Viruses

23 pages, 7013 KiB

Open AccessArticle

Characterization of Human Norovirus Nonstructural Protein NS1.2 Involved in the Induction of the Filamentous Endoplasmic Reticulum, Enlarged Lipid Droplets, LC3 Recruitment, and Interaction with NTPase and NS4

by Chien-Hui Hung, Ju-Bei Yen, Pey-Jium Chang, Lee-Wen Chen, Tsung-Yu Huang, Wan-Ju Tsai and Yu-Chin Tsai

Viruses 2023, 15(3), 812; https://doi.org/10.3390/v15030812 - 22 Mar 2023

Cited by 2 | Viewed by 2446

Abstract

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is [...] Read more.

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is accompanied by a distorted-filamentous ER morphology and aggregated-enlarged LDs. LC3 was recruited to the NS1.2-localized membrane through an autophagy-independent pathway. NS1.2, expressed from a cDNA clone of GII.4 norovirus, formed complexes with NTPase and NS4, which exhibited aggregated vesicle-like structures that were also colocalized with LC3 and LDs. NS1.2 is structurally divided into three domains from the N terminus: an inherently disordered region (IDR), a region that contains a putative hydrolase with the H-box/NC catalytic center (H-box/NC), and a C-terminal 251–330 a.a. region containing membrane-targeting domain. All three functional domains of NS1.2 were required for the induction of the filamentous ER. The IDR was essential for LC3 recruitment by NS1.2. Both the H-Box/NC and membrane-targeting domains are required for the induction of aggregated-enlarged LDs, NS1.2 self-assembly, and interaction with NTPase. The membrane-targeting domain was sufficient to interact with NS4. The study characterized the NS1.2 domain required for membrane targeting and protein–protein interactions, which are crucial for forming a viral replication complex. Full article

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

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Figure 1
The domain of NS1.2 required for ER targeting and reorganization. (A) The schematic diagram shows the name and structure of Flag-tagged NS1.2 and its deletion mutants. (B) A7 cells were transfected with plasmids expressing Flag-tagged NS1.2, which is denoted as F-NS1.2, or its derivative mutants. Cells were fixed at 24h after transfection. Indirect immunofluorescence staining was performed using anti-Flag and anti-PDI antibodies and shown as green and red color, respectively. (C) The amino acid sequences between 261 and 278 of NS1.2 (1–280) and substituted mutant NS1.2 (1–280LF/ED) are shown on the top of the panel. The hydrophobic and acidic amino acids were highlighted in yellow and red, respectively. The helical wheel plot between 261 and 278 of NS1.2 predicted from computational analysis (accessed on 13 July 2021, <a href="https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py" target="_blank">https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py</a>) was shown. The hydrophobic and basic residues were shown in yellow and blue, respectively. The acidic residues are shown in red. The serine and threonine residues are shown in purple. The small amino acids are shown in grey. Proline is shown in green. The arrow represents the direction and magnitude of the hydrophobic moment. A7 cell were transfected with F-tagged NS1.2 (1–280), denoted as F-1–280, and substituted mutant, F-1–280(LF/ED). Cells were fixed at 24 h after transfection. Indirect immunofluorescence staining was performed using anti-Flag and anti-PDI antibodies. (D) A7 cells were transfected with plasmids expressing GFP-tagged NS1.2, (251–330), (251–290), or (280–330), which are denoted as GFP-NS1.2, GFP-251–330, GFP-250–290 and GFP-280–330, respectively. Cells were fixed at 24h after transfection, and indirect immunofluorescence staining was performed using anti-PDI or anti-GM130 antibodies, as indicated and shown in red color. LD was stained with LipidTox red neutral lipid dye. (E) The A7, HeLa, and HEK293T cells expressed Flag-tagged NS1.2 (117–330) were examined by immunofluorescent analysis using anti-Flag and anti-GM130 antibodies. 4′, 6-diamidino-2-pheylindole (DAPI) staining revealed the nucleus. Scale bar: 10 μm. Full article ">Figure 2
The domain of NS1.2 is required for LDs targeting and induced enlarged LDs. (A) The schematic diagram shows the name and structure of Flag-tagged NS1.2 and its deletion mutants. (B) A7 cells were transfected with plasmids expressing Flag-tagged NS1.2 and its deletion mutants. Cells were fixed at 24h after transfection. Indirect immunofluorescence staining was performed using anti-Flag and counterstain LD with LipidTox red neutral lipid dye. The transfected cells were marked with an asterisk; and the control cells were marked with a white arrowhead. The dashed boxes in the merged images were enlarged and shown on the right. 4′, 6-diamidino-2-pheylindole (DAPI) staining revealed the nucleus. Scale bar: 10 μm. (C) Quantification of the number and average size of LDs in panel B. The diameter of LD is represented as the size of LD. Each dot represents the quantitated data from a single cell (n > 20). The results were analyzed statistically through a t-test. ** p < 0.01; NS, not statistically significant. C: control cells that did not express F-tagged NS1.2 and its deletion mutants. Full article ">Figure 3
LC3 is recruited to NS1.2 localized ER filamentous membrane. (A) A7 cells, (B) MEF Atg5+/+ and MEF Atg5−/−, (C) AD293(GFP-LC3) were transfected with control plasmid ((C), 1st row) or plasmid expressing Flag-tagged NS1.2 or NS1.2 (117–330), denoted as F-NS1.2 and F-117–330, as indicated. Cells were fixed at 24 h after transfection. Indirect immunofluorescence staining was performed using anti-Flag and anti-PDI ((A), 1st row) or anti-Flag and anti-LC3 ((A), 2nd and 3rd row and (B)) or anti-Flag (C) antibodies. *: cell without expressing F-NS1.2. 4′, 6-diamidino-2-pheylindole (DAPI) staining revealed the nucleus. Scale bar: 10 μm. Full article ">Figure 4
The domain involved in the dimerization/oligomerization of NS.1.2. (A) The schematic diagram shows the structure of NS1.2 and deletion mutants of NS1.2 and the summary of coimmunoprecipitation results. The “+”, “++”, and “+++” represent the relative level of Myc-tagged protein coimmunoprecipitated with Flag-tagged protein by anti-Flag antibody in each experiment. (B) HEK293T cells were cotransfected with plasmids expressing GFP or GFP-NS1.2 and Flag-NS1.2. Flagtagged NS1.2 is denoted as F-NS1.2. Proteins in the lysates were immunoprecipitated (IP) at 48 h after transfection using GFP-Trap, or anti-Flag conjugated magnetic beads and analyzed by immunoblotting using anti-GFP or anti-Flag antibodies as indicated. (C–E) HEK293T cells were cotransfected with plasmids expression of Myc-tagged NS1.2 (C), Myc-tagged H-box/NC domain (117–250) (D), or Myc-tagged membrane-targeting domain (233–330) (E) with a control plasmid, Flag-NS1.2 or NS1.2 deletion mutants as indicated. The control plasmid is denoted as C. Proteins in the lysates were immunoprecipitated (IP) at 48 h after transfection using anti-Myc or anti-Flag conjugated magnetic beads and analyzed by immunoblotting using anti-Myc or anti-Flag antibodies as indicated. “*”: suggested dimer form of F-117–250. Full article ">Figure 5
NS1.2 interacts and colocalizes with NS4 and NTPase. (A) 293T cell were co-transfected with plasmids expressing Flag-NS1.2 and Myc-tagged NS1.2, or NTPase, or NS4, or VPg, or Protease, or RdRP. Proteins in the lysates were immunoprecipitated (IP) 48 h after transfection using anti-Flag or anti-Myc antibodies and analyzed by immunoblotting using anti-Flag or anti-Myc antibodies as indicated. (B) Upper panel: The schematic figure of the Flag-NV plasmid: a cDNA expression plasmid of GII.4 HuNV. Lower panel: 293T cells were transfected with a control plasmid (C) or plasmid Flag-NV. The cell lysate was harvested at 48 h after transfection and examined by immunoblotting using antibodies as indicated. The arrows indicate the mature NS1.2, NTPase and NS4. *: nonspecific bands; arrowhead: truncated form of NS1.2. (C) 293T cells were transfected with plasmids p-F-NV for 48 h. Proteins in the lysates were immunoprecipitated (IP) using anti-Flag antibodies or control IgG. Proteins that bound on beads were analyzed by immunoblotting using antibodies as indicated. (D,E) A7 cells were transfected with plasmid Flag-NV. Cells were fixed at 48 h after transfection. Indirect immunofluorescence staining was performed using anti-Flag (NS1.2), anti-NTPase (a1,a2) or anti-NS4 (b1,b2), anti-LC3 (c1,c2) antibodies or counterstained LD with LipidTox red neutral lipid dye (d1,d2). (F) AD293(GFP-LC3) cells were transfected with control plasmids (1st row) or plasmids expressing Flag-tagged NS1.2 (2nd row) and were fixed at 24 h after transfection. Indirect immunofluorescence staining was performed using anti-Flag antibodies. *: cells expressing polyprotein of GII.4 HuNoV. DAPI staining revealed the nucleus. Full article ">Figure 6
The domain of NS.1.2 interacts with NS4. (A) The schematic diagram shows the deletion mutants of NS1.2 and the summary of coimmunoprecipitation results. (B,C) 293T cells were cotransfected with plasmids expression Myc-tagged NS4 with control plasmid (C), Flag-tagged NS1.2, or NS1.2 deletion mutants as indicated. Proteins in the lysates were immunoprecipitated (IP) at 48 h after transfection using anti-Flag or anti-Myc conjugated magnetic beads and analyzed by immunoblotting using anti-Flag or anti-Myc antibodies. (D) 293T cells were cotransfected with plasmids expressing Myc-tagged NS4 and Flag-tagged NS1.2 or its deletion mutants as described. Indirect immunofluorescence staining was performed at 48 h after transfection using anti-Flag and anti-Myc antibodies. Cells coexpressing NS1.2 and NTPase were marked with white arrow; cells expressing NS4 were marked with an asterisk. 4′, 6-diamidino-2-pheylindole (DAPI) staining revealed the nucleus. Full article ">Figure 7
The domain of NS1.2 interacts with NTPase. (A) The schematic diagram shows the deletion mutants of NS1.2 and the summary of coimmunoprecipitation results. The “+”, “++”, and “+++” represent the relative level of protein coimmunoprecipitated with Flag-NS1.2 or NTPase-Myc by anti-Flag or anti-Myc antibody, respectively. (B) 293T cells were cotransfected with plasmids expression Myc-tagged NTPase with control plasmid (C), Flag-tagged NS1.2 or NS1.2 deletion mutants as indicated. Proteins in the lysates were immunoprecipitated (IP) at 48 h after transfection by anti-Flag (middle panel) or anti-Myc (right panel) conjugated magnetic beads and analyzed by immunoblotting with anti-Flag or anti-Myc antibodies. The input lanes were loaded with 4% of lysate. White arrow: suggested dimer form of Flag-tagged protein. Arrow: NTPase. *: nonspecific band. (C) 293T cells were cotransfected with plasmids expressing Myc-tagged NS4 and Flag-tagged NS1.2 or its deletion mutants as indicated. Indirect immunofluorescence staining was performed 48 h after transfection using anti-Flag and anti-Myc antibodies. Cells coexpressing NS1.2 and NTPase were marked with white arrow; cells expressing NTPase alone were marked with an asterisk. 4′, 6-diamidino-2-pheylindole (DAPI) staining revealed the nucleus. Full article ">Figure 8
Summary of the structural domains of HuNoV NS1.2 involved in specific functions. The schematic diagram on top of the figure shows the structural domain of NS1.2. The domain required for various functions of NS1.2 determined in this study was shown in the button of the schematic diagram. Full article ">

10 pages, 855 KiB

Open AccessArticle

Real-World Experience of the Comparative Effectiveness and Safety of Molnupiravir and Nirmatrelvir/Ritonavir in High-Risk Patients with COVID-19 in a Community Setting

by Yoshikazu Mutoh, Takumi Umemura, Takeshi Nishikawa, Kaho Kondo, Yuta Nishina, Kazuaki Soejima, Yoichiro Noguchi, Tomohiro Bando, Sho Ota, Tatsuki Shimahara, Shuko Hirota, Satoshi Hagimoto, Reoto Takei, Jun Fukihara, Hajime Sasano, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Toshiaki Matsuda, Tomoki Kimura, Toshihiko Ichihara and Yasuhiro Kondoh Show full author list Hide full author list

Viruses 2023, 15(3), 811; https://doi.org/10.3390/v15030811 - 22 Mar 2023

Cited by 10 | Viewed by 3608

Abstract

Molnupiravir (MOV) and nirmatrelvir/ritonavir (NMV/r) are efficacious oral antiviral agents for patients with the 2019 coronavirus (COVID-19). However, little is known about their effectiveness in older adults and those at high risk of disease progression. This retrospective single-center observational study assessed and compared [...] Read more.

Molnupiravir (MOV) and nirmatrelvir/ritonavir (NMV/r) are efficacious oral antiviral agents for patients with the 2019 coronavirus (COVID-19). However, little is known about their effectiveness in older adults and those at high risk of disease progression. This retrospective single-center observational study assessed and compared the outcomes of COVID-19 treated with MOV and NMV/r in a real-world community setting. We included patients with confirmed COVID-19 combined with one or more risk factors for disease progression from June to October 2022. Of 283 patients, 79.9% received MOV and 20.1% NMV/r. The mean patient age was 71.7 years, 56.5% were men, and 71.7% had received ≥3 doses of vaccine. COVID-19-related hospitalization (2.8% and 3.5%, respectively; p = 0.978) or death (0.4% and 3.5%, respectively; p = 0.104) did not differ significantly between the MOV and NMV/r groups. The incidence of adverse events was 2.7% and 5.3%, and the incidence of treatment discontinuation was 2.7% and 5.3% in the MOV and NMV/r groups, respectively. The real-world effectiveness of MOV and NMV/r was similar among older adults and those at high risk of disease progression. The incidence of hospitalization or death was low. Full article

(This article belongs to the Collection Coronaviruses)

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10 pages, 386 KiB

Open AccessArticle

Acute Bronchitis and Bronchiolitis Infection in Children with Asthma and Allergic Rhinitis: A Retrospective Cohort Study Based on 5,027,486 Children in Taiwan

by Fung-Chang Sung, Chang-Ching Wei, Chih-Hsin Muo, Shan P. Tsai, Chao W. Chen, Dennis P. H. Hsieh, Pei-Chun Chen and Chung-Yen Lu

Viruses 2023, 15(3), 810; https://doi.org/10.3390/v15030810 - 22 Mar 2023

Cited by 1 | Viewed by 2729

Abstract

This study evaluated the risks of childhood acute bronchitis and bronchiolitis (CABs) for children with asthma or allergic rhinitis (AR). Using insurance claims data of Taiwan, we identified, from children of ≤12 years old in 2000–2016, cohorts with and without asthma (N = [...] Read more.

