victoria/3/1975; PDB entry 4gms), TX77 (A/Texas/1/1977, PDB entry 6mxu) and BK79 (A/Netherlands/209/1980, PDB entry 6n08, A/Philippines/2/1982, PDB entry 6mym) contain Lys189 and Asn193. In these structures, there is no interaction with neighbouring Asn193 and Lys189 is able to sample many conformations. (b) In the structure of the SI87 virus (A/Sichuan/2/1987; PDB entry 6p6p), the interaction of Arg189 with neighbouring Asn193 reduces the conformations observed. Full article ">Figure 5
(a) In many structures, such as WI05 (A/Hong-Kong/4443/2005; PDB entry 2yp7, orange), Asn145 is seen to interact with Lys140. As seen in the structure of the FU02 virus, A/Wyoming/3/2003(PDB entry 6bko, blue), the substitution responsible for cluster transitions 6 and 8, Asn145Lys, disrupts this interaction resulting in changes in charge and a change in shape and volume at both positions 140 and 145. (b) The substitution Lys140Ile occurs between viruses in the WI05 and BR07 clusters. Comparing the experimental structures for WI05 (A/Hong-Kong/4443/2005; PDB entry 2yp7, orange) and BR07 (A/Brisbane/10/2007; PDB entry 6aop, blue) the interaction between 140 and 145 is lost and causes a change in volume at position 140. Full article ">Figure 6
Predicted N-glycosylation sites in HA. There is a progressive increase over time in the number of N-glycosylation sites in HA. With the exception of position 158, these have little effect on antigenic properties of the viruses. Those sites are highlighted for the following genetic clusters (a) HK68 (b) BE92 (c) CAL04 (d) 3C.1 (e) 3C.2A1 (f) 3C.3A. Full article ">Figure 6 Cont.
Predicted N-glycosylation sites in HA. There is a progressive increase over time in the number of N-glycosylation sites in HA. With the exception of position 158, these have little effect on antigenic properties of the viruses. Those sites are highlighted for the following genetic clusters (a) HK68 (b) BE92 (c) CAL04 (d) 3C.1 (e) 3C.2A1 (f) 3C.3A. Full article ">

21 pages, 3830 KiB

Open AccessArticle

VirClust—A Tool for Hierarchical Clustering, Core Protein Detection and Annotation of (Prokaryotic) Viruses

by Cristina Moraru

Viruses 2023, 15(4), 1007; https://doi.org/10.3390/v15041007 - 19 Apr 2023

Cited by 14 | Viewed by 2695

Abstract

Recent years have seen major changes in the classification criteria and taxonomy of viruses. The current classification scheme, also called “megataxonomy of viruses”, recognizes six different viral realms, defined based on the presence of viral hallmark genes (VHGs). Within the realms, viruses are [...] Read more.

Recent years have seen major changes in the classification criteria and taxonomy of viruses. The current classification scheme, also called “megataxonomy of viruses”, recognizes six different viral realms, defined based on the presence of viral hallmark genes (VHGs). Within the realms, viruses are classified into hierarchical taxons, ideally defined by the phylogeny of their shared genes. To enable the detection of shared genes, viruses have first to be clustered, and there is currently a need for tools to assist with virus clustering and classification. Here, VirClust is presented. It is a novel, reference-free tool capable of performing: (i) protein clustering, based on BLASTp and Hidden Markov Models (HMMs) similarities; (ii) hierarchical clustering of viruses based on intergenomic distances calculated from their shared protein content; (iii) identification of core proteins and (iv) annotation of viral proteins. VirClust has flexible parameters both for protein clustering and for splitting the viral genome tree into smaller genome clusters, corresponding to different taxonomic levels. Benchmarking on a phage dataset showed that the genome trees produced by VirClust match the current ICTV classification at family, sub-family and genus levels. VirClust is freely available, as a web-service and stand-alone tool. Full article

(This article belongs to the Section Bacterial Viruses)

