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The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MJM is a founder and shareholder in the company Vaxdyn, S.L. Vaxdyn played no role in the present study. No other competing interest is declared for the remaining co-authors.

A harmonized balance between positive and negative regulation of pattern recognition receptor (PRR)-initiated immune responses is required to achieve the most favorable outcome for the host. This balance is crucial because it must not only ensure activation of the first line of defense against viral infection but also prevent inappropriate immune activation, which results in autoimmune diseases. Recent studies have shown how signal transduction pathways initiated by PRRs are positively and negatively regulated by diverse modulators to maintain host immune homeostasis. However, viruses have developed strategies to subvert the host antiviral response and establish infection. Viruses have evolved numerous genes encoding immunomodulatory proteins that antagonize the host immune system. This review focuses on the current state of knowledge regarding key host factors that regulate innate immune signaling molecules upon viral infection and discusses evidence showing how specific viral proteins counteract antiviral responses via immunomodulatory strategies., (© 2021. The Author(s).)

Future HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4 + T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1. IMPORTANCE The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically-intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Our investigations into the possible cellular mechanisms maintaining the HIV-1 reservoir in different T-cell subsets have revealed a link between the half-lives of T-cells and the level of proviruses they contain. Taken together, we believe our study shows that more differentiated and proliferative cells, such as transitional and effector memory T-cells, contain the highest levels of genetically-intact proviruses, and the rapid turnover rate of these cells contributes to the expansion of genetically-intact proviruses within them. Therefore, our study delivers an in-depth assessment of the cellular mechanisms, such as cellular proliferation and half-life, that contribute to and maintain the latent HIV-1 reservoir.

Recent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community. IMPORTANCE Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen. These findings show that the frequent oscillation in the immune status in immunocompromised individuals can trigger an accelerated virus evolution, thus consolidating this study model as an accelerated pathway to better understand SARS-CoV-2 adaptive traits and anticipate the emergence of variants of concern.

Over the last two decades, there have been three deadly human outbreaks of coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years caused by yet another spillover of a bat-derived SARS-like coronavirus (SL-CoV) into humans. Determining the Ag and the human B cells, CD4 + and CD8 + T cell epitope landscapes that are conserved among human and animal coronaviruses should inform in the development of future pan-coronavirus vaccines. In the current study, using several immunoinformatics and sequence alignment approaches, we identified several human B cell and CD4 + and CD8 + T cell epitopes that are highly conserved in 1) greater than 81,000 SARS-CoV-2 genome sequences identified in 190 countries on six continents; 2) six circulating CoVs that caused previous human outbreaks of the common cold; 3) nine SL-CoVs isolated from bats; 4) nine SL-CoV isolated from pangolins; 5) three SL-CoVs isolated from civet cats; and 6) four MERS strains isolated from camels. Furthermore, the identified epitopes: 1) recalled B cells and CD4 + and CD8 + T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2, and 2) induced strong B cell and T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice. The findings pave the way to develop a preemptive multiepitope pan-coronavirus vaccine to protect against past, current, and future outbreaks., (Copyright © 2021 by The American Association of Immunologists, Inc.)

A new pandemic caused by the betacoronavirus SARS-CoV-2 originated in China in late 2019. Although often asymptomatic, a relevant percentage of affected people can develop severe pneumonia. Initial evidence suggests that dysregulation of the immune response could contribute to the pathogenesis, as previously demonstrated for SARS-CoV. The presence of genome composition features involved in delaying viral recognition is herein investigated for human coronaviruses (HCoVs), with a special emphasis on SARS-CoV-2. A broad collection of HCoVs polyprotein, envelope, matrix, nucleocapsid and spike coding sequences was downloaded and several statistics representative of genome composition and codon bias were investigated. A model able to evaluate and test the presence of a significant under- or over-representation of dinucleotide pairs while accounting for the underlying codon bias and protein sequence was also implemented. The study revealed the significant under-representation of CpG dinucleotide pair in all HcoV, but especially in SARS-CoV and even more in SARS-CoV-2. The presence of forces acting to minimize CpG content was confirmed by relative synonymous codon usage pattern. Codons containing the CpG pair were severely under-represented, primarily in the polyprotein and spike coding sequences of SARS-CoV-2. Additionally, a significant under-representation of the TpA pair was observed in the N and S region of SARS-CoV and SARS-CoV-2. Increasing experimental evidence has proven that CpG and TpA are targeted by innate antiviral host defences, contributing both to RNA degradation and RIG-1 mediated interferon production. The low content of these dinucleotides could contribute to a delayed interferon production, dysregulated immune response, higher viral replication and poor outcome. Significantly, the RIG-1 signalling pathway was proven to be defective in elderlies, suggesting a likely interaction between limited viral recognition and lower responsiveness in interferon production that could justify the higher disease severity and mortality in older patients.