This study evaluated the risks of childhood acute bronchitis and bronchiolitis (CABs) for children with asthma or allergic rhinitis (AR). Using insurance claims data of Taiwan, we identified, from children of ≤12 years old in 2000–2016, cohorts with and without asthma (N = 192,126, each) and cohorts with and without AR (N = 1,062,903, each) matched by sex and age. By the end of 2016, the asthma cohort had the highest bronchitis incidence, AR and non-asthma cohorts followed, and the lowest in the non-AR cohort (525.1, 322.4, 236.0 and 169.9 per 1000 person-years, respectively). The Cox method estimated adjusted hazard ratios (aHRs) of bronchitis were 1.82 (95% confidence interval (CI), 1.80–1.83) for the asthma cohort and 1.68 (95% CI, 1.68–1.69) for the AR cohort, relative to the respective comparisons. The bronchiolitis incidence rates for these cohorts were 42.7, 29.5, 28.5 and 20.1 per 1000 person-years, respectively. The aHRs of bronchiolitis were 1.50 (95% CI, 1.48–1.52) for the asthma cohort and 1.46 (95% CI, 1.45–1.47) for the AR cohort relative to their comparisons. The CABs incidence rates decreased substantially with increasing age, but were relatively similar for boys and girls. In conclusion, children with asthma are more likely to develop CABs than are children with AR. Full article

(This article belongs to the Special Issue Pediatric Respiratory Viral Infection)

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10 pages, 1183 KiB

Open AccessCommunication

Molecular Screening for High-Risk Human Papillomaviruses in Patients with Periodontitis

by Kalina Shishkova, Raina Gergova, Elena Tasheva, Stoyan Shishkov and Ivo Sirakov

Viruses 2023, 15(3), 809; https://doi.org/10.3390/v15030809 - 22 Mar 2023

Cited by 2 | Viewed by 1687

Abstract

Members of the Papillomaviridae family account for 27.9–30% of all infectious agents associated with human cancer. The aim of our study was to investigate the presence of high-risk HPV (human papilloma virus) genotypes in patients with periodontitis and a pronounced clinical picture. To [...] Read more.

Members of the Papillomaviridae family account for 27.9–30% of all infectious agents associated with human cancer. The aim of our study was to investigate the presence of high-risk HPV (human papilloma virus) genotypes in patients with periodontitis and a pronounced clinical picture. To achieve this goal, after proving the bacterial etiology of periodontitis, the samples positive for bacteria were examined for the presence of HPV. The genotype of HPV is also determined in samples with the presence of the virus proven by PCR (polymerase chain reaction). All positive tests for bacteria associated with the development of periodontitis indicated the presence of HPV. There was a statistically significant difference in HPV positive results between the periodontitis positive target group and the control group. The higher presence of high-risk HPV genotypes in the target group, which was also positive for the presence of periodontitis-causing bacteria, has been proven. A statistically significant relationship was established between the presence of periodontitis-causing bacteria and high-risk strains of HPV. The most common HPV genotype that tests positive for bacteria associated with the development of periodontitis is HPV58. Full article

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

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13 pages, 3870 KiB

Open AccessArticle

Establishment of an In Vitro Model of Pseudorabies Virus Latency and Reactivation and Identification of Key Viral Latency-Associated Genes

by Li Pan, Mingzhi Li, Xinyu Zhang, Yu Xia, Assad Moon Mian, Hongxia Wu, Yuan Sun and Hua-Ji Qiu

Viruses 2023, 15(3), 808; https://doi.org/10.3390/v15030808 - 22 Mar 2023

Cited by 2 | Viewed by 2117

Abstract

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the [...] Read more.

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the latent viruses to reactivate and cause recurrent diseases. The current strategies of antiviral drug therapy and vaccine immunization are ineffective in eliminating these viruses from the infected host. Moreover, overspecialized and complex models are also a major obstacle to the elucidation of the mechanisms involved in the latency and reactivation of the PRV. Here, we present a streamlined model of the latent infection and reactivation of the PRV. A latent infection established in N2a cells infected with the PRV at a low multiplicity of infection (MOI) and maintained at 42 °C. The latent PRV was reactivated when the infected cells were transferred to 37 °C for 12 to 72 h. When the above process was repeated with a UL54-deleted PRV mutant, it was observed that the UL54 deletion did not affect viral latency. However, viral reactivation was limited and delayed. This study establishes a powerful and streamlined model to simulate PRV latency and reveals the potential role of temperature in PRV reactivation and disease. Meanwhile, the key role of the early gene UL54 in the latency and reactivation of PRV was initially elucidated. Full article

(This article belongs to the Special Issue Pseudorabies Virus, Volume II)

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Figure 1
Generation and characterization of rPRV-EGFP. (A) Schematic diagram of the construction of the recombinant virus. Using the PRV genomic fosmid library, the EGFP sequence was inserted nondestructively downstream of the US9 gene in the PRV genome. The relative position of the EGFP sequence in the WT-PRV genome is shown on top of the green box. (B) PCR amplification of the EGFP and gB genes from the genomes of rPRV-EGFP and WT-PRV. (C) The green fluorescence and cytopathic effects (CPEs) in the PK-15 cells infected with rPRV-EGFP and WT-PRV at 48 h postinfection (hpi). (D) Plaques of rPRV-EGFP and WT-PRV in the PK-15 cells. ns: not significant (p ≥ 0.05). The diameters of plaques were averaged for three independent experiments. ns: not significant. (E) Transmission electron microscopy photographs of rPRV-EGFP and WT-PRV. Scale bar = 200 nm. (F) One-step growth curves of rPRV-EGFP and WT-PRV in PK-15 cells. Full article ">Figure 2
Screening for appropriate cell types and optimal infectious dose for in vitro PRV latent infection models. N2a, BHK-21, and PK-15 cells were seeded into 35 mm dishes at 5 × 105 per dish and were cultured with complete medium. rPRV-EGFP was added to the three cell types at an MOI of 1, 0.1, 0.01, or 0.001. The infected cells were incubated continuously at 37 or 42 °C for 72 h. Fluorescence was monitored at 72 h postinfection. Full article ">Figure 3
Optimal incubation time for screening and detection of latency indicators. (A) Optimal incubation time screening. N2a cells were infected with rPRV-EGFP (MOI = 0.01) and were incubated at 37 °C for 2, 2.5, 3, 3.5, 4, 4.5, 5, and 5.5 h. The cells were then transferred to 42 °C for 120 h, and EGFP fluorescence was monitored. (B) Identification of the optimal conditions for latent PRV infection. N2a cells were infected at an MOI of 0.01, were incubated at 37 °C for 2 h, then transferred to 42 °C for 120 h. (C) Latent and lytic viral DNA detection. N2a cells were infected with rPRV-EGFP at an MOI of 0.01 and were incubated at 37 °C for 2 h. After rPRV-EGFP was cultured at 42 °C for 120 h, the samples were then collected, and viral DNA was extracted; additionally, the gB gene was detected through real-time PCR to quantify genomic DNA. (D) Quantification of transcripts of latent viral genes. After rPRV-EGFP was cultured at 42 °C for 120 h, the samples were then collected, and the mRNAs of IE180, EP0, LAT, and gB were detected through RT-qPCR. (E) Detection of late viral protein expression levels at two culture temperatures. N2a cells were infected with different doses of rPRV-EGFP (MOI = 1, 0.1, or 0.01) and were incubated at 37 or 42 °C for 120 h, respectively. At 120 h postinfection (hpi), the cells were harvested, and samples were prepared for Western blotting. The homemade mouse anti-gB and -gD monoclonal antibodies were used as primary antibodies, and goat antimouse FITC antibodies were used as secondary antibodies. Bars represent the means ± SDs of three independent experiments. ns: not significant (p ≥ 0.05). * p < 0.05, *** p < 0.001, and **** p < 0.0001. Full article ">Figure 4
Reactivation of latent rPRV-EGFP. (A) Fluorescence spots of rPRV-EGFP during reactivation. N2a cells were infected with rPRV-EGFP (MOI = 0.01), were incubated at 42 °C for 120 h, and were then transferred to 37 °C for additional incubation. EGFP fluorescence was monitored at 12, 24, 48, and 72 h postreactivation (hpr). (B) Detection of viral genomic DNA during reactivation. The viral DNA was extracted, and the gB gene was detected at 0, 12, 24, 48, and 72 hpr. (C) Identification of expression levels of viral late proteins following rPRV-EGFP reactivation. N2a cells were infected with rPRV-EGFP (MOI = 0.01), were incubated at 42 °C for 120 h, and were then transferred to 37 °C. At 0, 12, 24, 48, and 72 hpr, the cells were harvested, and samples were prepared for Western blotting. The homemade mouse anti-gB and -gD monoclonal antibodies were used as primary antibodies, and goat antimouse FITC antibodies were used as secondary antibodies. Bars represent the means ± SDs of three independent experiments. ns: not significant (p ≥ 0.05). * p < 0.05, *** p < 0.001, and **** p < 0.0001. Full article ">Figure 5
Generation and characterization of rPRV-ΔUL54-EGFP. (A) Schematic diagram of the construction of rPRV-ΔUL54-EGFP. Using the PRV genomic fosmid library, the UL54 deletion mutant was constructed using rPRV-EGFP as the parental strain. The relative position of the UL54 gene in the WT-PRV genome is shown on top of the red box. (B) PCR amplification of the UL54, EGFP, and gB genes from the genomes of rPRV-ΔUL54-EGFP and WT-PRV. (C) The green fluorescence and cytopathic effects (CPEs) in the PK-15 cells infected with rPRV-ΔUL54-EGFP and WT-PRV at 60 h postinfection (hpi). (D) Plaques of rPRV-ΔUL54-EGFP and WT-PRV in the PK-15 cells. The diameters of plaques were averaged for three independent experiments. (E) Transmission electron microscopy imagingof rPRV-ΔUL54-EGFP and WT-PRV. Scale bar = 200 nm. (F) Multistep growth curves of rPRV-ΔUL54-EGFP and WT-PRV in PK-15 cells. PK-15 cells were infected with rPRV-ΔUL54-EGFP and WT-PRV (MOI = 0.01). Bars represent the means ± SDs of three independent experiments. ns: not significant (p ≥ 0.05). ** p < 0.01, and *** p < 0.001. Full article ">Figure 6
Latent infection and reactivation of rPRV-ΔUL54-EGFP. Based on the established model, rPRV-ΔUL54-EGFP was tested. (A) Identification of the effect of UL54 deletion on latent infection. (B) Latent and lytic viral DNA copy detection. N2a cells were infected with rPRV-ΔUL54-EGFP at an MOI of 0.01 and were incubated at 37 °C for 2 h. After rPRV-ΔUL54-EGFP was cultured at 42 °C for 120 h, the samples were then collected, and viral DNA was extracted; additionally, the gB gene was detected through real-time PCR to quantify genomic DNA. (C) Quantification of the transcripts of the latency-associated viral genes. After rPRV-ΔUL54-EGFP was cultured at 42 °C for 120 h, the samples were then collected, and total RNA was extracted; additionally, the mRNA of the IE180, EP0, LAT, and gB genes was detected through RT-qPCR. (D) Inability of latent rPRV-UL54-EGFP to express lytic viral proteins. (E) Fluorescence spots of rPRV-EGFP and rPRV-ΔUL54-EGFP during reactivation. N2a cells were infected with rPRV-EGFP and rPRV-ΔUL54-EGFP (MOI = 0.01), were incubated at 42 °C for 120 h, and were then transferred to 37 °C for culture. EGFP fluorescence was monitored at 12, 24, 48, and 72 h postreactivation (hpr). (F) Detection of viral genomic DNA during reactivation. N2a cells were infected with rPRV-ΔUL54-EGFP and rPRV-ΔUL54-EGFP (MOI = 0.01). The viral DNA was extracted, and the gB gene was quantified at 0, 12, 24, 48, and 72 hpr. (G) Identification of expression levels of viral envelope proteins following rPRV-ΔUL54-EGFP reactivation. Bars represent the means ± SDs of three independent experiments. ns: not significant (p ≥ 0.05). * p < 0.05, *** p < 0.001, and **** p < 0.0001. Full article ">

12 pages, 1904 KiB

Open AccessArticle

Phage vs. Phage: Direct Selections of Sandwich Binding Pairs

by Emily C. Sanders, Alicia M. Santos, Eugene K. Nguyen, Aidan A. Gelston, Sudipta Majumdar and Gregory A. Weiss

Viruses 2023, 15(3), 807; https://doi.org/10.3390/v15030807 - 22 Mar 2023

Cited by 2 | Viewed by 2926

Abstract

The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) [...] Read more.