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Figure 1
VirClust—branch and module organization. The three branches are marked in different colors. For each branch, the individual steps are labeld with a number followed by a letter (A, B or C, corresponding to each branch). Full article ">Figure 2
Example of a heatmap showing pairwise intergenomic similarities (%). Full article ">Figure 3
Integrated visualization of the viral clustering outputted by VirClust for the Crz_DB dataset. The genome clustering was performed based on PCs. The resulting tree was split into VGCs using a 0.9 intergenomic distance threshold. The visual components are described further. 1. Hierarchical tree calculated in step 3A, using PC-based intergenomic distances. 2. Silhouette width, color-coded in a range from −1 (red) to 1 (green). 3. VGC ID, as outputted in the genome statistic table from step 4A. 4. Heatmap representation of the PC distribution in the viral genomes. Rows are represented by individual viral genomes. Columns are represented by individual PCs. The ID of each PC can be read at the bottom of the heatmap at image magnification. Colors encode the number of each PC per genome, with white signifying the PC absence, and the other colors signifying various degrees of replication (from 1 to n, see legend). 5. Viral genome-specific statistics: genome length, the proportion of PC shared (dark grey) with any other genomes in the dataset, reported to the total PCs in the genome (light grey bar), the proportion of PC shared in its own VGC, the proportion of PCs shared only in its own VGC, the proportion of PCs shared also outside its own VGC, and the proportion of PC shared only outside own VGC. For more details about these stats, see materials and methods. 6. Virus name (here including the GenBank accession number as a suffix). Full article ">Figure 4
PSC-based genome tree for the Fam_DB dataset. The annotation circles encode the ICTV taxonomy for each phage genome, as follows, from inner to outer circles: family, order, class, phylum, kingdom, and realm. The colors in the circles encode the different taxons (see <a href="#app1-viruses-15-01007" class="html-app">SI File S8</a> for taxon names) The tree was split using a distance threshold of 0.9 and the resulting VGCs are encoded by different branch colors. The extended tree is available in <a href="#app1-viruses-15-01007" class="html-app">SI File S8</a>. Full article ">Figure 5
Relationship between the number of VGCs and taxons for Duplodnaviria, when the PC- and PSC-based genomic trees of the Fam_Db dataset were cut with different intergenomic distances. The red arrows indicate the recommended distance-hreshold to use for tree cutting, when creating VGCs. Extended figures, for all distances, are found in <a href="#app1-viruses-15-01007" class="html-app">SI File S9</a>. Full article ">Figure 6
Relationship between the number of VGCs and taxons for Varidnaviria, when the PC- and PSC-based genomic trees of the Fam-DB dataset were cut with different intergenomic distances. The red lines indicate the recommended distance-hreshold to use for tree cutting, when creating VGCs. Extended figures, for all distances, are found in <a href="#app1-viruses-15-01007" class="html-app">SI File S9</a>. Full article ">Figure 7
A road-map for using VirClust to enable virus taxonomy. Full article ">

18 pages, 1573 KiB

Open AccessArticle

RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples

by Rachel L. Tulloch, Karan Kim, Chisha Sikazwe, Alice Michie, Rebecca Burrell, Edward C. Holmes, Dominic E. Dwyer, Philip N. Britton, Jen Kok and John-Sebastian Eden

Viruses 2023, 15(4), 1006; https://doi.org/10.3390/v15041006 - 19 Apr 2023

Cited by 2 | Viewed by 2085

Abstract

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing [...] Read more.

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations. Full article

(This article belongs to the Section General Virology)

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Figure 1

Figure 1
RAPIDprep development experiments. All results here are derived from the same sample extracts (RESP01-RESP03) run in duplicate and presented as mean values and error as standard deviation (SD). (A) The shaded bars are representative of the percentage of residual rRNA reads in the library following rRNA depletion with either an in-reaction cDNA synthesis method (grey) or a pre-cDNA hybridization approach (orange). The bars are clustered with respect to the sample they are derived from, labeled on the X-axis. (B) A comparison in total library yield, in nanomolar generated using Tapestation values, following a parallel experiment with a one-step and two-step second strand synthesis step using the Sequenase enzyme. The grey and orange shaded bars are representative of the one-step and two-step protocols, respectively. (C) Grey-shaded bars represent the total library yield of each sample under different library amplification cycling conditions. The X-axis is marked with the number of amplification cycles and is sub-grouped by source sample. (D) The duplication rate of reads generated in the final libraries following cycle titration; the number of cycles for each sample is indicated on the X-axis, and is sub-grouped by source sample. Full article ">Figure 2
Filtered read distribution and classification across forty RAPIDprep libraries. The sequence reads were classified into five categories: low-quality reads (blue), human rRNA reads (red), human non-rRNA (pink), non-human rRNA reads (green), and non-human non-rRNA reads (light green). Low-quality, human rRNA, human non-rRNA, and non-human rRNA were excluded from downstream analysis, and the non-human non-rRNA reads were the sole target reads for pathogen detection. Relative distribution was calculated by dividing the number of reads mapping to the relative category by the total number of reads for the individual library, before conversion into a percentage by multiplying the value by 100. The results were ordered by library number and grouped by sample type with a further key in grey shaded indicating the sample extraction platform used. Full article ">Figure 3
Quantitative detection of SARS-COV-2 and RSV sequences. A simple linear-regression model was applied to both SARS-CoV-2 (A) and RSV (B) data sets with a line of best fit estimating the relationship between log-transformed reads per million (logRPM) and cycle threshold (CT) values. The linear-regression slope coefficient and the intercept parameter are printed on the top right of each plot, with R2 calculated to measure the goodness of fit. Full article ">Figure 4
Comparison of RAPIDprep to commercial RNA library preparation kit. Using previously generated data for the kids SARI cohort, we compared the 24 most abundant species identified across both protocols for the same set of samples. An unclustered heatmap of microbial abundance (Z-score) is shown, with differences between samples identified by a deeper blue shading, while organisms conserved across samples are lighter blue through to red. A frequency histogram is overlayed on the color key and signifies the count of each Z score at any given point. Tick labels on the X-axis in the ICUXX format represent deep-RNA sequencing generated previously, while tick labels in the RAPIDXX format represent sequencing data generated in this study using the RAPIDprep assay for the corresponding samples. Full article ">