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS-CoV-2 and SARS-CoV share a high degree of genetic and pathologic identity and share safety and immune-enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus-based) preparations of SARS-CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS-CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person-to-person transmission. Furthermore, 'S' protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T-cell responses against the SARS-CoV-2 'S' protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid-19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS-CoV-2 viral infection. This manuscript reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine., (© 2020 John Wiley & Sons Ltd.)

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Linder, Bothe, Linder, Schwarzlmueller, Dahlström, Bartenhagen, Dugas, Pandey, Thorn-Seshold, Boehmer, Koenig, Kobold, Schnurr, Raedler, Spielmann, Karimzadeh, Schmidt, Endres and Rothenfusser.)

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ryan, Carr, Furtado, Ma, Ashander, Simões, Oliver, Granado, Dawson, Michael, Appukuttan, Lynn and Smith.)

SARS-CoV-2 has a high transmission rate and shows frequent mutations, thus making vaccine development an arduous task. However, researchers around the globe are working hard to find a solution e.g. synthetic vaccine. Here, we have performed genome-wide analysis of 566 Indian SARS-CoV-2 genomes to extract the potential conserved regions for identifying peptide based synthetic vaccines, viz. epitopes with high immunogenicity and antigenicity. In this regard, different multiple sequence alignment techniques are used to align the SARS-CoV-2 genomes separately. Subsequently, consensus conserved regions are identified after finding the conserved regions from each aligned result of alignment techniques. Further, the consensus conserved regions are refined considering that their lengths are greater than or equal to 60nt and their corresponding proteins are devoid of any stop codons. Subsequently, their specificity as query coverage are verified using Nucleotide BLAST. Finally, with these consensus conserved regions, T-cell and B-cell epitopes are identified based on their immunogenic and antigenic scores which are then used to rank the conserved regions. As a result, we have ranked 23 consensus conserved regions that are associated with different proteins. This ranking also resulted in 34 MHC-I and 37 MHC-II restricted T-cell epitopes with 16 and 19 unique HLA alleles and 29 B-cell epitopes. After ranking, the consensus conserved region from NSP3 gene is obtained that is highly immunogenic and antigenic. In order to judge the relevance of the identified epitopes, the physico-chemical properties and binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The 2019 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak has caused a large number of deaths, with thousands of confirmed cases worldwide. The present study followed computational approaches to identify B- and T-cell epitopes for the spike (S) glycoprotein of SARS-CoV-2 by its interactions with the human leukocyte antigen alleles. We identified 24 peptide stretches on the SARS-CoV-2 S protein that are well conserved among the reported strains. The S protein structure further validated the presence of predicted peptides on the surface, of which 20 are surface exposed and predicted to have reasonable epitope binding efficiency. The work could be useful for understanding the immunodominant regions in the surface protein of SARS-CoV-2 and could potentially help in designing some peptide-based diagnostics. Also, identified T-cell epitopes might be considered for incorporation in vaccine designs., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mammalian orthoreovirus (reovirus) is a dsRNA virus, which has long been used as a model system to study host-virus interactions. One of the earliest interactions during virus infection is the detection of the viral genomic material, and the consequent induction of an interferon (IFN) based antiviral response. Similar to the replication of related dsRNA viruses, the genomic material of reovirus is thought to remain protected by viral structural proteins throughout infection. Thus, how innate immune sensor proteins gain access to the viral genomic material, is incompletely understood. This review summarizes currently known information about the innate immune recognition of the reovirus genomic material. Using this information, we propose hypotheses about host detection of reovirus.

