Viruses

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability. Full article

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America. Full article

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6/7) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB. Full article

Equine encephalosis virus (EEV) is a neglected virus endemic to South Africa and is considered to generally result in mild disease in equines. Specimens were analyzed from live horses that presented with undefined neurological, febrile, or respiratory signs, or sudden and unexpected death. Between 2010 and 2017, 111 of 1523 (7.3%) horse samples tested positive for EEV using a nested real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Clinical signs were reported in 106 (7.2%) EEV positive and 1360 negative horses and included pyrexia (77/106, 72.6%), icterus (20/106, 18.9%) and dyspnea (12/106, 11.3%). Neurological signs were inversely associated with EEV infection (OR < 1, p < 0.05) relative to EEV negative cases despite a high percentage of animals presenting with neurological abnormalities (51/106, 48.1%). Seventeen of the EEV positive horses also had coinfections with either West Nile (5/106, 4.7%), Middelburg (4/106, 3.8%) or African Horse sickness virus (8/106, 7.6%). To investigate a possible genetic link between EEV strains causing the observed clinical signs in horses, the full genomes of six isolates were compared to the reference strains. Based on the outer capsid protein (VP2), serotype 1 and 4 were identified as the predominant serotypes with widespread reassortment between the seven different serotypes. Full article

Rhesus macaques can be readily infected with chimeric simian-human immunodeficiency viruses (SHIV) as a suitable virus challenge system for testing the efficacy of HIV vaccines. Three Chinese-origin rhesus macaques (ChRM) were inoculated intravenously (IV) with SHIVC109P4 in a rapid serial in vivo passage. SHIV recovered from the peripheral blood of the final ChRM was used to generate a ChRM-adapted virus challenge stock. This stock was titrated for the intrarectal route (IR) in 8 ChRMs using undiluted, 1:10 or 1:100 dilutions, to determine a suitable dose for use in future vaccine efficacy testing via repeated low-dose IR challenges. All 11 ChRMs were successfully infected, reaching similar median peak viraemias at 1–2 weeks post inoculation but undetectable levels by 8 weeks post inoculation. T-cell responses were detected in all animals and Tier 1 neutralizing antibodies (Nab) developed in 10 of 11 infected ChRMs. All ChRMs remained healthy and maintained normal CD4⁺ T cell counts. Sequence analyses showed >98% amino acid identity between the original inoculum and virus recovered at peak viraemia indicating only minimal changes in the env gene. Thus, while replication is limited over time, our adapted SHIV can be used to test for protection of virus acquisition in ChRMs. Full article

The circulation in Europe of novel reassortant strains of infectious bursal disease virus (IBDV), containing a unique genetic background composition, represents a serious problem for animal health. Since the emergence of this novel IBDV mosaic was first described in Poland, this scenario has become particularly attractive to uncover the evolutionary forces driving the genetic diversity of IBDV populations. This study additionally addressed the phenotypic characterization of these emergent strains, as well as the main features affecting the viral fitness during the competition process of IBDV lineages in the field. Our results showed how different evolutionary mechanisms modulate the genetic diversity of co-existent IBDV lineages, leading to the error catastrophe effect, Muller ratchet effect, or prevalence, depending on their genetic compositions. We also determined that the action of the positive selection pressure, depending on the genomic segment on which it is acting, can drive two main phenotypes for IBDV: immune-escaping strains from the selection on segment A or strains with functional advantages from the selection on segment B. This last group seems to possess an increased fitness landscape in the viral quasispecies composition, presenting better adaptability to dissimilar environmental conditions and likely becoming the dominant population. The reassortant strains also exhibited a lower mortality rate compared with the well-known vvIBDV strains, which can facilitate their spreading. Full article

The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1. Full article

Understanding SARS-CoV-2 evolution is a fundamental effort in coping with the COVID-19 pandemic. The virus genomes have been broadly evolving due to the high number of infected hosts world-wide. Mutagenesis and selection are two inter-dependent mechanisms of virus diversification. However, which mechanisms contribute to the mutation profiles of SARS-CoV-2 remain under-explored. Here, we delineate the contribution of mutagenesis and selection to the genome diversity of SARS-CoV-2 isolates. We generated a comprehensive phylogenetic tree with representative genomes. Instead of counting mutations relative to the reference genome, we identified each mutation event at the nodes of the phylogenetic tree. With this approach, we obtained the mutation events that are independent of each other and generated the mutation profile of SARS-CoV-2 genomes. The results suggest that the heterogeneous mutation patterns are mainly reflections of host (i) antiviral mechanisms that are achieved through APOBEC, ADAR, and ZAP proteins, and (ii) probable adaptation against reactive oxygen species. Full article