15 pages, 300 KiB

Open AccessArticle

Prevalence of Human Papillomavirus in Different Mucous Membranes in HIV Concordant Couples in Rwanda

by Schifra Uwamungu, Bethelehem Nigussie, Claude Mambo Muvunyi, Bengt Hasséus, Maria Andersson and Daniel Giglio

Viruses 2023, 15(4), 1005; https://doi.org/10.3390/v15041005 - 19 Apr 2023

Viewed by 1912

Abstract

Background: The prevalence of human papillomavirus (HPV) infections in other anatomical sites besides the uterine cervix is unknown in East Africa. Here, we assessed the prevalence and concordance of HPVs in different anatomical sites in HIV concordant couples in Rwanda. Methods: Fifty HIV-positive [...] Read more.

Background: The prevalence of human papillomavirus (HPV) infections in other anatomical sites besides the uterine cervix is unknown in East Africa. Here, we assessed the prevalence and concordance of HPVs in different anatomical sites in HIV concordant couples in Rwanda. Methods: Fifty HIV-positive concordant male-female couples at the HIV clinic at the University Teaching Hospital of Kigali in Rwanda were interviewed, swabbed from the oral cavity (OC), oropharynx (OP), anal canal (AC), vagina (V), uterine cervix (UC) and penis. A pap smear test and a self-collected vaginal swab (Vself) were taken. Twelve high-risk (HR)-HPVs were analyzed. Results: HR-HPVs occurred in 10%/12% in OC, 10%/0% in OP and 2%/24% in AC (p = 0.002) in men and women, respectively. HR-HPVs occurred in 24% of UC, 32% of Vself, 30% of V and 24% of P samples. Only 22.2% of all HR-HPV infections were shared by both partners (κ −0.34 ± 0.11; p = 0.004). The type-specific HR-HPV concordance was significant between male to female OC-OC (κ 0.56 ± 0.17), V-VSelf (κ 0.70 ± 0.10), UC-V (κ 0.54 ± 0.13), UC-Vself (κ 0.51 ± 0.13) and UC-female AC (κ 0.42 ± 0.15). Conclusions: HPV infections are prevalent in HIV-positive couples in Rwanda but concordance within couples is low. Vaginal self-sampling for HPV is representative of cervical HPV status. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

13 pages, 1306 KiB

Open AccessArticle

Whole Genomic Sequence Analysis of Human Adenovirus Species C Shows Frequent Recombination in Tianjin, China

by Yue Lei, Zhichao Zhuang, Yang Liu, Zhaolin Tan, Xin Gao, Xiaoyan Li and Dongjing Yang

Viruses 2023, 15(4), 1004; https://doi.org/10.3390/v15041004 - 19 Apr 2023

Cited by 3 | Viewed by 1734

Abstract

Human adenovirus species C (HAdV-C) is frequently detected in China and worldwide. For the first time, 16 HAdV-C strains were isolated from sewage water (14 strains) and hospitalised children with diarrhoea (2 strains,) in Tianjin, China. Nearly complete genome data were successfully obtained [...] Read more.