Marek's disease virus (MDV), a causative agent of Marek's disease, has evolved its virulence partly because the current control strategies fail to provide sterilizing immunity. Gallid alphaherpesvirus 3 (GaHV-3) and turkey herpesvirus have been developed as bivalent vaccines to improve upon the level of protection elicited by single formulations. Since the in vitro passage of vaccines can result in attenuation, a GaHV-3 strain 301B/1 was cloned as a bacterial artificial chromosome (BAC) by inserting the mini -F replicon into the virus genome. A fully infectious virus, v301B-BAC, was reconstituted from the 301B/1 BAC clone and had similar growth kinetics comparable to that of the parental 301B/1 virus with strong reactivity against anti-301B/1 chicken sera. Protective efficacies of v301B-BAC, parental 301B/1, and SB-1 vaccine were evaluated against a very virulent MDV Md5 challenge. Clinical signs were significantly lower in the v301B-BAC vaccinated groups (24-25 %), parental 301B/1 (29 %) compare to that of non-vaccinated control (100%) and the removal of BAC sequences from v301B-BAC genome further reduced this to 17 %. The protective indices of v301B-BACs (75-76 %) were comparable with those of both the 301B/1 and the SB-1 vaccine (71%). Removal of the mini -F replicon resulted in a reconstituted virus with a protective index of 83 %. The shedding of challenge virus was notably lower in the v301B-BAC, and v301B-delBAC vaccinated groups. Overall, the protective efficacy of the 301B-BAC-derived vaccine virus against a very virulent MDV challenge was comparable to that of the parental 301B/1 virus as well as the SB-1 vaccine virus.

Internal N 6 -methyladenosine (m 6 A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m 6 A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m 6 A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m 6 A methylated and that viral m 6 A methylation promotes HMPV replication and gene expression. Inactivating m 6 A addition sites with synonymous mutations or demethylase resulted in m 6 A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m 6 A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m 6 A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m 6 A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m 6 A can serve as a target to attenuate HMPV for vaccine purposes.

We subjected various open reading frames (ORFs) in the genome of respiratory syncytial virus (RSV) to codon pair optimization (CPO) by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (Δ1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multicycle virus replication in vitro , temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response. IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon pair optimized (CPO) by increasing the content of codon pairs that are overrepresented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response., (Copyright © 2020 Le Nouën et al.)

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

Human cytomegalovirus (HCMV) is ubiquitously prevalent. HCMV infection is typically asymptomatic and controlled by the immune system in healthy individuals, yet HCMV can be severely pathogenic for the fetus during pregnancy and in immunocompromised persons, such as transplant recipients or HIV infected patients. HCMV has co-evolved with the hosts, developed strategies to hide from immune effector cells and to successfully survive in the human organism. One strategy for evading or delaying the immune response is maintenance of the viral genome to establish the phase of latency. Furthermore, HCMV immune evasion involves the downregulation of human leukocyte antigens (HLA)-Ia molecules to hide infected cells from T-cell recognition. HCMV expresses several proteins that are described for downregulation of the HLA class I pathway via various mechanisms. Here, we review the wide range of immune evasion mechanisms of HCMV. Understanding the mechanisms of HCMV immune evasion will contribute to the development of new customized therapeutic strategies against the virus.

Reticuloendotheliosis virus (REV), an avian retrovirus is able to infect a variety of birds and can cause immunosuppression. The aim of this study was to investigate the relationship of thymic lymphocytes apoptosis, proliferation and T cell subtype with immunosuppression. In this study, a hundred and twenty one-day old SPF chickens were randomly divided into control groups (group C) and a REV infection groups (group I). The chickens of group I received intraperitoneal injections of REV with 10 4.62 /0.1 ml TCID 50 . On day 14, 21, 28 and 35 post-inoculation, the chickens of C group and I group were sacrificed by cardiac puncture blood collection, and the thymic lymphocytes was sterile collected. The proliferation ability of lymphocytes was tested by Cell Counting Kit-8. Flow cytometry was performed to detect apoptosis, cell cycle stage and the change in T cell subtype. The RNA genome copy numbers of REV virus were detected using real-time PCR. Real-time PCR and western blotting were performed to analyze the expression of CyclinD1 and Bcl-2. Our results showed that REV genome copy number steadily declined, the proliferation potential of thymic lymphocytes was inhibited, lymphocytes apoptosed, the ratio of CD4+/CD8+ decreased and the expression of CyclinD1 and Bcl-2 were firstly inhibited, then rapidly recovered. Thus, immunosuppression lead by REV is closely related to the change of T cell subtype, apoptosis, and proliferation of thymic lymphocytes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