The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers’ control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide–peptide and one Fab–peptide sandwich for the cancer and Parkinson’s disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays. Full article

(This article belongs to the Special Issue Phage Display in Cancer Research)

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Graphical abstract

Graphical abstract
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Phage vs. phage selection strategy. (1) Biotinylated phage displaying the known binding partner are adsorbed to a microtiter plate and exposed to the target. Next, the phage library is added and allowed to bind. (2) Nonbinding phages are removed by washing. (3) Sandwich assemblies are eluted from the microtiter plate by treatment with low pH solution and sonication. (4) In the eluate, biotinylated phage display with the known binding partner can removed from the solution through binding to magnetic streptavidin beads. (5) The sandwich forming pair of phages are used to infect E. coli and amplified. (6) The process is repeated with the amplified selectants as the phage library for the next round of selection. (7) After three to four rounds of selection, the selectants are identified by DNA sequencing. Full article ">Figure 2
Efficient phage biotinylation and its effects on target binding. (A) Biotinylation of phages (bDPep1Φ) was confirmed via direct detection ELISA with peroxidase-conjugated streptavidin (Strep-HRP) assay. (B) An indirect phage ELISA with DJ-1 verified that both biotinylated and non-biotinylated page (DPep1Φ) could bind the target antigen despite biotinylation. ELISA data were fit to a four-parameter logistic curve. Error bars indicate standard deviation (n = 3); each data point includes error bars, although most are quite small. HRP = horseradish peroxidase. Full article ">Figure 3
Sensitivity and specificity determination of sandwich peptide selectants. (A) A dose-dependent direct phage assay with sandwich peptide selectants binding to DJ-1. Sub-micromolar phage EC50 values were observed. NO = not observed. (B) An indirect phage assay with Hb, HEWL, HSA, E. coli lysate, and BSA determined the specificity of the sandwich selectants. Data are normalized to the DJ-1 signal. (C) Streptavidin-bound DPep1 peptide assay for sandwiching the target DJ-1 with DPep3Φ. The resultant dose-dependent response yielded an EC50 for binding to DJ-1 of 249 pM. (D) The DPep1-DPep3Φ sandwich interaction also demonstrated the interaction’s improved selectivity for DJ-1. ANOVA with Sidak’s multiple comparisons yielded p-values of <0.01 (**) and <0.001 (***). ELISA data were fit to a four-parameter logistic curve. Error bars, included for every data point, depict standard error of the mean (SEM) (n = 3). Full article ">Figure 4
Sensitivity and specificity determination of DFab1. (A) A dose-dependent direct phage assay with DFab1Φ and NegΦ. Data were fit to a four-parameter logistic curve and yielded picomolar EC50 values for the DFab1Φ interaction with DJ-1. Error bars represent SEM (n = 3). NO = not observed. (B) An indirect phage assay with DJ-1, Hb, HEWL, HSA, E. coli lysate, and BSA determined the specificity of the sandwich selectants. ANOVA with Tukey’s multiple comparisons yielded p-values of <0.0001 (****). (C) A similar dose-dependent response with an EC50 of 1.53 μM was observed in an identical sandwich ELISA with DFab1Φ. (D) The DPep1-DFab1Φ also demonstrated strong selectivity for DJ-1. ELISA data were fit to a four-parameter logistic curve. Data are normalized to DFab1Φ binding to DJ-1. Error is represented as SEM (n = 3). NO = not observed. ANOVA with Tukey’s multiple comparisons yielded p-values of <0.01 (**), <0.001 (***), <0.0001 (****). Full article ">

18 pages, 872 KiB

Open AccessReview

Interleukins, Chemokines, and Tumor Necrosis Factor Superfamily Ligands in the Pathogenesis of West Nile Virus Infection

by Emna Benzarti, Kristy O. Murray and Shannon E. Ronca

Viruses 2023, 15(3), 806; https://doi.org/10.3390/v15030806 - 22 Mar 2023

Cited by 6 | Viewed by 2667

Abstract

West Nile virus (WNV) is a mosquito-borne pathogen that can lead to encephalitis and death in susceptible hosts. Cytokines play a critical role in inflammation and immunity in response to WNV infection. Murine models provide evidence that some cytokines offer protection against acute [...] Read more.

West Nile virus (WNV) is a mosquito-borne pathogen that can lead to encephalitis and death in susceptible hosts. Cytokines play a critical role in inflammation and immunity in response to WNV infection. Murine models provide evidence that some cytokines offer protection against acute WNV infection and assist with viral clearance, while others play a multifaceted role WNV neuropathogenesis and immune-mediated tissue damage. This article aims to provide an up-to-date review of cytokine expression patterns in human and experimental animal models of WNV infections. Here, we outline the interleukins, chemokines, and tumor necrosis factor superfamily ligands associated with WNV infection and pathogenesis and describe the complex roles they play in mediating both protection and pathology of the central nervous system during or after virus clearance. By understanding of the role of these cytokines during WNV neuroinvasive infection, we can develop treatment options aimed at modulating these immune molecules in order to reduce neuroinflammation and improve patient outcomes. Full article

(This article belongs to the Special Issue Innate Immunity to Virus Infection 2023)

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9 pages, 271 KiB

Open AccessOpinion

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome

by Antti Vaheri, Teemu Smura, Hanna Vauhkonen, Jussi Hepojoki, Tarja Sironen, Tomas Strandin, Johanna Tietäväinen, Tuula Outinen, Satu Mäkelä, Ilkka Pörsti and Jukka Mustonen

Viruses 2023, 15(3), 805; https://doi.org/10.3390/v15030805 - 22 Mar 2023

Cited by 5 | Viewed by 1979

Abstract

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70–80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as [...] Read more.

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70–80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, “Why this variation?” remains largely unanswered. Full article

(This article belongs to the Special Issue Hantavirus Ecology, Epidemiology, and Disease, in Memory of Dr. Ho Wang Lee)

24 pages, 3647 KiB

Open AccessArticle

The Antiviral Compound PSP Inhibits HIV-1 Entry via PKR-Dependent Activation in Monocytic Cells

by Eduardo Alvarez-Rivera, Madeline Rodríguez-Valentín and Nawal M. Boukli

Viruses 2023, 15(3), 804; https://doi.org/10.3390/v15030804 - 22 Mar 2023

Cited by 3 | Viewed by 3476

Abstract

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring [...] Read more.

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring Enzyme-1α (IRE1α) and interferon-induced protein (IFN-IP) double-stranded RNA- activated protein kinase (PKR) are reported to overlap with actin components. In our published findings, Coriolus versicolor bioactive extract polysaccharide peptide (PSP) has demonstrated anti-HIV replicative properties in THP1 monocytic cells. However, its involvement towards viral infectivity has not been elucidated before. In the present study, we examined the roles of PKR and IRE1α in cofilin-1 phosphorylation and its HIV-1 restrictive roles in THP1. HIV-1 p24 antigen was measured through infected supernatant to determine PSP’s restrictive potential. Quantitative proteomics was performed to analyze cytoskeletal and UPR regulators. PKR, IRE1α, and cofilin-1 biomarkers were measured through immunoblots. Validation of key proteome markers was done through RT-qPCR. PKR/IRE1α inhibitors were used to validate viral entry and cofilin-1 phosphorylation through Western blots. Our findings show that PSP treatment before infection leads to an overall lower infectivity. Additionally, PKR and IRE1α show to be key regulators in cofilin-1 phosphorylation and viral restriction. Full article

(This article belongs to the Special Issue Novel Antiviral Agents: Synthesis, Molecular Modelling Studies and Biological Investigation)

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19 pages, 8910 KiB

Open AccessArticle

Design of Phage-Cocktail–Containing Hydrogel for the Treatment of Pseudomonas aeruginosa–Infected Wounds

by Fatemeh Shafigh Kheljan, Farzam Sheikhzadeh Hesari, Mohammad Sadegh Aminifazl, Mikael Skurnik, Sophia Goladze and Gholamreza Zarrini

Viruses 2023, 15(3), 803; https://doi.org/10.3390/v15030803 - 21 Mar 2023

Cited by 10 | Viewed by 3550

Abstract

Recently, the treatment of infected wounds has become a global problem due to increased antibiotic resistance in bacteria. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is often present in chronic skin infections, and it has become a threat to public health as it is [...] Read more.

Recently, the treatment of infected wounds has become a global problem due to increased antibiotic resistance in bacteria. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is often present in chronic skin infections, and it has become a threat to public health as it is increasingly multidrug resistant. Due to this, new measures to enable treatment of infections are necessary. Treatment of bacterial infections with bacteriophages, known as phage therapy, has been in use for a century, and has potential with its antimicrobial effect. The main purpose of this study was to create a phage-containing wound dressing with the ability to prevent bacterial infection and rapid wound healing without side effects. Several phages against P. aeruginosa were isolated from wastewater, and two polyvalent phages were used to prepare a phage cocktail. The phage cocktail was loaded in a hydrogel composed of polymers of sodium alginate (SA) and carboxymethyl cellulose (CMC). To compare the antimicrobial effects, hydrogels containing phages, ciprofloxacin, or phages plus ciprofloxacin were produced, and hydrogels without either. The antimicrobial effect of these hydrogels was investigated in vitro and in vivo using an experimental mouse wound infection model. The wound-healing process in different mouse groups showed that phage-containing hydrogels and antibiotic-containing hydrogels have almost the same antimicrobial effect. However, in terms of wound healing and pathological process, the phage-containing hydrogels performed better than the antibiotic alone. The best performance was achieved with the phage–antibiotic hydrogel, indicating a synergistic effect between the phage cocktail and the antibiotic. In conclusion, phage-containing hydrogels eliminate efficiently P. aeruginosa in wounds and may be a proper option for treating infectious wounds. Full article

(This article belongs to the Special Issue Phage and Antibiotic Combination Therapy against MDR Bacteria)

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12 pages, 1260 KiB

Open AccessArticle

Molecular Characterization and Cluster Analysis of SARS-CoV-2 Viral Isolates in Kahramanmaraş City, Turkey: The Delta VOC Wave within One Month

by Nadia Marascio, Merve Cilburunoglu, Elif Gulsum Torun, Federica Centofanti, Elida Mataj, Michele Equestre, Roberto Bruni, Angela Quirino, Giovanni Matera, Anna Rita Ciccaglione and Kezban Tulay Yalcinkaya

Viruses 2023, 15(3), 802; https://doi.org/10.3390/v15030802 - 21 Mar 2023

Cited by 2 | Viewed by 1992

Abstract

The SARS-CoV-2 pandemic has seriously affected the population in Turkey. Since the beginning, phylogenetic analysis has been necessary to monitor public health measures against COVID-19 disease. In any case, the analysis of spike (S) and nucleocapsid (N) gene mutations was crucial in determining [...] Read more.

The SARS-CoV-2 pandemic has seriously affected the population in Turkey. Since the beginning, phylogenetic analysis has been necessary to monitor public health measures against COVID-19 disease. In any case, the analysis of spike (S) and nucleocapsid (N) gene mutations was crucial in determining their potential impact on viral spread. We screened S and N regions to detect usual and unusual substitutions, whilst also investigating the clusters among a patient cohort resident in Kahramanmaraş city, in a restricted time span. Sequences were obtained by Sanger methods and genotyped by the PANGO Lineage tool. Amino acid substitutions were annotated comparing newly generated sequences to the NC_045512.2 reference sequence. Clusters were defined using phylogenetic analysis with a 70% cut-off. All sequences were classified as Delta. Eight isolates carried unusual mutations on the S protein, some of them located in the S2 key domain. One isolate displayed the unusual L139S on the N protein, while few isolates carried the T24I and A359S N substitutions able to destabilize the protein. Phylogeny identified nine monophyletic clusters. This study provided additional information about SARS-CoV-2 epidemiology in Turkey, suggesting local transmission of infection in the city by several transmission routes, and highlighting the necessity to improve the power of sequencing worldwide. Full article

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

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18 pages, 7178 KiB

Open AccessArticle

Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture

by Maria Clara da Costa Simas, Sara Mesquita Costa, Priscila da Silva Figueiredo Celestino Gomes, Nádia Vaez Gonçalves da Cruz, Isadora Alonso Corrêa, Marcos Romário Matos de Souza, Marcos Dornelas-Ribeiro, Tatiana Lucia Santos Nogueira, Caleb Guedes Miranda dos Santos, Luísa Hoffmann, Amilcar Tanuri, Rodrigo Soares de Moura-Neto, Clarissa R. Damaso, Luciana Jesus da Costa and Rosane Silva

Viruses 2023, 15(3), 801; https://doi.org/10.3390/v15030801 - 21 Mar 2023

Cited by 4 | Viewed by 2890

Abstract

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of [...] Read more.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions. Full article

(This article belongs to the Collection Coronaviruses)