Human adenovirus species C (HAdV-C) is frequently detected in China and worldwide. For the first time, 16 HAdV-C strains were isolated from sewage water (14 strains) and hospitalised children with diarrhoea (2 strains,) in Tianjin, China. Nearly complete genome data were successfully obtained for these viruses. Subsequently, genomic and bioinformatics analyses of the 16 HAdV-C strains were performed. A phylogenetic tree of the complete HAdV-C genome divided these strains into three types: HAdV-C1, HAdV-C2, HAdV-C5. Phylogenetic analysis based on the fiber gene showed similar outcomes to analyses of the hexon gene and complete HAdV-C genomes, whereas the penton gene sequences showed more variation than previously reported. Furthermore, analysis of the whole-genome sequencing revealed seven recombination patterns transmitted in Tianjin, of which at least four patterns have not been previously reported. However, the penton base gene sequences of the HAdV-C species had significantly lower heterogeneity than those of the hexon and fiber gene sequences of recombinant isolates; that is, many strains were distinct in origin, but shared hexon and fiber genes. These data illustrate the importance of frequent recombination in the complexity of the HAdV-C epidemic in Tianjin, thus emphasising the necessity for HAdV-C sewage and virological monitoring in China. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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Figure 1
Phylogenetic network built using the complete genomes of 16 Tianjin human adenovirus (HAdV) (represented by black dots) and 52 human adenovirus species C (HAdV-C) strains obtained from the GenBank database. Full article ">Figure 2
Phylogenetic trees based on the complete genome (A), fiber gene (B), hexon gene (C) and penton gene (D) of 68 HAdV-C sequences, including 16 Tianjin strains in this study and 52 reference strains from the GenBank. Tianjin sequences isolated from faeces are indicated with a black square, whereas sequences isolated from sewage are indicated with a black dot. Each prototype sequence is indicated with a black triangle. The trees were constructed using the Neighbour-joining method of MEGA 7.0 with 1000 bootstraps. HAdV-C, human adenovirus species C. Full article ">Figure 3
Schematic representation of recombination events in 13 genomes within 7 recombination patterns. A genetic map of human adenovirus species C (HAdV-C) is shown at the top. Analysed genomes are represented by a blue rectangle, the major parents are represented by a green rectangle, while the minor parents are represented by a purple rectangle. Breakpoints are identified based on the recombination detection program (RPD) version 4 output. Full article ">Figure 4
Bootscanning analysis of the 13 Tianjin HAdV-C genomes using a sliding window of 200 nt moving in 20-nt steps. For each bootscanning analysis, the names of viruses of the query sequence were indicated in the plot. Full article ">

22 pages, 2524 KiB

Open AccessArticle

Stabilization of the Quadruplex-Forming G-Rich Sequences in the Rhinovirus Genome Inhibits Uncoating—Role of Na⁺ and K⁺

by Antonio Real-Hohn, Martin Groznica, Georg Kontaxis, Rong Zhu, Otávio Augusto Chaves, Leonardo Vazquez, Peter Hinterdorfer, Heinrich Kowalski and Dieter Blaas

Viruses 2023, 15(4), 1003; https://doi.org/10.3390/v15041003 - 19 Apr 2023

Cited by 1 | Viewed by 2064

Abstract

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither [...] Read more.

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na⁺ but not in K⁺-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na⁺ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published. Full article

(This article belongs to the Special Issue Enteroviruses 2023)