APOBEC3G (A3G) is a host enzyme that mutates the genomes of retroviruses like HIV. Since A3G is expressed pre-infection, it has classically been considered an agent of innate immunity. We and others previously showed that the impact of A3G-induced mutations on the HIV genome extends to adaptive immunity also, by generating cytotoxic T cell (CTL) escape mutations. Accordingly, HIV genomic sequences encoding CTL epitopes often contain A3G-mutable "hotspot" sequence motifs, presumably to channel A3G action toward CTL escape. Here, we studied the depths and consequences of this apparent viral genome co-evolution with A3G. We identified all potential CTL epitopes in Gag, Pol, Env, and Nef restricted to several HLA class I alleles. We simulated A3G-induced mutations within CTL epitope-encoding sequences, and flanking regions. From the immune recognition perspective, we analyzed how A3G-driven mutations are predicted to impact CTL-epitope generation through modulating proteasomal processing and HLA class I binding. We found that A3G mutations were most often predicted to result in diminishing/abolishing HLA-binding affinity of peptide epitopes. From the viral genome evolution perspective, we evaluated enrichment of A3G hotspots at sequences encoding CTL epitopes and included control sequences in which the HIV genome was randomly shuffled. We found that sequences encoding immunogenic epitopes exhibited a selective enrichment of A3G hotspots, which were strongly biased to translate to non-synonymous amino acid substitutions. When superimposed on the known mutational gradient across the entire length of the HIV genome, we observed a gradient of A3G hotspot enrichment, and an HLA-specific pattern of the potential of A3G hotspots to lead to CTL escape mutations. These data illuminate the depths and extent of the co-evolution of the viral genome to subvert the host mutator A3G.

We have previously reported that human herpesvirus 6 (HHV-6) infection of endothelial cells (ECs) induces the loss of angiogenic properties, through the expression of HHV-6 U94, possibly associated to the release of a soluble mediator. It is also known that the soluble isoform of HLA-G exhibits an anti-angiogenic function, important in implantation, transplantation and neoplastic development. In this study, we analyzed the expression of HLA-G in HHV-6 infected ECs, showing that both HHV-6A and HHV-6B infection induce a potent up-modulation of HLA-G, including both membrane and soluble isoforms. Interestingly, HHV-6A and HHV-6B induced different isoforms of HLA-G. The virus-induced increase of HLA-G was likely due to the expression of the U94 viral gene, that by itself was able to reproduce the effect of whole virus. The effect of U94 was mediated by human transcription factor ATF3, that induced HLA-G activation by recognizing a consensus sequence on its promoter. Virus-induced inhibition of ECs angiogenic ability directly correlated to HLA-G expression and release, and the addition of anti-HLA-G antibody restored the angiogenic properties of HHV6-infected ECs. The induction of HLA-G expression in ECs might represent an important mediator of HHV-6 induced effects.

Group C orthobunyaviruses (GRCVs) are a complex of viruses in the genus Orthobunyavirus and are associated with human febrile disease in tropical and subtropical areas of South and Central America. While numerous GRCVs have been isolated from mosquitoes, animals, and humans, genetic analysis of these viruses is limited. In this study, we characterized 65 GRCV isolates from febrile patients identified through clinic-based surveillance in the northern and southern Peruvian Amazon. A 500 base pair region of the S segment and 750 base pair regions of the M and L segments were sequenced. Pairwise sequence analysis of the clinical isolates showed nucleotide identities ranging from 68% to 100% and deduced amino acid sequence identities ranging from 72% to 100%. Sequences were compared with reference strains of the following GRCVs: Caraparu virus (CARV), Murutucu virus (MURV), Oriboca virus (ORIV), Marituba virus (MTBV), Itaqui virus (ITQV), Apeu virus (APEUV), and Madrid virus (MADV). Sequence comparison of clinical isolates with the prototype strains based on the S and L segments identified two clades; clade I included isolates with high genetic association with CARV-MADV, and clade II included isolates with high genetic association with MURV, ORIV, APEUV, and MTBV. Genetic relationships based on the M segment were at time inconsistent with those based on the S and L segments. However, clade groupings based on the M segment were highly consistent with relationships based on microneutralization assays. These results advance our understanding of the genetic and serologic relationships of GRCVs circulating in the Peruvian Amazon., Competing Interests: The authors have declared that no competing interests exist.