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A 190-nucleotide deletion in the ORF7a gene (ORF7a∆190) and impact on the protein structure. (A) Top: Amplification and confirmation of 190-nt deletions amongst family members using primers forward (M_27145 F) and reverse (ORF8_28003R) (see scheme) indicating the positions on the SARS-CoV-2 genome. Bottom: ORF7a 190-nt deletions visualization in five family members on agarose gel (2%) stained with ethidium bromide revealed that the PCR products target the ORF7a gene. 100bp Molecular Weight Marker (lane M), 11785_ORF7a_wt, 58301_ORF7a_∆190, 59977_ORF7a_∆190, 59980_ORF7a_∆190, 58306_ORF7a∆190, 57817_ORF7a∆190. (B) Electropherograms from Sanger sequencing of the ORF7a PCR products from samples with 190-nt deletions: 59980, 59977, 58306, 58301 and 57817, the wt (without 190-nt deletion) 11785 aligned to the SARS-CoV-2 reference sequence (NCBI RefSeq SARS-CoV-2 genome sequence, NC_045512.2). (C) ORF7a deletions and impact on the protein structure. Left: Multiple sequence alignment of the reported ORF7a deletion (58301_ORF7a_∆190), and the corresponding sequences for SARS-CoV-2 and SARS-CoV ectodomain structures (PDB IDs: 7ci3 and 1xak, respectively) in respect of the reference sequence (NCBI RefSeq for SARS-CoV-2 ORF7a protein, YP_009724395.1). Identical residues are shaded in red; mutations from SARS-CoV to SARS-CoV-2 are colored in black and ORF7a altered residues are colored in grey. The labeled arrows indicate the secondary structure observed on the crystal structures. The different protein domains are indicated by the color bars below the alignment: N-terminal signaling region (residues 1–15, blue), Ig-like ectodomain (residues 16–96, yellow), hydrophobic transmembrane domain (residues 97–116, green) and ER retention motif (residues 117–121, brown). Right: Ribbon representation of the tridimensional SARS-CoV-2 ORF7a structure. The full-length structure of the ectodomain is colored in cyan. The β-strands are assigned numerically from the N-terminus to the C-terminus, corresponding to the alignment above. The deletion presented by the 58301_ORF7a_∆190 sample is colored in magenta, in both structures, covering a significant portion of the protein ectodomain, comprising the β sheets 3 to 7, the full transmembrane and ER retention motif. (D) Sars-CoV-2 whole genome sequencing of samples belonging to the five family members mapped to the reference sequence, aligned and visualized on IGV viewer. ORF7a region displays gaps evidencing the 190-nt deletion in all five samples. Full article ">Figure 1 Cont.
A 190-nucleotide deletion in the ORF7a gene (ORF7a∆190) and impact on the protein structure. (A) Top: Amplification and confirmation of 190-nt deletions amongst family members using primers forward (M_27145 F) and reverse (ORF8_28003R) (see scheme) indicating the positions on the SARS-CoV-2 genome. Bottom: ORF7a 190-nt deletions visualization in five family members on agarose gel (2%) stained with ethidium bromide revealed that the PCR products target the ORF7a gene. 100bp Molecular Weight Marker (lane M), 11785_ORF7a_wt, 58301_ORF7a_∆190, 59977_ORF7a_∆190, 59980_ORF7a_∆190, 58306_ORF7a∆190, 57817_ORF7a∆190. (B) Electropherograms from Sanger sequencing of the ORF7a PCR products from samples with 190-nt deletions: 59980, 59977, 58306, 58301 and 57817, the wt (without 190-nt deletion) 11785 aligned to the SARS-CoV-2 reference sequence (NCBI RefSeq SARS-CoV-2 genome sequence, NC_045512.2). (C) ORF7a deletions and impact on the protein structure. Left: Multiple sequence alignment of the reported ORF7a deletion (58301_ORF7a_∆190), and the corresponding sequences for SARS-CoV-2 and SARS-CoV ectodomain structures (PDB IDs: 7ci3 and 1xak, respectively) in respect of the reference sequence (NCBI RefSeq for SARS-CoV-2 ORF7a protein, YP_009724395.1). Identical residues are shaded in red; mutations from SARS-CoV to SARS-CoV-2 are colored in black and ORF7a altered residues are colored in grey. The labeled arrows indicate the secondary structure observed on the crystal structures. The different protein domains are indicated by the color bars below the alignment: N-terminal signaling region (residues 1–15, blue), Ig-like ectodomain (residues 16–96, yellow), hydrophobic transmembrane domain (residues 97–116, green) and ER retention motif (residues 117–121, brown). Right: Ribbon representation of the tridimensional SARS-CoV-2 ORF7a structure. The full-length structure of the ectodomain is colored in cyan. The β-strands are assigned numerically from the N-terminus to the C-terminus, corresponding to the alignment above. The deletion presented by the 58301_ORF7a_∆190 sample is colored in magenta, in both structures, covering a significant portion of the protein ectodomain, comprising the β sheets 3 to 7, the full transmembrane and ER retention motif. (D) Sars-CoV-2 whole genome sequencing of samples belonging to the five family members mapped to the reference sequence, aligned and visualized on IGV viewer. ORF7a region displays gaps evidencing the 190-nt deletion in all five samples. Full article ">Figure 2
Amplification and confirmation of 339 and 365-nt deletion amongst co-workers. (A) ORF7a 339-nt and 365-nt deletions visualization in a group of co-workers on agarose gel (2%) stained with ethidium bromide revealed the PCR products targeting ORF7a gene: 16305_ORF7a_wt, 16553_ORF7a_wt, 16538_ORF7a_∆365, 16991_ORF7a_∆339; 100bp Molecular Weight Marker (lane M). (B) Prevalence of viral genomic deletion analyzed through ORF7a PCR products from serial dilutions (10–1–10−4, lane 2–5, respectively) of sample 16538_ORF7a_∆365 viral stock and visualized on agarose gel (2%) stained with ethidium bromide. The two PCR fragments are present in 10−1 and 10−2, and only the non-deleted fragment is present in 10−3 and 10−4. (C) Eleven virus plaques were isolated from sample 16538. RNA extraction, reverse transcription, and PCR using primers that target the ORF7a region were performed. PCR products were visualized on agarose gel (2%) stained with ethidium bromide. Each lane corresponds to a virus plaque (P1–P11) derived from sample 16538. Virus plaques P2, P4 and P11 correspond to the virus without deletion in the ORF7a region, and virus plaques P5, P6 and P7 correspond to the virus with 365-nt deletions in the ORF7a region (ORF7a_∆365). M = 100 bp Molecular Weight Marker. (D) Electropherograms from Sanger sequencing of the ORF7a PCR products from sample 16991_ORF7a_∆339 aligned to SARS-CoV-2 reference and its deletion-resulting sequence (E) Electropherograms of both ORF7a PCR products isolated on a gel, from clinical samples and viruses isolated from cell culture (lanes P2 and P5, (B)) of 16538 ORF7a WT and the ORF7a_∆ 365 of 16538_ORF7a_∆365 and the non-deleted counterpart of 16538 * Samples submitted to NGS sequencing † Samples submitted to Sanger sequencing. Full article ">Figure 2 Cont.
Amplification and confirmation of 339 and 365-nt deletion amongst co-workers. (A) ORF7a 339-nt and 365-nt deletions visualization in a group of co-workers on agarose gel (2%) stained with ethidium bromide revealed the PCR products targeting ORF7a gene: 16305_ORF7a_wt, 16553_ORF7a_wt, 16538_ORF7a_∆365, 16991_ORF7a_∆339; 100bp Molecular Weight Marker (lane M). (B) Prevalence of viral genomic deletion analyzed through ORF7a PCR products from serial dilutions (10–1–10−4, lane 2–5, respectively) of sample 16538_ORF7a_∆365 viral stock and visualized on agarose gel (2%) stained with ethidium bromide. The two PCR fragments are present in 10−1 and 10−2, and only the non-deleted fragment is present in 10−3 and 10−4. (C) Eleven virus plaques were isolated from sample 16538. RNA extraction, reverse transcription, and PCR using primers that target the ORF7a region were performed. PCR products were visualized on agarose gel (2%) stained with ethidium bromide. Each lane corresponds to a virus plaque (P1–P11) derived from sample 16538. Virus plaques P2, P4 and P11 correspond to the virus without deletion in the ORF7a region, and virus plaques P5, P6 and P7 correspond to the virus with 365-nt deletions in the ORF7a region (ORF7a_∆365). M = 100 bp Molecular Weight Marker. (D) Electropherograms from Sanger sequencing of the ORF7a PCR products from sample 16991_ORF7a_∆339 aligned to SARS-CoV-2 reference and its deletion-resulting sequence (E) Electropherograms of both ORF7a PCR products isolated on a gel, from clinical samples and viruses isolated from cell culture (lanes P2 and P5, (B)) of 16538 ORF7a WT and the ORF7a_∆ 365 of 16538_ORF7a_∆365 and the non-deleted counterpart of 16538 * Samples submitted to NGS sequencing † Samples submitted to Sanger sequencing. Full article ">Figure 3
Amplification and examination of sgRNAs from samples with and without deletions on ORF7a. (A) Schematic illustration of SARS-CoV-2 genome encompassing TRS-L leader sequence (light orange box) and the primers’ position for sgRNAs amplification. Green dots represent the TRS-B sequences at the 5′ of each ORF of the structural and accessory proteins. (B) Sanger sequencing visualization of sgRNAs fragments from samples ORF7a_wt, ORF7a_∆190 and ORF7a_∆339 as illustrated in <a href="#app1-viruses-15-00801" class="html-app">Figure S2</a>. The light orange box indicates the 75-nt leader sequence, the red bar represents the forward primer SARS-Leader 18-41F, yellow boxes represent coding sequences (ORF7a, ORF8 and N), the reverse primers are not shown. Full article ">Figure 4
SARS-CoV-2 ORF7a 190-nt deletion reduces virus infectivity in Vero E6 cells. (A) Vero E6 cells were infected at MOI of 0.1 of SARS-CoV-2_wt (11785_wt) or SARS-CoV-2_ORF7a_∆190 (58301_ORF7a_∆190). (B) Cell supernatant was collected at 24, 48 and 72 hpi and used for virus titration in Vero E6 cells. Virus titration (mean ± SD) was plotted in a graphic. The virus titles at each time point were compared using multiple unpaired t-tests. PFU = plaque forming units (C) Cell supernatant was collected at 24, 48 and 72 hpi and used to perform RT-qPCR for SARS-CoV-2 N. SARS-CoV-2 RNA equivalent to PFU (mean ± SD) was plotted in the graphic. The virus titrations at each time point were compared using multiple unpaired t-tests. Results are from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; ns indicates no significant difference (p > 0.05). Full article ">

12 pages, 3151 KiB

Open AccessArticle

Two Point Mutations in the Glycoprotein of SFTSV Enhance the Propagation Recombinant Vesicular Stomatitis Virus Vectors at Assembly Step

by Qiang Hu, Yuhang Zhang, Jiafu Jiang and Aihua Zheng

Viruses 2023, 15(3), 800; https://doi.org/10.3390/v15030800 - 21 Mar 2023

Cited by 2 | Viewed by 2019

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen for which approved therapeutic drugs or vaccines are not available. We previously developed a recombinant vesicular stomatitis virus-based vaccine candidate (rVSV-SFTSV) by replacing the original glycoprotein with Gn/Gc from SFTSV, which [...] Read more.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen for which approved therapeutic drugs or vaccines are not available. We previously developed a recombinant vesicular stomatitis virus-based vaccine candidate (rVSV-SFTSV) by replacing the original glycoprotein with Gn/Gc from SFTSV, which conferred complete protection in a mouse model. Here, we found that two spontaneous mutations, M749T/C617R, emerged in the Gc glycoprotein during passaging that could significantly increase the titer of rVSV-SFTSV. M749T/C617R enhanced the genetic stability of rVSV-SFTSV, and no further mutations appeared after 10 passages. Using immunofluorescence analysis, we found that M749T/C617R could increase glycoprotein traffic to the plasma membrane, thus facilitating virus assembly. Remarkably, the broad-spectrum immunogenicity of rVSV-SFTSV was not affected by the M749T/C617R mutations. Overall, M749T/C617R could enhance the further development of rVSV-SFTSV into an effective vaccine in the future. Full article

(This article belongs to the Special Issue Advances in Antiviral Immunity and Virus Vaccines)

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Characterization of rVSV-SFTSV variants. (a) Schematic of the mutations that emerged in the SFTSV Gc protein. Mutation (Mut). Mutated nucleotides (Red) (b) Gc expression in the supernatants and cell lysates of rVSV-SFTSV WT- and variant-infected Vero cells (MOI = 0.01). Forty-eight hours post-infection, purified particles and cell lysates were blotted with anti-SFTSV Gc polyclonal antibodies (GAPDH as control). Con. indicates mock-infected cells. (c) Quantification of (b). (d) Growth kinetics of rVSVs (MOI = 0.01). *** t test, p < 0.001; **** t test, p < 0.0001. (e) Representative image of plaques formed using rVSV-SFTSV variants (6 days), rVSV-HRTV (6 days), and rVSV-G (3 days) at indicated days post-infection in Vero cells. Scale bar: 500 μm. (f) Genetic stability of rVSV-WT and rVSV-M749T + C617R during passages (P) in Vero cells. The above data are representative of three independent experiments. Full article ">Figure 2
The effect of M749T and C617R mutations on the plasma membrane localization of Gc. (a) Vero cells were infected with rVSV-WT and variants 24 h after transfection with a plasmid encoding the ER reporter DsRed-KDEL (red) and stained with anti-Gc polyclonal antibodies (green) 24 h post-infection. (b) Live Vero cells were infected with rVSVs for 16 h and stained with anti-Gc polyclonal antibodies (green). (c) Quantification of the density of Gc in the whole cell from (a) (n = 60). (d) Quantification of Gc in plasma membrane from (b) (n = 60). Statistical significance was determined using an unpaired Student’s t-test. ns, p > 0.05; **** p < 0.0001. The above data are representative of three independent experiments. Full article ">Figure 3
The humoral responses in mice elicited using rVSV-WT and rVSV-M749T + C617R. Three groups of C57/BL6 IFNAR−/− mice (n = 5 per group) were i.p. immunized with rVSV-WT, rVSV-M749T + C617R, and DMEM (2 × 104 PFU). (a) Mouse body weight changes after immunization. (b) NAb titers were determined against rVSV-GFP-SFTSV AH12 (b), rVSV-GFP-SFTSV YG1 (c), rVSV-GFP-HRTV (d), and rVSV-GFP-G (e) 28 days after immunization. Titers were calculated using the Reed–Muench method. The above data are representative of two independent experiments. The p-value was determined using a two-sided multiple t-test; **** t-test, p < 0.0001. Full article ">Figure 4
rVSV-WT and rVSV-M749T + C617R confer complete protection against lethal SFTSV Wuhan strain challenge in mice. (a) Survival rate of the mice after challenge. Immunized mice were i.p. challenged with a lethal dose of SFTSV (2 × 104 PFU) 30 days after immunization. (b) Body weight changes in mice after SFTSV challenge. Data are presented as the means ± SD. (c) SFTSV viral RNAs in the mice after challenge as measured using real-time PCR. The black bar is the mock-immunized control (con.); The dotted line represents the detection limit. (d) Viremia in the sera was measured using a plaque assay in Vero cells. Statistical significance was determined using multiple t-test. The above data are representative of two independent experiments. The p-value was determined using a two-sided multiple t-test; **** t-test, p < 0.0001; *** t-test, p < 0.001. Full article ">

13 pages, 2421 KiB

Open AccessArticle

Dynamic of Mayaro Virus Transmission in Aedes aegypti, Culex quinquefasciatus Mosquitoes, and a Mice Model

by Larissa Krokovsky, Carlos Ralph Batista Lins, Duschinka Ribeiro Duarte Guedes, Gabriel da Luz Wallau, Constância Flávia Junqueira Ayres and Marcelo Henrique Santos Paiva

Viruses 2023, 15(3), 799; https://doi.org/10.3390/v15030799 - 21 Mar 2023

Cited by 5 | Viewed by 2097

Abstract

Mayaro virus (MAYV) is transmitted by Haemagogus spp. mosquitoes and has been circulating in Amazon areas in the North and Central West regions of Brazil since the 1980s, with an increase in human case notifications in the last 10 years. MAYV introduction in [...] Read more.