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Figure 1

Figure 1
All RV genomes harbor conserved sequences with propensity to form GQs. Complete RNA sequences of 120 RVs available in the NCBI database were analyzed for putative GQ-forming sequences using the QGRS mapper software. G-scores ≥ 10 are shown as rectangles with their lengths corresponding to the number of bases making up the putative GQ. The color bar indicates the respective G-scores. Note that the sequences were used without alignment optimization, i.e., neither deletions nor insertions were taken into account. Also, note the four vertical lines indicating strong conservation. Species RV-A, RV-B, and RV-C are indicated. Full article ">Figure 2
Impact of Na+ and K+ on ThT binding and ThT displacement by PDS. Ribooligonucleotides dissolved at 5 µM in 100 mM Na+ (A) or K+ (B) phosphate buffer (both at pH 7.4) as indicated, were incubated with ThT, and the fluorescence was measured at 490 nm (n = 3; left panel). Right panels; as above, but fluorescence was measured in the presence of increasing concentrations of PDS added in steps. Values are normalized to the initial fluorescence signal (i.e., without PDS = 100%; n = 3). Full article ">Figure 3
GQ-binding compounds decrease the temperature of RV-A2 capsid permeability onset, impair uncoating, and decrease infectivity. (A) Scatter plot of the temperature of onset (Ton) of capsid permeability (i.e., RNA accessibility for SYTO 82) determined by PaSTRy with purified RV-A2 that had been pre-incubated +/−200 µM PDS at 34 °C for 4 h. ‘***’, note the strong reduction in the temperature of RNA accessibility-onset upon pretreatment with 200 µM PDS at 34 °C. (B,C) HeLa cells were infected with purified RV-A2 pre-incubated +/−PDS as in (A). Subviral A- and B-particles were immunoprecipitated with mAb-2G2. (B) Viral protein was quantified by using mAb 8F5 and a goat anti-mouse IgG HRP-conjugated secondary antibody, followed by measuring VP2 by densitometry. (C) RV-A2 RNA levels were quantified by qPCR and normalized relative to a known amount of AiV seed virus spiked into samples prior to RNA isolation. (D) Cleared supernatants prepared as above were separated by 10–40% (w/v) sucrose density gradient ultra-centrifugation. Dot blots were prepared from each fraction, and VP2-containing viral material was quantified with mAb 8F5 and IRDye 680RD goat anti-mouse IgG secondary antibodies (n = 3). The obtained signal intensity was plotted against fractions from top to bottom. Note that VP2 is present in all viral and subviral particles. Native virus (150S) and subviral B-particles (80S) were used as sedimentation controls and run on separate gradients. Their positions are indicated in the plot; the position of 14S pentamers was inferred from the literature. ‘#’ indicates the approximate position of 135S A particles (E) Purified RV-A2 was incubated with +/−PDS or PhenDC3 at the concentrations indicated for 4 h at 34 °C. Free compounds were removed by centrifugal ultrafiltration, and the retained material was used to infect HeLa cells. The percentage of infected cells was determined at 9 h pi by FACS analysis of the intracellularly produced VP2 with mAb 8F5 and an anti-mouse AlexaFluor 488 conjugated secondary antibody and is indicated as bars (n = 3; * p ≤ 0.05; ** p ≤ 0.01). Full article ">Figure 4
PDS treatment of protein-free RNA cores results in a drastic reorganization in the Na+ but not the K+ phosphate buffer. (A) The RV-A2 capsid proteins were digested via gentle proteolysis in 100 mM Na+ phosphate buffer or 100 mM K+ phosphate buffer, as indicated. The protein-free RNA cores were then mixed with ThT (final concentration of 5 µM), and the ThT fluorescence signal was acquired at 30 °C. (B) RV-A2 RNA (prepared as in (A)) was incubated with 20 µM PDS for 10 min at room temperature and subjected to rotary shadowing followed by TEM. Insets depicting representative images of non-PDS-treated ex-virion RNA in Na+ or K+ phosphate buffers are displayed. (C) Samples similarly treated as in (B) were analyzed by AFM in the presence of 100 mM Na+ or K+ phosphate buffer. (D) Purified RV-A2 was diluted in the above buffers and incubated overnight with or without 20 µM PDS at room temperature. After ultrafiltration, the infectivity of the respective retentates was determined by end-point titration. Viral titer is expressed as TCID50 in the bar graph (n = 3). The significance of the differences was evaluated by the two-way ANOVA; NS—statistically not significant (p ≥ 0.05). (E) HeLa cells were challenged with RV-A2 (MOI = 10) for 30 min at 4 °C, allowing virus attachment. A synchronized virus entry was triggered by a transfer to 34 °C. Immediately before (=0 min pi), 180 min, and 300 min pi after the temperature shift, the medium in the respective well was adjusted to 20 µM PDS, and incubation of cells continued for 9 h to allow for one cycle of infection. Non-infected cells without PDS treatment were used as controls. Cells were immunostained with mAb 8F5 and analyzed by flow cytometry. Non-infected and infected populations are displayed at the left and right, respectively, in the fluorescence intensity histogram, and the corresponding percentage is provided on top. MFI is the mean value of fluorescence intensity calculated for each sample. ‘*’ p ≤ 0.05. Full article ">

17 pages, 1893 KiB

Open AccessArticle

The Role of Airborne Particles in the Epidemiology of Clade 2.3.4.4b H5N1 High Pathogenicity Avian Influenza Virus in Commercial Poultry Production Units

by Joe James, Caroline J. Warren, Dilhani De Silva, Thomas Lewis, Katherine Grace, Scott M. Reid, Marco Falchieri, Ian H. Brown and Ashley C. Banyard

Viruses 2023, 15(4), 1002; https://doi.org/10.3390/v15041002 - 19 Apr 2023

Cited by 8 | Viewed by 3920

Abstract

Since October 2021, Europe has experienced the largest avian influenza virus (AIV) epizootic, caused by clade 2.3.4.4b H5N1 high pathogenicity AIV (HPAIV), with over 284 poultry infected premises (IPs) and 2480 dead H5N1-positive wild birds detected in Great Britain alone. Many IPs have [...] Read more.