The properties of the human immunodeficiency virus (HIV) pose serious difficulties for the development of an effective prophylactic vaccine. Here we describe the construction and characterization of recombinant (r), replication-competent forms of rhesus monkey rhadinovirus (RRV), a gamma-2 herpesvirus, containing a near-full-length (nfl) genome of the simian immunodeficiency virus (SIV). A 306-nucleotide deletion in the pol gene rendered this nfl genome replication-incompetent as a consequence of deletion of the active site of the essential reverse transcriptase enzyme. Three variations were constructed to drive expression of the SIV proteins: one with SIV's own promoter region, one with a cytomegalovirus (cmv) immediate-early promoter/enhancer region, and one with an RRV dual promoter (p26 plus PAN). Following infection of rhesus fibroblasts in culture with these rRRV vectors, synthesis of the early protein Nef and the late structural proteins Gag and Env could be demonstrated. Expression levels of the SIV proteins were highest with the rRRV-SIVcmv-nfl construct. Electron microscopic examination of rhesus fibroblasts infected with rRRV-SIVcmv-nfl revealed numerous budding and mature SIV particles and these infected cells released impressive levels of p27 Gag protein (>150 ng/ml) into the cell-free supernatant. The released SIV particles were shown to be incompetent for replication. Monkeys inoculated with rRRV-SIVcmv-nfl became persistently infected, made readily-detectable antibodies against SIV, and developed T-cell responses against all nine SIV gene products. Thus, rRRV expressing a near-full-length SIV genome mimics live-attenuated strains of SIV in several important respects: the infection is persistent; >95% of the SIV proteome is naturally expressed; SIV particles are formed; and CD8+ T-cell responses are maintained indefinitely in an effector-differentiated state. Although the magnitude of anti-SIV immune responses in monkeys infected with rRRV-SIVcmv-nfl falls short of what is seen with live-attenuated SIV infection, further experimentation seems warranted., Competing Interests: The authors have declared that no competing interests exist.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess., (Copyright © 2018 American Society for Microbiology.)

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4 + and CD8 + T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Background: The first licensed dengue vaccine is a recombinant, live, attenuated, tetravalent dengue virus vaccine (CYD-TDV; Sanofi Pasteur). This study assessed the biodistribution, shedding, and toxicity of CYD-TDV in a non-human primate model as part of the nonclinical safety assessment program for the vaccine., Methods: Cynomolgus monkeys were given one subcutaneous injection of either one human dose (5log 10 CCID 50 /serotype) of CYD-TDV or saline control. Study endpoints included clinical observations, body temperature, body weight, food consumption, clinical pathology, immunogenicity, and post-mortem examinations including histopathology. Viral load, distribution, persistence, and shedding in tissues and body fluids were evaluated by quantitative reverse transcriptase polymerase chain reaction., Results: The subcutaneous administration of CYD-TDV was well tolerated. There were no toxicological findings other than expected minor local reactions at the injection site. A transient low level of CYD-TDV viral RNA was detected in blood and the viral genome was identified primarily at the injection site and in the draining lymph nodes following immunization., Conclusions: These results, together with other data from repeat-dose toxicity and neurovirulence studies, confirm the absence of toxicological concern with CYD-TDV and corroborate clinical study observations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: To determine the diagnostic value of different clinical, laboratory, and instrumental signs in the diagnosis of myocarditis in patients with the picture of idiopathic arrhythmias, dilated cardiomyopathy (DCM) and in a comparison group when comparing with myocardial morphological examination., Subjects and Methods: A study group included 100 patients (35 women; mean age, 44.7±12.5 years) with idiopathic arrhythmias (n=20) and DCM as a syndrome (n=100). All underwent myocardial morphological examination: endomyocardial biopsy (EMB) (n=71), intraoperative biopsy (n=13), study of the explanted heart (n=6), and autopsy (n=11). A comparison group consisted of 50 patients (25 women; mean age, 53.7±11.7 years) with non-inflammatory diseases of the heart (left ventricular end-diastolic dimension <6.0 cm, ejection fraction >50%) who underwent open-heart surgery (n=47), EMB (n=2), or autopsy (n=1). The investigators also performed polymerase chain reaction for cardiotropic viral DNA in the blood and myocardium, anticardiac antibody (ACA) identification, myocardial scintigraphy (n=26), coronary angiography (n=47), magnetic resonance imaging (MRI) (n=25), and multislice computed tomography of the heart (n=45). The diagnostic value of the extended spectrum of clinical, laboratory, and instrumental markers for myocarditis was estimated., Results: Active/borderline myocarditis was diagnosed in 76% of the patients in the study group (75.5% in the arrhythmia subgroup and 76.3% in the DCM one) and in 24.3% of those in the comparison group (p<0.001). A viral genome in the myocardium was detected statistically significantly less frequently in the study group than that in the comparison one (40.2 and 65%, respectively; p<0.01): in 46.6% in the DCM subgroup and 15.8% in the arrhythmia one. An ACA set (sensitivity, specificity, and predictive value of positive and negative test results (45.7, 80, 80.4, and 45%, respectively)) was of the greatest diagnostic importance in identifying myocarditis; antibodies to cardiomyocyte nuclei in a titer of 1:160-1:320 had the highest specificity (93.3%). A specificity above 70% was seen for a full medical history triad (acute onset, an association between onset and infection, a symptom duration of less than one year), systemic immune manifestations, anginas in the history and elevated anti-O-streptolysin levels, systemic blood changes, Q waves/QS complexes on ECGs, local hypokinesias, pericardial effusion, atriomegalia (in arrhythmias), angina/ischemia with intact coronary arteries, and focal perfusion defects during myocardial scintigraphy. A sensitivity higher than 50% was observed for age over 40 years (differential diagnosis with genetic forms), acute onset, a correlation with infection, and delayed contrast agent accumulation, as evidenced by MSCT/MRI., Conclusion: When the incidence of myocarditis is similar in the arrhythmia and DCM subgroups, the viral genome detection rate is statistically significantly higher in DCM. Among the non-invasive markers, an ACA set (high sensitivity and specificity) is of the greatest diagnostic value in the diagnosis of myocarditis. The diagnostic rule based on counting the number of scores has been developed, which makes it possible to individually establish the risk of myocarditis in patients with idiopathic arrhythmias and DCM for both the determination of indications for biopsy and the lack of the possibility of its performance. The risk of myocarditis is high if there are 5-7 scores; that is close to 100% if there are 8 scores or more.