Mayaro virus (MAYV) is transmitted by Haemagogus spp. mosquitoes and has been circulating in Amazon areas in the North and Central West regions of Brazil since the 1980s, with an increase in human case notifications in the last 10 years. MAYV introduction in urban areas is a public health concern as infections can cause severe symptoms similar to other alphaviruses. Studies with Aedes aegypti have demonstrated the potential vector competence of the species and the detection of MAYV in urban populations of mosquitoes. Considering the two most abundant urban mosquito species in Brazil, we investigated the dynamics of MAYV transmission by Ae. aegypti and Culex quinquefasciatus in a mice model. Mosquito colonies were artificially fed with blood containing MAYV and infection (IR) and dissemination rates (DR) were evaluated. On the 7th day post-infection (dpi), IFNAR BL/6 mice were made available as a blood source to both mosquito species. After the appearance of clinical signs of infection, a second blood feeding was performed with a new group of non-infected mosquitoes. RT-qPCR and plaque assays were carried out with animal and mosquito tissues to determine IR and DR. For Ae. aegypti, we found an IR of 97.5–100% and a DR reached 100% in both 7 and 14 dpi. While IR and DR for Cx. quinquefasciatus was 13.1–14.81% and 60% to 80%, respectively. A total of 18 mice were used (test = 12 and control = 6) for Ae. aegypti and 12 (test = 8 and control = 4) for Cx. quinquefasciatus to evaluate the mosquito–mice transmission rate. All mice that were bitten by infected Ae. aegypti showed clinical signs of infection while all mice exposed to infected Cx. quinquefasciatus mosquitoes remained healthy. Viremia in the mice from Ae. aegypti group ranged from 2.5 × 10⁸ to 5 × 10⁹ PFU/mL. Ae. aegypti from the second blood feeding showed a 50% IR. Our study showed the applicability of an efficient model to complete arbovirus transmission cycle studies and suggests that the Ae. aegypti population evaluated is a competent vector for MAYV, while highlighting the vectorial capacity of Ae. aegypti and the possible introduction into urban areas. The mice model employed here is an important tool for arthropod–vector transmission studies with laboratory and field mosquito populations, as well as with other arboviruses. Full article

(This article belongs to the Special Issue Animal Flaviviruses and Alphaviruses)

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Figure 1
Illustration of the vector competence study design of Mayaro virus transmission in a mouse model. (A) Vector competence study design in a timeline. (B) Mayaro transmission cycle model in a timeline. MID—midguts; SG—salivary glands; and MAYV—Mayaro virus. The illustration was created by the authors using the software Inkscape v 1.2. Full article ">Figure 2
Infection and dissemination rates of Aedes aegypti (RecLab) and Culex quinquefasciatus (CqSLab) mosquitoes experimentally fed with blood containing Mayaro. (A) Representative graph of infection rates found in Aedes aegypti (RecLab) and Culex quinquefasciatus (CqSLab). (B) Representative graph of dissemination rates found in Aedes aegypti (RecLab) and Culex quinquefasciatus (CqSLab). Dpi—day post-infection; ns—not significant. Statistical analysis was performed using R software (R DEVELOPMENT CORE TEAM) (*** p < 0.001). Full article ">Figure 3
Positive midgut and salivary glands of Aedes aegypti and Culex quinquefasciatus by RT-qPCR for Mayaro virus. (A,B) Quantification of RNA viral copy numbers in the positive midguts and salivary glands of Aedes aegypti and Culex quinquefasciatus mosquitoes experimentally fed with blood containing MAYV. Dpi—day post-infection; ns—not significant. Statistical analysis was performed using the R software (R DEVELOPMENT CORE TEAM) ** p < 0.01, and *** p < 0.001). Full article ">Figure 4
Animal experimentation panel with results of IFNAR BL/6 mice infected with Mayaro and mosquitoes Aedes aegypti. (A) Foot edema presented in mice hind foot after Mayaro virus infection compared with mock hind foot showed in bottom view; (B) foot edema presented in mice hind foot after Mayaro infection and mock hind foot showed in top view; (C) visualization of plaque-forming units in different mice samples at 10−5 dilution in VERO cell plaque assay; (D) Mayaro titer in mice tissues infected with Mayaro virus after Aedes aegypti blood feeding; (E) RNA copy number of mice tissues and Aedes aegypti whole-body sample infected with Mayaro virus after Aedes aegypti blood feeding. Gray circles—mice serum samples; Red circles—mice brain samples; Green diamond—mice liver samples; Blue triangle—mice gonad samples; Black circles—mosquito body samples; ns—not significant. Statistical analysis was performed using Graph Pad Prism 9 (* p < 0.05, ** p < 0.01). Full article ">

14 pages, 1392 KiB

Open AccessReview

Elucidating the Implications of Norovirus N- and O-Glycosylation, O-GlcNAcylation, and Phosphorylation

by Chia-Chi Cheng, Guan-Ming Ke, Pei-Yu Chu and Liang-Yin Ke

Viruses 2023, 15(3), 798; https://doi.org/10.3390/v15030798 - 21 Mar 2023

Cited by 1 | Viewed by 2680

Abstract

Norovirus is the most common cause of foodborne gastroenteritis, affecting millions of people worldwide annually. Among the ten genotypes (GI–GX) of norovirus, only GI, GII, GIV, GVIII, and GIX infect humans. Some genotypes reportedly exhibit post-translational modifications (PTMs), including N- and O [...] Read more.

Norovirus is the most common cause of foodborne gastroenteritis, affecting millions of people worldwide annually. Among the ten genotypes (GI–GX) of norovirus, only GI, GII, GIV, GVIII, and GIX infect humans. Some genotypes reportedly exhibit post-translational modifications (PTMs), including N- and O-glycosylation, O-GlcNAcylation, and phosphorylation, in their viral antigens. PTMs have been linked to increased viral genome replication, viral particle release, and virulence. Owing to breakthroughs in mass spectrometry (MS) technologies, more PTMs have been discovered in recent years and have contributed significantly to preventing and treating infectious diseases. However, the mechanisms by which PTMs act on noroviruses remain poorly understood. In this section, we outline the current knowledge of the three common types of PTM and investigate their impact on norovirus pathogenesis. Moreover, we summarize the strategies and techniques for the identification of PTMs. Full article

(This article belongs to the Special Issue Norovirus and Foodborne Diseases)

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Figure 1
Replication cycle of noroviruses and protein post-translational modifications. (1) Attachment: Human norovirus (HuNoV) attaches to the HBGAs on the host cell surface, allowing viral entrance. Note that fucosylation of HBGA by fucosyltransferase 2 (FUT2) is required for certain genotypes of norovirus infection. In contrast, deamination on Asn373 of norovirus capsid protein VP1 impairs the recognition of HBGAs. (2) Binding. (3) Uncoating through undefined pathways [<a href="#B80-viruses-15-00798" class="html-bibr">80</a>]. (4) Translation: The positive-sense RNA genome may serve as a template for viral protein translation. After translation, viral proteins could undergo N- or O-glycosylation at the endoplasmic reticulum or Golgi apparatus. Mechanisms of noroviral protein glycosylation remain unclear. (5) Assembly: viral proteins assemble to form new viral particles. (6) Release: viral particle release from host cell [<a href="#B81-viruses-15-00798" class="html-bibr">81</a>]. Abbreviations: HBGA, histo-blood group antigen. Full article ">Figure 2
Replication cycle of noroviruses and post-translational modification crosstalk between phosphorylation and O-GlcNAcylation. Co-occurring PTMs on proteins are common, and PTM crosstalk between phosphorylation and O-GlcNAcylation is the most common since they will compete for the same residues (serine/threonine residues). Phosphorylation is required for viral function. In contrast, O-GlcNAcylation of norovirus capsid protein VP1 could interfere with receptor binding. Studies on the O-GlcNAcylation of noroviruses are few and lack direct evidence from receptor binding assays. Abbreviations: HBGA, histo-blood group antigen; VP1, major capsid protein VP1; ATP, adenosine triphosphate; ADP, adenosine diphosphate; OGT, O-linked N-acetylglucosamine (GlcNAc) transferase; OGA, O-GlcNAcase; O-GlcNAc, O-linked N-acetylglucosamine; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine. Full article ">Figure 3
PTM crosstalk between O-GlcNAcylation and phosphorylation. Phosphorylation occurs when protein kinase attaches a phosphate group from ATP to the substrate; in contrast, PPase removes a phosphate group from a phosphoprotein. Similarly, OGT adds O-GlcNAc from UDP-GlcNAc to the substrates for O-GlcNAcylation. Conversely, OGA removes O-GlcNAc. These two processes are both reversible. Because phosphorylation and O-GlcNAcylation can compete for serine and threonine residues, PTM crosstalk occurs [<a href="#B83-viruses-15-00798" class="html-bibr">83</a>]. Abbreviations: O-GlcNAc, O-linked β-N-acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; Ser, serine; Thr, threonine; ATP, adenosine triphosphate; ADT, adenosine diphosphate; UPD, uridine diphosphate; PPase, protein phosphatase. Full article ">

22 pages, 1977 KiB

Open AccessReview

B and T Cell Epitopes of the Incursionary Foot-and-Mouth Disease Virus Serotype SAT2 for Vaccine Development

by Qian Li, Ashenafi Kiros Wubshet, Yang Wang, Livio Heath and Jie Zhang

Viruses 2023, 15(3), 797; https://doi.org/10.3390/v15030797 - 21 Mar 2023

Cited by 7 | Viewed by 3247

Abstract

Failure of cross-protection among interserotypes and intratypes of foot-and-mouth disease virus (FMDV) is a big threat to endemic countries and their prevention and control strategies. However, insights into practices relating to the development of a multi-epitope vaccine appear as a best alternative approach [...] Read more.

Failure of cross-protection among interserotypes and intratypes of foot-and-mouth disease virus (FMDV) is a big threat to endemic countries and their prevention and control strategies. However, insights into practices relating to the development of a multi-epitope vaccine appear as a best alternative approach to alleviate the cross-protection-associated problems. In order to facilitate the development of such a vaccine design approach, identification and prediction of the antigenic B and T cell epitopes along with determining the level of immunogenicity are essential bioinformatics steps. These steps are well applied in Eurasian serotypes, but very rare in South African Territories (SAT) Types, particularly in serotype SAT2. For this reason, the available scattered immunogenic information on SAT2 epitopes needs to be organized and clearly understood. Therefore, in this review, we compiled relevant bioinformatic reports about B and T cell epitopes of the incursionary SAT2 FMDV and the promising experimental demonstrations of such designed and developed vaccines against this serotype. Full article

(This article belongs to the Special Issue Foot-and-Mouth Disease Virus and Other Vesicular Disease Viruses)

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This simple schematic diagram shows the genome organization of FMDV, the encoded polyprotein, and matured proteins. It describes the long process of protein cleavage. The single ORF is illustrated in the box, and the viral proteins are named according to Rueckert and Wimmer’s nomenclature of picornavirus proteins [<a href="#B18-viruses-15-00797" class="html-bibr">18</a>]. Inside the boxes, there is a leader protein (L pro), four structural viral proteins (VP1-4), and seven non-structural proteins (2A-C, 3A-D). In addition, cleaved protein products and the cascade of cleavage are also shown in the diagram. The mature functional protein elements after cleavage are categorized as structural and non-structural proteins. The main cleavage sites are also shown in the box at the left side. The right and left untranslated regions (UTR) of the open reading frame are 3’ UTR and 5’ UTR respectively. The right extreme flank is shorter than the left one. Full article ">Figure 2
Incursionary features of FMDV serotype SAT2. In this map, SAT2 topotype VII was taken as an example. The directional arrows on the map show the epidemiological dynamics of SAT2 across different African regions, jumping the WOAH demarcated pools. Full article ">Figure 3
The illustration describes successive steps in epitope-based vaccine designing (this figure was adopted from Aryandra Arya and Sunil K Arora [<a href="#B103-viruses-15-00797" class="html-bibr">103</a>]. (1) In vitro antigenic epitope selection, identification, and analysis by using multiple immunoinformatic software from proteomic databases; (2) immunogenic B and T cell epitope selection by epitope insertion and sequence analysis; (3) synthesis of the designed neutralizing epitopes in the form of particulate, and evaluation of humoral and cellular immune responses by in vitro testing; (4) In vivo animal immunization to analyze the antibody responses to functionally characterize the anticipated neutralizing Abs- and cell-mediated immune responses. Full article ">Figure 4
Adjuvants targeting both innate and adaptive immunity and the mechanism action. (A) Innate immunity; molecular adjuvants (cytokines, chemokines, and dendritic stimulating molecules) incorporated as a gene in the same plasmid and, when co-delivered with the vaccine, can elicit efficient DC recruitment, activation, and Ag presentation to T cells. The CD8+ T lymphocytes are authorized by dendritic cells to become effector CTLs. For this, antigens need to be taken up, processed, and presented by dendritic cells (DCs) in association with MHC molecules. Cytokine adjuvants help sustain CD25+ regulatory T cells (T-regs) of the CD4+ cells. This can significantly improve the immune response. (B) Vaccine with adjuvants, such as oil-in-water emulsions and almunium hydroxide, can stimulate the adaptive arm of the immune system. The maturation of DCs are firstly recognized by CD4+ and CD8+ T cells. CD4+ T lymphocytes undergo a clonal expansion into two distinct T-helper subpopulations following contact with the MHC I-peptide complex. TH1 and TH2 cells stimulate the humoral and cell-mediated immune response through a different cytokine expression pattern. TLR4 agonists are considered to be the main ligand to activate DCs. This kind of adjuvant elicits a TH2-type functional immune response with preferential IgG1 antibody production [<a href="#B157-viruses-15-00797" class="html-bibr">157</a>,<a href="#B158-viruses-15-00797" class="html-bibr">158</a>,<a href="#B159-viruses-15-00797" class="html-bibr">159</a>]. CTL: cytotoxic T lymphocyte; DC: dendritic cell; PAMP: pathogen-associated molecular pattern; PRR: pattern recognition receptor; TLR: toll-like receptor (TLR is a receptor family belonging to PRRs); Treg: regulatory T cell; Th: T helper cell. Full article ">

13 pages, 1954 KiB

Open AccessArticle

Antibody Immunity to Zika Virus among Young Children in a Flavivirus-Endemic Area in Nicaragua

by Omar Zepeda, Daniel O. Espinoza, Evelin Martinez, Kaitlyn A. Cross, Sylvia Becker-Dreps, Aravinda M. de Silva, Natalie M. Bowman, Lakshmanane Premkumar, Elizabeth M. Stringer, Filemón Bucardo and Matthew H. Collins

Viruses 2023, 15(3), 796; https://doi.org/10.3390/v15030796 - 21 Mar 2023

Cited by 2 | Viewed by 2600

Abstract

Objective: To understand the dynamics of Zika virus (ZIKV)-specific antibody immunity in children born to mothers in a flavivirus-endemic region during and after the emergence of ZIKV in the Americas. Methods: We performed serologic testing for ZIKV cross-reactive and type-specific IgG in two [...] Read more.