Diarrhoeal disease is considered an important cause of morbidity and mortality in developing areas, and a large contributor to the burden of disease in children younger than five years of age. This study investigated the prevalence and genogroups of human sapovirus (SV) in children ≤5 years of age in rural communities of Vhembe district, South Africa. Between 2017 and 2020, a total of 284 stool samples were collected from children suffering with diarrhoea (n = 228) and from children without diarrhoea (n = 56). RNA extraction using Boom extraction method, and screening for SV using real-time PCR were done in the lab. Positive samples were subjected to conventional RT-PCR targeting the capsid fragment. Positive sample isolates were genotyped using Sanger sequencing. Overall SV were detected in 14.1% (40/284) of the stool samples (16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples). Significant correlation between SV positive cases and water sources was noted. Genogroup-I was identified as the most prevalent strain comprising 81.3% (13/16), followed by SV-GII 12.5% (2/16) and SV-GIV 6.2% (1/16). This study provides valuable data on prevalence of SV amongst outpatients in rural and underdeveloped communities, and highlights the necessity for further monitoring of SV circulating strains as potential emerging strains. Full article

The novel SARS-CoV-2 Variant of Concern (VOC)-202012/01 (also known as B.1.1.7), first collected in United Kingdom on 20 September 2020, is a rapidly growing lineage that in January 2021 constituted 86% of all SARS-CoV-2 genomes sequenced in England. The VOC has been detected in 40 out of 46 countries that reported at least 50 genomes in January 2021. We have estimated that the replicative advantage of the VOC is in the range 1.83–2.18 [95% CI: 1.71–2.40] with respect to the 20A.EU1 variant that dominated in England in November 2020, and in range 1.65–1.72 [95% CI: 1.46–2.04] in Wales, Scotland, Denmark, and USA. As the VOC strain will likely spread globally towards fixation, it is important to monitor its molecular evolution. We have estimated growth rates of expanding mutations acquired by the VOC lineage to find that the L18F substitution in spike has initiated a fast growing VOC substrain. The L18F substitution is of significance because it has been found to compromise binding of neutralizing antibodies. Of concern are immune escape mutations acquired by the VOC: E484K, F490S, S494P (in the receptor binding motif of spike) and Q677H, Q675H (in the proximity of the polybasic cleavage site at the S1/S2 boundary). These mutants may hinder efficiency of existing vaccines and expand in response to the increasing after-infection or vaccine-induced seroprevalence. Full article

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections. Full article

The APOBEC family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes – APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new host-pathogen conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation. Full article

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture. Full article

Adenoviruses have served as a model for investigating viral-cell interactions and discovering different cellular processes, such as RNA splicing and DNA replication. In addition, the development and evaluation of adenoviruses as the viral vectors for vaccination and gene therapy has led to detailed investigations about adenovirus biology, including the structure and function of the adenovirus encoded proteins. While the determination of the structure and function of the viral capsid proteins in adenovirus biology has been the subject of numerous reports, the last few years have seen increased interest in elucidating the structure and function of the adenovirus core proteins. Here, we provide a review of research about the structure and function of the adenovirus core proteins in adenovirus biology. Full article

Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research. Full article

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity. Full article

The aquatic virus, infectious pancreatic necrosis virus (IPNV), is known to infect various farmed fish, in particular salmonids, and is responsible for large economic losses in the aquaculture industry. Common practices to detect the virus include qPCR tests based on specific primers and serum neutralization tests for virus serotyping. Following the potential presence of IPNV viruses in a fish farm in Scotland containing vaccinated and IPNV-resistant fish, the common serotyping of the IPNV isolates was not made possible. This led us to determine the complete genome of the new IPNV isolates in order to investigate the cause of the serotyping discrepancy. Next-generation sequencing using the Illumina technology along with the sequence-independent single primer amplification (SISPA) approach was conducted to fully characterize the new Scottish isolates. With this approach, the full genome of two isolates, V1810–4 and V1810–6, was determined and analyzed. The potential origin of the virus isolates was investigated by phylogenetic analyses along with tridimensional and secondary protein structure analyses. These revealed the emergence of a new variant from one of the main virus serotypes, probably caused by the presence of selective pressure exerted by the vaccinated IPNV-resistant farmed fish. Full article

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development. Full article

Studies of viruses that coevolved with lemurs provide an opportunity to understand the basal traits of primate viruses and provide an evolutionary context for host-virus interactions. Germline integration of endogenous retroviruses (ERVs) are fossil evidence of past infections. Hence, characterization of novel ERVs provides insight into the ancient precursors of extant viruses and the evolutionary history of their hosts. Here, we report the discovery of a novel endogenous retrovirus present in the genome of a lemur, Coquerel’s sifaka (Propithecus coquereli). Using next-generation sequencing, we identified and characterized the complete genome sequence of a retrovirus, named prosimian retrovirus 1 (PSRV1). Phylogenetic analyses indicate that PSRV1 is a gamma-type betaretrovirus basal to the other primate betaretroviruses and most closely related to simian retroviruses. Molecular clock analysis of PSRV1 long terminal repeat (LTR) sequences estimated the time of endogenization within 4.56 MYA (± 2.4 MYA), placing it after the divergence of Propithecus species. These results indicate that PSRV1 is an important milestone of lemur evolution during the radiation of the Propithecus genus. These findings may have implications for both human and animal health in that the acquisition of a gamma-type env gene within an endogenized betaretrovirus could facilitate a cross-species jump between vertebrate class hosts. Full article