Virus vaccines have to be immunogenic, sufficiently stable, safe, and suitable to induce long-lasting immunity. To meet these requirements, vaccine studies need to provide a comprehensive understanding of (i) the protective roles of antiviral B and T-cell-mediated immune responses, (ii) the complexity and plasticity of major viral antigens, and (iii) virus molecular biology and pathogenesis. There are many types of vaccines including subunit vaccines, whole-inactivated virus, vectored, and live-attenuated virus vaccines, each of which featuring specific advantages and limitations. While nonliving virus vaccines have clear advantages in being safe and stable, they may cause side effects and be less efficacious compared to live-attenuated virus vaccines. In most cases, the latter induce long-lasting immunity but they may require special safety measures to prevent reversion to highly virulent viruses following vaccination. The chapter summarizes the recent progress in the development of coronavirus (CoV) vaccines, focusing on two zoonotic CoVs, the severe acute respiratory syndrome CoV (SARS-CoV), and the Middle East respiratory syndrome CoV, both of which cause deadly disease and epidemics in humans. The development of attenuated virus vaccines to combat infections caused by highly pathogenic CoVs was largely based on the identification and characterization of viral virulence proteins that, for example, interfere with the innate and adaptive immune response or are involved in interactions with specific cell types, such as macrophages, dendritic and epithelial cells, and T lymphocytes, thereby modulating antiviral host responses and viral pathogenesis and potentially resulting in deleterious side effects following vaccination., (© 2016 Elsevier Inc. All rights reserved.)

The biological arms race generally involves the rapid co-evolution of anti-virus systems in host organisms and of anti-anti-virus systems in their viral parasites. The CRISPR-Cas system is an example of a prokaryotic immune system in which such co-evolution occurs, as was recently demonstrated by the characterization of a set of viral anti-CRISPR proteins.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Viruses efficiently block the host antiviral response in order to replicate and spread before host intervention. The mechanism initiating antiviral immunity during stealth viral replication is unknown, but recent data demonstrate that defective viral genomes generated at peak virus replication are critical for this process in vivo. This article summarizes the supporting evidence and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory defective viral genomes generated during natural infections., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ΔK3 ΔK5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1α-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP(high) and pK8.1-driven blue fluorescent protein-positive (BFP(+)) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages., Importance: Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

Background: Studies of Merkel cell polyomavirus (MCPyV) in nonmelanoma skin cancers (NMSC) other than Merkel cell carcinoma (MCC) produced controversial results. Therefore, we studied the prevalence of MCPyV in basal cell carcinoma (BCC) and in squamous cell carcinoma (SCC)., Methods: Tissue specimens were analyzed for the presence of MCPyV DNA by conventional polymerase chain reaction (PCR). Expression of MCPyV large T protein was determined by immunohistochemistry., Results: MCPyV DNA was frequently detected in skin cancers by PCR, in 36 of 88 BCCs, in 21 of 75 SCCs and in 10 of 47 normal skin samples. In BCC, a significant difference in the detection rate compared to normal skin was observed. In contrast, weak reactivity for MCPyV large T antigen was detected only sporadically in immunosuppressed patients (2 of 88 BCCs, 1 of 75 SCCs). Mutations of the large T antigen of MCPyV were more frequently observed in MCC than in BCC/SCC., Conclusions: Our results suggest that the frequent detection of the MCPyV genome in NMSC by PCR reflects ubiquitous spread of the virus. However, the low immunohistochemical detection rate of MCPyV and the lack of MCC-specific MCPyV mutations argue against an essential role of MCPyV in the development of skin cancers other than MCC., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