Objective: To understand the dynamics of Zika virus (ZIKV)-specific antibody immunity in children born to mothers in a flavivirus-endemic region during and after the emergence of ZIKV in the Americas. Methods: We performed serologic testing for ZIKV cross-reactive and type-specific IgG in two longitudinal cohorts, which enrolled pregnant women and their children (PW1 and PW2) after the beginning of the ZIKV epidemic in Nicaragua. Quarterly samples from children over their first two years of life and maternal blood samples at birth and at the end of the two-year follow-up period were studied. Results: Most mothers in this dengue-endemic area were flavivirus-immune at enrollment. ZIKV-specific IgG (anti-ZIKV EDIII IgG) was detected in 82 of 102 (80.4%) mothers in cohort PW1 and 89 of 134 (66.4%) mothers in cohort PW2, consistent with extensive transmission observed in Nicaragua during 2016. ZIKV-reactive IgG decayed to undetectable levels by 6–9 months in infants, whereas these antibodies were maintained in mothers at the year two time point. Interestingly, a greater contribution to ZIKV immunity by IgG3 was observed in babies born soon after ZIKV transmission. Finally, 43 of 343 (13%) children exhibited persistent or increasing ZIKV-reactive IgG at ≥9 months, with 10 of 30 (33%) tested demonstrating serologic evidence of incident dengue infection. Conclusions: These data inform our understanding of protective and pathogenic immunity to potential flavivirus infections in early life in areas where multiple flaviviruses co-circulate, particularly considering the immune interactions between ZIKV and dengue and the future possibility of ZIKV vaccination in women of childbearing potential. This study also shows the benefits of cord blood sampling for serologic surveillance of infectious diseases in resource-limited settings. Full article

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

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Seroprevalence of ZIKV-reactive IgG in cord blood. ZIKV IgG (A) and ZIKV EDIII (B) ELISA were performed on cord blood plasma diluted 1:200 from Cohort 1 (n = 104) and Cohort 2 (n = 135). The cutoff for positivity was determined on each plate by running negative control plasma. The percent seropositivity for each assay is shown beneath the cohort names, and the geometric mean (GM) of OD for positive samples is shown beneath that. (C) The OD value from the ZIKV EDIII ELISA for available paired samples (n = 102) from mother peripheral blood and cord blood are graphed in a scatter plot to assess the efficiency of ZIKV-reactive IgG transfer across the placenta. Pearson correlation analysis was performed, and the R2 and p values are included in the upper left corner of each graph. ns, not significant; ****, p < 0.0001 according to an unpaired Student’s t-test comparing differences between GM of Cohort 1 vs. Cohort 2. Full article ">Figure 2
ZIKV-reactive antibody kinetics and IgG subtype. Decay of IgG reactive to ZIKV (A) and ZIKV EDIII (B) is shown for infants in both PW1 and PW2 cohorts. Spaghetti plots of trajectories were generated from the OD from each ELISA as a ratio to the LLOD, using a continuous time scale of weeks of life for each infant. Data from individual infants are shown as semi-transparent lines. Fit lines summarizing all data were calculated using an exponential decay (red dashed line) and a quadratic polynomial (blue dashed line) model. (C) IgG3 reactivity to ZIKV (left panel) and ZIKV EDIII (right panel) is shown in a subset of cord blood samples (n = 10 per subgroup) from subgroups indicated on the x-axis. OD, optical density; LLOD, lower limit of detection. X-axis legend of left graph of panel C: needs space between LOW and EDIII in the 2 group from left. Full article ">Figure 3
Incident flavivirus infection detected after initial decay of maternal-derived anti-ZIKV IgG in early life. Paired samples available from time points before and after the observed increase in anti-ZIKV IgG were selected for NAb testing. (A) Representative raw FRNT data measuring NAb to ZIKV (left) and DENV2 (right) are shown for three subjects. (B,C) eFRNT50 values for ZIKV (left) and DENV2 (right) are shown for suspected cases of incident flavivirus infection in Cohort 1 (B) and Cohort 2 (C). For nine samples with suspected incident flavivirus infection., and eFRNT50 values for paired samples are linked by a solid line. Full article ">

14 pages, 2788 KiB

Open AccessArticle

Tissue and Time Optimization for Real-Time Detection of Apple Mosaic Virus and Apple Necrotic Mosaic Virus Associated with Mosaic Disease of Apple (Malus domestica)

by Sajad Un Nabi, Javid Iqbal Mir, Salwee Yasmin, Ambreena Din, Wasim H. Raja, G. S. Madhu, Shugufta Parveen, Sheikh Mansoor, Yong Suk Chung, Om Chand Sharma, Muneer Ahmad Sheikh, Fahad A. Al-Misned and Hamed A. El-Serehy

Viruses 2023, 15(3), 795; https://doi.org/10.3390/v15030795 - 21 Mar 2023

Cited by 5 | Viewed by 2379

Abstract

Besides apple mosaic virus (ApMV), apple necrotic mosaic virus (ApNMV) has also been found to be associated with apple mosaic disease. Both viruses are unevenly distributed throughout the plant and their titer decreases variably with high temperatures, hence requiring proper tissue and time [...] Read more.

Besides apple mosaic virus (ApMV), apple necrotic mosaic virus (ApNMV) has also been found to be associated with apple mosaic disease. Both viruses are unevenly distributed throughout the plant and their titer decreases variably with high temperatures, hence requiring proper tissue and time for early and real-time detection within plants. The present study was carried out to understand the distribution and titer of ApMV and ApNMV in apple trees from different plant parts (spatial) during different seasons (temporal) for the optimization of tissue and time for their timely detection. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) was carried out to detect and quantify both viruses in the various plant parts of apple trees during different seasons. Depending on the availability of tissue, both ApMV and ApNMV were detected in all the plant parts during the spring season using RT-PCR. During the summer, both viruses were detected only in seeds and fruits, whereas they were detected in leaves and pedicel during the autumn season. The RT-qPCR results showed that during the spring, the ApMV and ApNMV expression was higher in leaves, whereas in the summer and autumn, the titer was mostly detected in seeds and leaves, respectively. The leaves in the spring and autumn seasons and the seeds in the summer season can be used as detection tissues through RT-PCR for early and rapid detection of ApMV and ApNMV. This study was validated on 7 cultivars of apples infected with both viruses. This will help to accurately sample and index the planting material well ahead of time, which will aid in the production of virus-free, quality planting material. Full article

(This article belongs to the Special Issue Plant Virus Epidemiology and Control 2022)

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Different plant parts/tissues with presence or absence of symptoms noted for virus detection and quantification. (a) Symptomatic leaf and pedicel, (b) petals, (c) anther, (d) flowers without petal, (e) bark, (f) fruit and (g) seed. Full article ">Figure 2
The symptoms of mosaic and necrotic mosaic on two cultivars: Golden Delicious (a) and Oregon Spur (b). Full article ">Figure 3
Amplicon of 260 bp amplified from ApMV-infected tissues of cultivar Golden Delicious (GD) plant tissues by conventional RT-PCR during the spring season (a), summer season (b) and autumn season (c), where L: ladder 100 bp, LF: Leaf, FL: Flowers, GD4:Bark, PT: Petals, PD: Pedicel, BK: Bark, FR: Fruit, SD: Seed, NC: Negative control, and PC: Positive control. Full article ">Figure 4
Amplicon of 670 bp amplified from ApNMV-infected tissues of cultivar Golden Delicious (GD) plant tissues by conventional RT-PCR during the spring season (a), summer season (b) and autumn season (c), where L: ladder 1 kb, LF: Leaf, FL: Flowers, GD4:Bark, PT: Petals, PD: Pedicel, BK: Bark, FR: Fruit, SD: Seed, NC: Negative control, and PC: Positive control. Full article ">Figure 5
Comparative fold change ofthe CP gene of ApMV and ApNMV in different tissues in cultivar Golden Delicious (GD) and Oregon spur (OS) during the spring season. Y-axis represents the fold change in the viral infection, whereas X-axis represents different tissues tested for both viruses. Full article ">Figure 6
Comparative fold change of the CP gene of ApMV and ApNMV in different tissues in cultivar Golden Delicious (GD) and Oregon spur (OS) during the summer season. Y-axis represents the fold change in the virus infection, whereas X-axis represents different tissues tested for both viruses. Full article ">Figure 7
Comparative fold change of the CP gene of APMV and ApNMV in different tissues in cultivar Golden Delicious (GD) and Oregon spur (OS) during the autumn season. Y-axis represents the fold change in the virus infection, whereas X-axis represents different tissues tested for both viruses. Full article ">

23 pages, 4901 KiB

Open AccessArticle

Circulating Plasma Exosomal Proteins of Either SHIV-Infected Rhesus Macaque or HIV-Infected Patient Indicates a Link to Neuropathogenesis

by Partha K. Chandra, Stephen E. Braun, Sudipa Maity, Jorge A. Castorena-Gonzalez, Hogyoung Kim, Jeffrey G. Shaffer, Sinisa Cikic, Ibolya Rutkai, Jia Fan, Jessie J. Guidry, David K. Worthylake, Chenzhong Li, Asim B. Abdel-Mageed and David W. Busija

Viruses 2023, 15(3), 794; https://doi.org/10.3390/v15030794 - 21 Mar 2023

Cited by 2 | Viewed by 2787

Abstract

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50–60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to [...] Read more.

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50–60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood–brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers—possibly associated with viral reactivation and neuropathogenesis—that may elucidate the etiology of HAND. Full article

(This article belongs to the Special Issue Viruses and Extracellular Vesicles 2023)

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11 pages, 1607 KiB

Open AccessCommunication

Correlation of SARS-CoV-2 Neutralization with Antibody Levels in Vaccinated Individuals

by Shazeda Haque Chowdhury, Sean Riley, Riley Mikolajczyk, Lauren Smith, Lakshmanan Suresh and Amy Jacobs

Viruses 2023, 15(3), 793; https://doi.org/10.3390/v15030793 - 21 Mar 2023

Cited by 1 | Viewed by 1858

Abstract

Neutralizing antibody titers are an important measurement of the effectiveness of vaccination against SARS-CoV-2. Our laboratory has set out to further verify the functionality of these antibodies by measuring the neutralization capacity of patient samples against infectious SARS-CoV-2. Samples from patients from Western [...] Read more.

Neutralizing antibody titers are an important measurement of the effectiveness of vaccination against SARS-CoV-2. Our laboratory has set out to further verify the functionality of these antibodies by measuring the neutralization capacity of patient samples against infectious SARS-CoV-2. Samples from patients from Western New York who had been vaccinated with the original Moderna and Pfizer vaccines (two doses) were tested for neutralization of both Delta (B.1.617.2) and Omicron (BA.5). Strong correlations between antibody levels and neutralization of the delta variant were attained; however, antibodies from the first two doses of the vaccines did not have good neutralization coverage of the subvariant omicron BA.5. Further studies are ongoing with local patient samples to determine correlation following updated booster administration. Full article

(This article belongs to the Special Issue RNA Viruses and Antibody Response)

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8 pages, 2600 KiB

Open AccessCommunication

Importance of Cellular Immunity and IFN-γ Concentration in Preventing SARS-CoV-2 Infection and Reinfection: A Cohort Study

by Dragan Primorac, Petar Brlek, Eduard Stjepan Pavelić, Jana Mešić, David Glavaš Weinberger, Vid Matišić, Vilim Molnar, Saša Srića and Renata Zadro

Viruses 2023, 15(3), 792; https://doi.org/10.3390/v15030792 - 20 Mar 2023

Cited by 5 | Viewed by 2247

Abstract

Recent studies have highlighted the underestimated importance of the cellular immune response after the emergence of variants of concern (VOCs) of SARS-CoV-2, and the significantly reduced neutralizing power of antibody titers in individuals with previous SARS-CoV-2 infection or vaccination. Our study included 303 [...] Read more.