Since October 2021, Europe has experienced the largest avian influenza virus (AIV) epizootic, caused by clade 2.3.4.4b H5N1 high pathogenicity AIV (HPAIV), with over 284 poultry infected premises (IPs) and 2480 dead H5N1-positive wild birds detected in Great Britain alone. Many IPs have presented as geographical clusters, raising questions about the lateral spread between premises by airborne particles. Airborne transmission over short distances has been observed for some AIV strains. However, the risk of airborne spread of this strain remains to be elucidated. We conducted extensive sampling from IPs where clade 2.3.4.4b H5N1 HPAIVs were confirmed during the 2022/23 epizootic, each representing a major poultry species (ducks, turkeys, and chickens). A range of environmental samples were collected inside and outside houses, including deposited dust, feathers, and other potential fomites. Viral RNA (vRNA) and infectious viruses were detected in air samples collected from inside and outside but in close proximity to infected houses, with vRNA alone being detected at greater distances (≤10 m) outside. Some dust samples collected outside of the affected houses contained infectious viruses, while feathers from the affected houses, located up to 80 m away, only contained vRNA. Together, these data suggest that airborne particles harboring infectious HPAIV can be translocated short distances (<10 m) through the air, while macroscopic particles containing vRNA might travel further (≤80 m). Therefore, the potential for airborne transmission of clade 2.3.4.4b H5N1 HPAIV between premises is considered low. Other factors, including indirect contact with wild birds and the efficiency of biosecurity, represent greater importance in disease incursion. Full article

(This article belongs to the Special Issue Aerosol Transmission of Viral Disease)

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Figure 1
Infected premises 1 (IP1): Environmental sampling and presence or absence of H5 HPAIV RNA at a duck IP. (A,B). Map showing the layout of the IP with the two affected houses indicated, wind direction at time of sampling, the site where carcasses were loaded onto trucks (red star), and mortality and culling indicated. (C–F). The location of the environmental sampling and H5 RRT-PCR results showing the presence (orange) or absence (blue) of H5 HPAIV RNA in the sample and the positive isolation of infectious virus (red outline) or negative isolation of infectious virus (black outline). Samples were collected from air filters (C), feathers (D), dust (E), and water (F). Locations of wet straw samples (n = 2) and an Op swab from a dead duck (n = 1) are not shown but are indicated in <a href="#app1-viruses-15-01002" class="html-app">Table S1</a>. Full article ">Figure 2
Infected premises 2 (IP2): Environmental sampling and presence or absence of H5 HPAIV RNA at a turkey IP. (A,B). Map showing the layout of the IP with the three affected houses indicated, wind direction at time of sampling, the site where carcasses were loaded onto trucks (red star), and mortality and culling indicated. (C–F). The location of the environmental sampling and H5 RRT-PCR results showing the presence (orange) or absence (blue) of H5 HPAIV RNA in the sample and the positive isolation of infectious virus (red outline) or negative isolation of infectious virus (black outline). Samples were collected from air filters (C), feathers (D), dust (E), and water (F). Op swabs from dead turkeys are not shown but indicated in <a href="#app1-viruses-15-01002" class="html-app">Table S2</a>. Full article ">Figure 3
Infected premises 3 (IP3): Environmental sampling and presence or absence of H5 HPAIV RNA at a turkey IP. (A,B). Map showing the layout of the IP with the three affected houses indicated, wind direction at time of sampling, and mortality indicated. (C–F). The location of the environmental sampling and H5 RRT-PCR results showing the presence (orange) or absence (blue) of H5 HPAIV RNA in the sample and the positive isolation of infectious virus (red outline) or negative isolation of infectious virus (black outline). Samples were collected from air filters (C), feathers (D), external dust (E), and internal dust (F). Water and Op and Cl swabs from dead chickens are not shown but indicated in <a href="#app1-viruses-15-01002" class="html-app">Table S3</a>. Full article ">

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