JC and BK polyomaviruses were discovered over 40 years ago and have become increasingly prevalent causes of morbidity and mortality in a variety of distinct, immunocompromised patient cohorts. The recent discoveries of eight new members of the Polyomaviridae family that are capable of infecting humans suggest that there are more to be discovered and raise the possibility that they may play a more significant role in human disease than previously understood. In spite of this, there remains a dearth of specific therapeutic options for human polyomavirus infections and an incomplete understanding of the relationship between the virus and the host immune system. This review summarises the human polyomaviruses with particular emphasis on pathogenesis in those directly implicated in disease aetiology and the therapeutic options available for treatment in the immunocompromised host.

The popularity of nonreplicating adenoviruses of chimpanzee origin (ChAdVs) as vectors for subunit vaccines is on the rise. This is mainly for their excellent safety and impressive immunogenicity observed in human studies to date. Here, we recloned the chimpanzee adenovirus sero type 68 (ChAdV-68), also designated SAdV-25 and AdC68, genome and demonstrated its straightforward genetic manipulation facilitated by the use of bacterial artificial chromosome recombineering. To generate the ChAdV68.GagB vaccine, the HIV-1 consensus clade B Gag-derived Tg was inserted into the E1 region. In part confirming previous observations, the ChAdV68.GagB vaccine alone and in heterologous prime-boost regimens with plasmid DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines induced robust polyfunctional HIV-1-specific CD8(+) and CD4(+) T-cell responses with a gut-homing phenotype. Importantly, we showed that when a single epitope is expressed as an immunodominant CD8(+) T-cell determinant, responses elicited by ChAdV68.GagB alone and in combination lowered surrogate challenge EcoHIV/NDK (where EcoHIV is chimeric ecotropic HIV) virus load in mice both at the peak T-cell frequencies 2 weeks after vaccination and 16 weeks later indicating development of protective effector memory. These results parallel the immunogenicity of similar vaccine regimens in macaques and an ongoing phase I/IIa trial in humans, and support further development of vaccines vectored by ChAdVs., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The toll-like receptor (TLR)-7 has been shown to sense the retroviral infection. However, a surrogate sensor has been implicated. We examined whether retrovirus serves as a TLR3 ligand in human cells by utilizing cell lines LNCaP and PC-3 lacking TLR7, and the xenotropic murine leukemia virus-relamoted virus (XMRV) insensitive to human tripartite motif-containing (TRIM) 5, a newly characterized pattern recognition receptor (PRR). A dominant-negative TLR3 or a chemical inhibitor of TLR3 attenuated the XMRV-induced IP-10/CXCL10 expression, a marker of TLR3 response. These data clearly indicated that retroviral infection exemplified by XMRV activates the TLR3 signal in human cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.

Unlabelled: To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells., Conclusion: In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection., (Copyright © 2012 American Association for the Study of Liver Diseases.)

The continued global spread and evolution of HIV diversity pose significant challenges to diagnostics and vaccine strategies. NIAID partnered with the FDA, WRAIR, academia, and industry to form a Viral Panel Working Group to design and prepare a panel of well-characterized current and diverse HIV isolates. Plasma samples that had screened positive for HIV infection and had evidence of recently acquired infection were donated by blood centers in North and South America, Europe, and Africa. A total of 80 plasma samples were tested by quantitative nucleic acid tests, p24 antigen, EIA, and Western blot to assign a Fiebig stage indicative of approximate time from initial infection. Evaluation of viral load using FDA-cleared assays showed excellent concordance when subtype B virus was tested, but lower correlations for subtype C. Plasma samples were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors to generate 30 viral isolates (50-80% success rate for samples with viral load >10,000 copies/ml), which were then expanded to 10(7)-10(9) virus copies per ml. Analysis of env sequences showed that sequences derived from cultured PBMCs were not distinguishable from those obtained from the original plasma. The pilot collection includes 30 isolates representing subtypes B, C, B/F, CRF04&#95;cpx, and CRF02&#95;AG. These studies will serve as a basis for the development of a comprehensive panel of highly characterized viral isolates that reflects the current dynamic and complex HIV epidemic, and will be made available through the External Quality Assurance Program Oversight Laboratory (EQAPOL).