Recent studies have highlighted the underestimated importance of the cellular immune response after the emergence of variants of concern (VOCs) of SARS-CoV-2, and the significantly reduced neutralizing power of antibody titers in individuals with previous SARS-CoV-2 infection or vaccination. Our study included 303 participants who were tested at St. Catherine Specialty Hospital using the Quan-T-Cell SARS-CoV-2 in combination with the Quan-T-Cell ELISA (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the analysis of IFN-γ concentration, and with Anti-SARS-CoV-2 QuantiVac ELISA IgG (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the detection of human antibodies of the immunoglobulin class IgG against the S1 domain of the SARS-CoV-2 spike protein. The statistical analysis showed a significant difference in the concentration of IFN-γ between reinfected participants and those without infection (p = 0.012). Participants who were not infected or reinfected with SARS-CoV-2 after vaccination and/or previous SARS-CoV-2 infection had a significantly higher level of cellular immunity. Furthermore, in individuals without additional vaccination, those who experienced infection/reinfection had significantly lower levels of IFN-γ compared to uninfected participants (p = 0.016). Our findings suggest a long-lasting effect of cellular immunity, measured by IFN-γ concentrations, which plays a key role in preventing infections and reinfections after the emergence of SARS-CoV-2 variants of concern. Full article

(This article belongs to the Special Issue SARS-CoV-2 and Other Vaccines: Immunogenicity Parameters and Protection 2nd Edition)

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35 pages, 1395 KiB

Open AccessArticle

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe

by Claude Saegerman, Marie-France Humblet, Marc Leandri, Gaëlle Gonzalez, Paul Heyman, Hein Sprong, Monique L’Hostis, Sara Moutailler, Sarah I. Bonnet, Nadia Haddad, Nathalie Boulanger, Stephen L. Leib, Thierry Hoch, Etienne Thiry, Laure Bournez, Jana Kerlik, Aurélie Velay, Solveig Jore, Elsa Jourdain, Emmanuelle Gilot-Fromont, Katharina Brugger, Julia Geller, Marie Studahl, Nataša Knap, Tatjana Avšič-Županc, Daniel Růžek, Tizza P. Zomer, René Bødker, Thomas F. H. Berger, Sandra Martin-Latil, Nick De Regge, Alice Raffetin, Sandrine A. Lacour, Matthias Klein, Tinne Lernout, Elsa Quillery, Zdeněk Hubálek, Francisco Ruiz-Fons, Agustín Estrada-Peña, Philippe Fravalo, Pauline Kooh, Florence Etore, Céline M. Gossner and Bethan Purse Show full author list Hide full author list

Viruses 2023, 15(3), 791; https://doi.org/10.3390/v15030791 - 20 Mar 2023

Cited by 11 | Viewed by 4763

Abstract

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over [...] Read more.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence. Full article

(This article belongs to the Special Issue Zoonotic Viral Diseases: Drivers, Causes, Prevention and Cure)

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Boxplot of the relative importance of the eight domains of possible drivers of the observed emergence or increasing incidence of TBE in humans (N = 40 European experts). Legend: The bold line represents the median of the score distribution between the different experts attributed to each domain; the solid lines at the top and bottom of each rectangle represent, respectively, the first and third quartiles; adjacent lines to the whiskers represent the limits of the 95% confidence interval; small circles represent outside values. The eight domains of drivers are: D1, disease/pathogen characteristics; D2, distance to Europe and the country of the expert (spatial-temporal scales); D3, ability to monitor, treat, and control the disease; D4, European farm characteristics; D5, global change; D6, wildlife interface; D7, human activity; and D8, economic and trade activities. Full article ">Figure 2
Ranking of the overall weighted score for each potential driver of the observed emergence or increasing incidence of TBE in humans (boxplot based on input from 40 European experts). Legend: The x-axis represents the drivers with the following codification: D1 to D8 refer to the eight domains of drivers, and D1_1 to D8_11 refer to a specific driver (for the codification, see <a href="#viruses-15-00791-t0A1" class="html-table">Table A1</a>). A relation to <a href="#viruses-15-00791-f003" class="html-fig">Figure 3</a> was provided by the group, which named “very high importance”, “high importance” and “less importance”, as possible drivers of the observed emergence or increasing incidence of TBE in humans. Full article ">Figure 3
Aggregation of drivers of the observed emergence or increasing incidence of TBE in humans into three homogenous groups using a regression tree analysis. Legend: N, number; SD, standard deviation. D1-01 to D8-11 refer to a specific driver (for the codification, see <a href="#viruses-15-00791-t0A1" class="html-table">Table A1</a>). Full article ">Figure 4
Level of uncertainty per domain of drivers. Legend: The bold line represents the median of the level of uncertainty attributed by experts using a scale from 0 (minimal uncertainty in the scoring) to 100 (maximum uncertainty in the scoring); the solid lines at the top and bottom of each rectangle represent, respectively, the first and third quartiles; adjacent lines to the whiskers represent the limits of the 95% confidence interval; small circles represent outside values. The eight domains of drivers are: D1, disease/pathogen characteristics; D2, distance to Europe and the country of the expert (spatial-temporal scales); D3, ability to monitor, treat, and control the disease; D4, European farm characteristics; D5, global change; D6, wildlife interface; D7, human activity; and D8, economic and trade activities. Full article ">

24 pages, 3139 KiB

Open AccessArticle

One Health Surveillance Highlights Circulation of Viruses with Zoonotic Potential in Bats, Pigs, and Humans in Viet Nam

by Alice Latinne, Nguyen Thi Thanh Nga, Nguyen Van Long, Pham Thi Bich Ngoc, Hoang Bich Thuy, PREDICT Consortium, Nguyen Van Long, Pham Thanh Long, Nguyen Thanh Phuong, Le Tin Vinh Quang, Nguyen Tung, Vu Sinh Nam, Vu Trong Duoc, Nguyen Duc Thinh, Randal Schoepp, Keersten Ricks, Ken Inui, Pawin Padungtod, Christine K. Johnson, Jonna A. K. Mazet, Chris Walzer, Sarah H. Olson and Amanda E. Fine Show full author list Hide full author list

Viruses 2023, 15(3), 790; https://doi.org/10.3390/v15030790 - 20 Mar 2023

Cited by 4 | Viewed by 7954

Abstract

A One Health cross-sectoral surveillance approach was implemented to screen biological samples from bats, pigs, and humans at high-risk interfaces for zoonotic viral spillover for five viral families with zoonotic potential in Viet Nam. Over 1600 animal and human samples from bat guano [...] Read more.

A One Health cross-sectoral surveillance approach was implemented to screen biological samples from bats, pigs, and humans at high-risk interfaces for zoonotic viral spillover for five viral families with zoonotic potential in Viet Nam. Over 1600 animal and human samples from bat guano harvesting sites, natural bat roosts, and pig farming operations were tested for coronaviruses (CoVs), paramyxoviruses, influenza viruses, filoviruses and flaviviruses using consensus PCR assays. Human samples were also tested using immunoassays to detect antibodies against eight virus groups. Significant viral diversity, including CoVs closely related to ancestors of pig pathogens, was detected in bats roosting at the human–animal interfaces, illustrating the high risk for CoV spillover from bats to pigs in Viet Nam, where pig density is very high. Season and reproductive period were significantly associated with the detection of bat CoVs, with site-specific effects. Phylogeographic analysis indicated localized viral transmission among pig farms. Our limited human sampling did not detect any known zoonotic bat viruses in human communities living close to the bat cave and harvesting bat guano, but our serological assays showed possible previous exposure to Marburg virus-like (Filoviridae), Crimean–Congo hemorrhagic fever virus-like (Bunyaviridae) viruses and flaviviruses. Targeted and coordinated One Health surveillance helped uncover this viral pathogen emergence hotspot. Full article

(This article belongs to the Special Issue Viruses and Bats 2023)

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Map of sampling locations in targeted provinces in Viet Nam. Full article ">Figure 2
Maximum Likelihood phylogenetic tree summarizing phylogenetic relationships among bat and pig coronavirus RdRp sequences (concatenated dataset including two fragments of the RdRp gene) identified in Viet Nam. Well-supported nodes (bootstrap > 75%) are indicated by a black dot. Virus sequences isolated in bat samples from Viet Nam are indicated by a bat symbol colored in green while bat viruses from other countries are black. Sequences isolated in pig samples in Viet Nam are indicated by a pig symbol colored in orange while pig viruses from other countries are grey. Full article ">Figure 3
Maximum Likelihood phylogenetic tree summarizing phylogenetic relationships among bat and pig paramyxovirus Pol gene sequences identified in Viet Nam. Well-supported nodes (bootstrap > 75%) are indicated by a black dot. Virus sequences isolated in bat samples from Viet Nam are indicated by a bat symbol colored in green, while bat viruses from other countries are black bat. Sequences isolated in pig samples in Viet Nam are indicated by a pig symbol colored in orange while pig viruses from other countries are grey. Full article ">Figure 4
Median-joining networks of (A) Betacoronavirus 1 (RdRp gene, [<a href="#B36-viruses-15-00790" class="html-bibr">36</a>], 393 bp), (B) porcine parainfluenza virus 1 (Pol gene, 546 bp), and (C) influenza A virus (PB1 gene, 384 bp). Circles correspond to distinct viral sequences and circle sizes are proportional to the number of identical sequences in the dataset. Small black circles represent median vectors (ancestral or unsampled intermediate sequences). The numbers of mutational steps between sequences are represented as hatch marks along branches. Full article ">Figure 5
Median-joining networks of (A) bat CoV 512/2005 (RdRp gene, [<a href="#B36-viruses-15-00790" class="html-bibr">36</a>], 393 bp), and (B) PREDICT_CoV-35 (RdRp gene, [<a href="#B36-viruses-15-00790" class="html-bibr">36</a>], 393 bp). Circles correspond to distinct viral sequences and circle sizes are proportional to the number of identical sequences in the dataset. Small black circles represent median vectors (ancestral or unsampled intermediate sequences). The numbers of mutational steps between sequences are represented as hatch marks along branches. Full article ">Figure 6
Spatiotemporal dispersal of bat coronavirus 512/2005 (grey shades) and PREDICT_CoV-35 (brown shades) in Southeast Asia inferred from a fragment of the RdRp gene (386 bp, [<a href="#B36-viruses-15-00790" class="html-bibr">36</a>]). Arrows indicate the direction of dispersal routes. Darker arrow colors indicate older dispersal events for both viruses. Full article ">

13 pages, 2294 KiB

Open AccessReview

Opportunities for CAR-T Cell Immunotherapy in HIV Cure

by Gerard Campos-Gonzalez, Javier Martinez-Picado, Talia Velasco-Hernandez and Maria Salgado

Viruses 2023, 15(3), 789; https://doi.org/10.3390/v15030789 - 19 Mar 2023

Cited by 9 | Viewed by 5924

Abstract

Chimeric antigen receptor (CAR) technology is having a huge impact in the blood malignancy field and is becoming a well-established therapy for many types of leukaemia. In recent decades, efforts have been made to demonstrate that CAR-T cells have potential as a therapy [...] Read more.

Chimeric antigen receptor (CAR) technology is having a huge impact in the blood malignancy field and is becoming a well-established therapy for many types of leukaemia. In recent decades, efforts have been made to demonstrate that CAR-T cells have potential as a therapy to achieve a sterilizing cure for human immunodeficiency virus (HIV) infection. However, translation of this technology to the HIV scenario has not been easy, as many challenges have appeared along the way that hinder the consolidation of CAR-T cells as a putative therapy. Here, we review the origin and development of CAR-T cells, describe the advantages of CAR-T cell therapy in comparison with other therapies, and describe the major obstacles currently faced regarding application of this technology in the HIV field, specifically, viral escape, CAR-T cell infectivity, and accessibility to hidden reservoirs. Nonetheless, promising results in successfully tackling some of these issues that have been obtained in clinical trials suggest a bright future for CAR-T cells as a consolidated therapy. Full article

(This article belongs to the Special Issue CAR-T Cell Therapy for HIV Cure 2023)

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13 pages, 4381 KiB

Open AccessArticle

Small RNA Profiling of Cucurbit Yellow Stunting Disorder Virus from Susceptible and Tolerant Squash (Cucurbita pepo) Lines

by Saritha Raman Kavalappara, Sudeep Bag, Alex Luckew and Cecilia E. McGregor

Viruses 2023, 15(3), 788; https://doi.org/10.3390/v15030788 - 19 Mar 2023

Cited by 3 | Viewed by 2706

Abstract

RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder [...] Read more.

RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder virus (CYSDV) were compared with those in Gold Star, a susceptible cultivar. The lower CYSDV symptom severity in PI 420328 correlated with lower virus titers and fewer sRNAs derived from CYSDV (vsRNA) compared to Gold Star. Elevated levels of 21- and 22-nucleotide (nt) size class vsRNAs were observed in PI 420328, indicating more robust and efficient RNA silencing in PI 420328. The distribution of vsRNA hotspots along the CYSDV genome was similar in both PI 420328 and Gold Star. However, the 3’ UTRs, CPm, and p26 were targeted at a higher frequency in PI 420328. Full article

(This article belongs to the Special Issue Crop Resistance to Viral Infections)

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Impact of CYSDV infection on squash line Gold Star and PI 420328, 30 days after inoculation: (A): mock-inoculated plants with non-viruliferous whiteflies displayed no phenotypic symptoms; (B): Gold Star developed prominent interveinal chlorosis and yellowing; (C) PI 420328 only displayed yellowing symptoms on the lower leaves; (D) estimated copy numbers of CYSDV in Gold Star (blue) and PI 420328 (red) at 30 DAI. Viral titer is represented as the mean Log concentration per ng total RNA. Each value is the average of three biological replicates with standard error bars; * indicates a significant difference (p < 0.05); (E) number of CYSDV-derived vsRNA reads per million total reads averaged over three replicates. Levels not connected by the same letter are significantly different (Student’s t-test, p < 0.05). Full article ">Figure 2
Relative abundance of different size classes in total vsRNAs 30 days after inoculation in susceptible (Gold Star) and tolerant (PI 420328) squash, systemically infected with cucurbit yellow stunting disorder virus (CYSDV). Each value is the average of three replicates with standard error bars. Asterix (*) indicates that corresponding values in Gold Star and PI 420328 are significantly different (t-test, p < 0.05). Full article ">Figure 3
Distribution of vsRNAs along the genomes of CYSDV in PI 420328 and Gold Star 30 days after inoculation with cucurbit yellow stunting disorder virus (CYSDV). The histograms plot the numbers of 21 to 24 nt viral siRNA reads at each nucleotide position of CYSDV genomic RNA 1 and RNA 2, (mapped with zero mismatches). The bars above the axis (blue) represent sense (forward) reads starting at each position, and those below (red) represent antisense (reverse) reads ending at the respective position. Full article ">Figure 4
Accumulation of 21–25 nt virus-derived sRNAs corresponding to UTRs and cistrons of CYSDV in Cucurbita pepo leaves at 30 DAI. Values are the average of three biological replicates and are represented as the frequency of vsRNA/100 bp of gene length/million total vsRNA reads. Asterix (*) indicates that corresponding values in Gold Star and PI 420328 are significantly different (t-test, p < 0.05). Full article ">Figure 5
The 5′-terminal nucleotide frequency of 20–24 nt CYSDV-derived small RNAs (sRNA) in Gold Star and PI 420328. The composite bar graphs indicate the percentage of 5′ U (red), 5′ G (orange), 5′ C (blue), and 5′ A (green) for each of the size classes of virus CYSDV-derived sRNAs. Full article ">

16 pages, 687 KiB

Open AccessReview

COVID-19 Pharmacotherapy in Pregnancy: A Literature Review of Current Therapeutic Choices

by Karolina Akinosoglou, Georgios Schinas, Emmanouil-Angelos Rigopoulos, Eleni Polyzou, Argyrios Tzouvelekis, George Adonakis and Charalambos Gogos

Viruses 2023, 15(3), 787; https://doi.org/10.3390/v15030787 - 19 Mar 2023

Cited by 9 | Viewed by 3087

Abstract

The clinical management of COVID-19 in pregnant women, who are considered a vulnerable population, remains uncertain even as the pandemic subsides. SARS-CoV-2 affects pregnant individuals in multiple ways and has been associated with severe maternal morbidity and mortality, as well as neonatal complications. [...] Read more.

The clinical management of COVID-19 in pregnant women, who are considered a vulnerable population, remains uncertain even as the pandemic subsides. SARS-CoV-2 affects pregnant individuals in multiple ways and has been associated with severe maternal morbidity and mortality, as well as neonatal complications. The unique anatomy and physiology of gestation make managing COVID-19 in this population a complex and challenging task, emphasizing the importance of spreading knowledge and expertise in this area. Therapeutic interventions require distinct clinical consideration, taking into account differences in pharmacokinetics, vertical transmission, drug toxicities, and postnatal care. Currently, there is limited data on antiviral and immunomodulating COVID-19 pharmacotherapy in pregnancy. Some medication has been shown to be safe and well tolerated among pregnant women with COVID-19; however, the lack of randomized clinical trials and studies in this patient population is evident. Available vaccines are considered safe and effective, with no evidence of harm to the fetus, embryo development, or short-term postnatal development. Pregnant women should be counseled about the risks of SARS-CoV-2 infection and informed of available ways to protect themselves and their families. Effective treatments for COVID-19 should not be withheld from pregnant individuals, and more research is needed to ensure the best outcomes. Full article

(This article belongs to the Special Issue COVID-19 Pharmacotherapy)

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13 pages, 6382 KiB

Open AccessArticle

Above-Standard Survival of Hepatocellular Carcinoma as the Final Outcome of Comprehensive Hepatology Care Programs in a Remote HCV-Endemic Area

by Wei-Ru Cho, Hui-Ling Huang, Nien-Tzu Hsu, Tung-Jung Huang and Te-Sheng Chang

Viruses 2023, 15(3), 786; https://doi.org/10.3390/v15030786 - 19 Mar 2023

Viewed by 1724

Abstract

Early detection and prompt linkage to care are critical for hepatocellular carcinoma (HCC) care. Chang Gung Memorial Hospital (CGMH) Yunlin branch, a local hospital in a rural area, undertakes health checkup programs in addition to its routine clinical service. Patients with HCC are [...] Read more.

Early detection and prompt linkage to care are critical for hepatocellular carcinoma (HCC) care. Chang Gung Memorial Hospital (CGMH) Yunlin branch, a local hospital in a rural area, undertakes health checkup programs in addition to its routine clinical service. Patients with HCC are referred to CGMH Chiayi branch, a tertiary referral hospital, for treatment. This study enrolled 77 consecutive patients with newly diagnosed HCCs between 2017 and 2022, with a mean age of 65.7 ± 11.1 years. The screening group included HCC patients detected through health checkups, and those detected by routine clinical service served as the control group. Compared to the 24 patients in the control group, the 53 patients in the screening group had more cases with early stage cancer (Barcelona Clinic Liver Cancer or BCLC stage 0 + A 86.8% vs. 62.5%, p = 0.028), better liver reserve (albumin–bilirubin or ALBI grade I 77.3% vs. 50%, p = 0.031) and more prolonged survival (p = 0.036). The median survival rates of the 77 patients were >5 years, 3.3 years, and 0.5 years in the BCLC stages 0 + A, B, and C, respectively, which were above the expectations of the BCLC guideline 2022 for stages 0, A, and B. This study provides a model of HCC screening and referral to high-quality care in remote viral-hepatitis-endemic areas. Full article

(This article belongs to the Special Issue Hepatitis-Associated Liver Cancer)

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23 pages, 1077 KiB

Open AccessReview

EV-A71 Mechanism of Entry: Receptors/Co-Receptors, Related Pathways and Inhibitors

by Kanghong Hu, Rominah Onintsoa Diarimalala, Chenguang Yao, Hanluo Li and Yanhong Wei

Viruses 2023, 15(3), 785; https://doi.org/10.3390/v15030785 - 18 Mar 2023

Cited by 6 | Viewed by 3728

Abstract

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first [...] Read more.

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as “co-receptors” because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71’s early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71. Full article

(This article belongs to the Special Issue Enteroviruses 2023)

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Enterovirus A71: mode of entry, virus–host interaction and viral life cycle. (A) Schematic illustration of the 3 different modes of entry identified to date for EV-A71 to invade cells. EV-A71 enters cells through receptor-mediated endocytosis, which includes clathrin-mediated (I), caveolae-mediated (II) and endophilin-A2-mediated (III). (I) In presence of the main receptor, hSCARB2, EV-A71 binds to it with or without the help of one or more co-receptors. The binding induces recruitment of adaptor proteins on the receptor cytoplasmic tail, which afterward bind to clathrin and form “a clathrin-coated pit” (CCP), leading to EV-A71 entry through clathrin-mediated endocytosis. hSCARB2 delivers β-GC from the ER to the lysosomes under physiological conditions. hSCARB2 is abundant in the lysosomal and endosomal compartments, and it also shuttles to the plasma membrane where it encounters EV-A71. After the binding of the virus on the cell surface, the virus–receptor complex is internalized. In the endosome or lysosome, where the pH is low, the virus initiates a conformational change that leads to uncoating. (II) In an alternative process, caveolins are the proteins binding to PSGL-1 cytoplasmic tail in the presence of actin cytoskeleton. Thus, this entrance way is called caveolae-mediated endocytosis. PSGL-1 can bind to EV-A71 and internalize via caveolin-mediated endocytosis, but PSGL-1 cannot initiate uncoating. (III) Recently, endophilin-A2-mediated endocytosis was identified. However, how receptor/co-receptor mediates this kind of entry is not yet elucidated. Once endocytosis initiates, the virus is internalized and delivered to early endosome for translocation, which is assured by the endosomal sorting complex required for transport to multivesicular bodies (ESCRT-MVBs). (B) EV-A71 promotes its production through interaction with intracellular signaling pathways. Once the virus is captured by main or co-receptors, several intracellular signaling pathways related to immune response are activated in order to eliminate the viral infection. However, EV-A71 has to escape immune response and overcome cellular apoptosis/autophagy for its survival by interfering with these pathways by interacting with host proteins. MAPK signaling cascade is activated after release of IL-2, IL-4, IL-10 and TNF-α. Subsequently, together with activated P13K/Akt pathway, MAPK downregulates GSK3, resulting in the delay of apoptosis. Multiple proteins involved in IFN-, apoptosis- and autophagy-related pathways are also regulated, such as JAK/STAT, TRAFs, p53, bax and mTOR. Consequently, many aspects of viral production are promoted, including viral polyprotein processing, RNA synthesis, assembly and maturation and release of new virions. Full article ">

13 pages, 2077 KiB

Open AccessArticle

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

by Huimin Bai, Yasushi Ami, Yuriko Suzaki, Yen Hai Doan, Masamichi Muramatsu and Tian-Cheng Li

Viruses 2023, 15(3), 784; https://doi.org/10.3390/v15030784 - 18 Mar 2023

Cited by 3 | Viewed by 2165

Abstract

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 [...] Read more.

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837C^RNA, T2885C^RNA, and T2837C/T2885C^RNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836T^RNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1. Full article

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

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Figure 1
HEV-1 (LC061267) genome organization and the position of the nucleotide mutations. ORF1 to ORF4 and the putative functional domains observed in ORF1 are depicted. Hel: helicase, HVR: hypervariable region, MT: methyltransferase, PCP: papain-like cysteine protease, RdRp: RNA-dependent RNA polymerase, X: X-domain, and Y: Y-domain. The numbers indicate the nucleotide position from the 5′-end. Mutated nucleotides are shown by bold and italics. Full article ">Figure 2
Generation and replication of the original and ORF4-defective HEV-1 in PLC/PRF/5 cells. PLC/PRF/5 cells were transfected with capped HEV-1RNA, A2836TRNA, T2837CRNA, T2885CRNA, and T2837RNA/T2885RNA, respectively. The culture supernatant was collected every 4 days, and the viral RNA was measured by RT-qPCR. The viral RNA copy numbers are shown in the HEV-1RNA-transfected cells (≤), A2836TRNA-transfected cells (○), T2837CRNA-transfected cells (◊), T2885CRNA-transfected cells (△), and T2837C/T2885CRNA-transfected cells (×). Full article ">Figure 3
Infectivity of the original and ORF4-defective HEV-1s in PLC/PRF/5 cells. PLC/PRF/5 cells were inoculated with HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 containing the same RNA copy number, respectively. The culture supernatant was collected every 4 days, and the viral RNA was measured by RT-qPCR. The RNA titers are shown in the HEV-1p0-infected cells (≤), A2836Tp0-infected cells (○), T2837Cp0-infected cells (◊), T2885Cp0-infected cells (△), and T2837C/T2885Cp0-infected cells (×). Full article ">Figure 4
Infection of original and ORF4-defective viruses in Mongolian gerbils. Fifteen Mongolia gerbils were randomly separated into five groups (n = 3 per group). Individual gerbils are indicated by ○, △, and ☐. Each group received HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, or T2837C/T2885Cp0 via intraperitoneal injection. The kinetics of the viral RNA in the fecal specimens were measured by RT-qPCR (a). The serum samples were collected at the end of the experiment (day 28 post-inoculation [p.i.]), and the anti-HEV-IgG antibody titers were determined by an ELISA with the virus-like particles (VLPs) of HEV-1 as the antigens (b). Dotted lines: the cut-off values. The minimum endpoints of the antibody titers are blackened. Full article ">Figure 5
Detection of viral proteins in HEV-1-infected PLC/PRF/5 cells. A cynomolgus monkey’s serum bearing the anti-HEV-1 IgG antibody titer 1:3,276,800 by ELISA was used for Western blot analyses. The minimum endpoints of the antibody titers are blackened (a). HEV-1p0-, T2837Cp0-, and T2837C/T2885Cp0-infected PLC/PRF/5 cells were harvested on day 48 p.i., and the virus proteins were detected by Western blot analysis (b). ORF4 was expressed by an E. coli expression system, and the related protein was analyzed by SDS-PAGE (c) and a Western blot analysis with monkey anti-HEV-1 serum (d) and anti-HEV-7 serum (e). M: molecular weight, NC: no-infected PLC/PRF/5 cells (b) or no-transformed BL21 (DE3) cells (c–e). pET32a (+): vector pET32a (+)-transformed BL21 (DE3). pET32aORF4: pET32aORF4-transformed BL21 (DE3). Full article ">

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Open AccessPerspective

Neurological Dysfunction in Long COVID Should Not Be Labelled as Functional Neurological Disorder

by Christina M. Van der Feltz-Cornelis, Andrew S. Moriarty and William David Strain

Viruses 2023, 15(3), 783; https://doi.org/10.3390/v15030783 - 18 Mar 2023

Cited by 2 | Viewed by 9892

Abstract

There have been suggestions that Long COVID might be purely functional (meaning psychological) in origin. Labelling patients with neurological dysfunction in Long COVID as having functional neurological disorder (FND) in the absence of proper testing may be symptomatic of that line of thought. [...] Read more.

There have been suggestions that Long COVID might be purely functional (meaning psychological) in origin. Labelling patients with neurological dysfunction in Long COVID as having functional neurological disorder (FND) in the absence of proper testing may be symptomatic of that line of thought. This practice is problematic for Long COVID patients, as motor and balance symptoms have been reported to occur in Long COVID frequently. FND is characterized by the presentation of symptoms that seem neurological but lack compatibility of the symptom with a neurological substrate. Although diagnostic classification according to the ICD-11 and DSM-5-TR is dependent predominantly on the exclusion of any other medical condition that could account for the symptoms, current neurological practice of FND classification allows for such comorbidity. As a consequence, Long COVID patients with motor and balance symptoms mislabeled as FND have no longer access to Long COVID care, whereas treatment for FND is seldom provided and is ineffective. Research into underlying mechanisms and diagnostic methods should explore how to determine whether motor and balance symptoms currently diagnosed as FND should be considered one part of Long COVID symptoms, in other words, one component of symptomatology, and in which cases they correctly represent FND. Research into rehabilitation models, treatment and integrated care are needed, which should take into account biological underpinnings as well as possible psychological mechanisms and the patient perspective. Full article

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

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Viruses, Volume 15, Issue 3 (March 2023) – 224 articles

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