Cross-reactive immunity occurs when infection with or vaccination against one virus protects against another related family member. A search for homologues of the HIV-1 envelope glycoprotein revealed that it is composed of thousands of intercalating and overlapping viral matches of pentapeptide or longer gapped consensi, belonging to over 70% of the currently sequenced virome, infecting all kingdoms from bacteria to man. It was also highly homologous to proteins from the Visna/Maedi and other ovine viruses, while other proteins (nef/tat/gag/pol) were homologous to proteins from the equine infectious anaemia virus and HTLV-2/HTLV-3 viruses. This phenomenon suggests that horizontal gene transfer from coinfecting RNA and DNA viruses to retroviruses is extensive, providing a route for the subsequent insertion of non-retroviral genes into human and other genomes via retroviral integration. This homology includes all viruses for which vaccines already exist. Cross-reactive immunity may be operative in AIDS, as Vaccinia vaccination decreases viral replication in HIV-1 infected patients' cells, for the CCR5 tropic form. Measles, Dengue virus, or GB virus C infections also decrease the HIV-1 viral load. A resumption of Vaccinia/smallpox vaccination might be expected to have a significant effect on the AIDS pandemic, and a careful study of the potential uses of other existing viral and bacterial vaccines merits close attention. This phenomenon may also be relevant to other recalcitrant viruses, bacteria, and parasites for which no vaccine exists and the armory of existing vaccines may have a role to play in diseases other than those for which they were designed.

Respiratory viral infections have been associated with an increased incidence of allergic asthma. However, the mechanisms by which respiratory infections facilitate allergic airway disease are incompletely understood. We previously showed that exposure to a low dose of house dust mite (HDM) resulted in enhanced HDM-mediated allergic airway inflammation, and, importantly, marked airway hyperreactivity only when allergen exposure occurred during an acute influenza A infection. In this study, we evaluated the impact of concurrent influenza infection and allergen exposure at the genomic level, using whole-genome microarray. Our data showed that, in contrast to exposure to a low dose of HDM, influenza A infection led to a dramatic increase in gene expression, particularly of TLRs, C-type lectin receptors, several complement components, as well as FcεR1. Additionally, we observed increased expression of a number of genes encoding chemokines and cytokines associated with the recruitment of proinflammatory cells. Moreover, HDM exposure in the context of an influenza A infection resulted in the induction of unique genes, including calgranulin A (S100a8), an endogenous damage-associated molecular pattern and TLR4 agonist. In addition, we observed significantly increased expression of serum amyloid A (Saa3) and serine protease inhibitor 3n (Serpina3n). This study showed that influenza infection markedly increased the expression of multiple gene classes capable of sensing allergens and amplifying the ensuing immune-inflammatory response. We propose that influenza A infection primes the lung environment in such a way as to lower the threshold of allergen responsiveness, thus facilitating the emergence of a clinically significant allergic phenotype.

Background: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse., Methods/principal Findings: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA., Conclusions/significance: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin-B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.

The polyomaviruses are small DNA viruses that can establish latency in the human host. The name polyomavirus is derived from the Greek roots poly-, which means "many," and -oma, which means "tumours." These viruses were originally isolated in mouse (mPyV) and in monkey (SV40). In 1971, the first human polyomaviruses BK and JC were isolated and subsequently demonstrated to be ubiquitous in the human population. To date, at least nine members of the Polyomaviridae family have been identified, some of them playing an etiological role in malignancies in immunosuppressed patients. Here, we describe the biology of human polyomaviruses, their nonmalignant and malignant potentials ability, and their relationship with the host immune response.

Intrinsic antiviral resistance is a branch of antiviral defence that involves constitutively expressed cellular proteins that act within individual infected cells. In recent years it has been discovered that components of cellular nuclear structures known as ND10 or PML nuclear bodies contribute to intrinsic resistance against a variety of viruses, notably of the herpesvirus family. Several ND10 components are rapidly recruited to sites that are closely associated with herpes simplex virus type 1 (HSV-1) genomes during the earliest stages of infection, and this property correlates with the efficiency of ND10 mediated restriction of HSV-1 replication. Similar but distinct recruitment of certain DNA damage response proteins also occurs during infection. These recruitment events are inhibited in a normal wild type HSV-1 infection by the viral regulatory protein ICP0. HSV‑1 mutants that do not express ICP0 are highly susceptible to repression through intrinsic resistance factors, but they replicate more efficiently in cells depleted of certain ND10 proteins or in which ND10 component recruitment is inefficient. This article presents the background to this recruitment phenomenon and summaries how it is conveniently studied by fluorescence microscopy.