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Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1–infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1–infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue., National Institutes of Health (U.S.) (NIH (2 P50 GM082545-06), Ragon Institute of MGH, MIT and Harvard (post-doctoral fellowship), Gordon and Betty Moore Foundation, Agouron Institute, National Institutes of Health (U.S.) (NIH K08 Award (K08 AI084546-04)), Burroughs Wellcome Fund (fellowship), Harvard University. Center for AIDS Research (P30 AI060354)

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 carriers. Higher levels of CC-chemokines were produced by KIR3DL1+ than KIR3DL1− NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines., Author Summary Natural Killer (NK) cells function in anti-tumor and anti-viral defenses, including those directed against HIV. HIV infected cells can activate NK cells, which, once activated, inhibit HIV replication in infected targets. NK cell activation levels depend on the interaction of cell surface receptors on NK cells with the molecules (or ligands) they recognize on neighboring target cells. One receptor-ligand combination has been identified to have a strong effect on slowing time to AIDS, HIV viral load control and NK cell activation potential. We compared anti-HIV NK cell responses in individuals with this NK receptor-ligand combination to those from subjects having NK receptor-ligand combinations associated a neutral effect on time to AIDS. NK cells inhibited HIV replication in autologous infected cells more potently when they came from individuals with NK receptor-ligand (KIR/HLA) gene combinations associated with slow versus typical time to AIDS. Inhibition of HIV replication was due to secretion of factors (chemokines) that bind and block the co-receptor HIV uses to enter susceptible target cells. NK cells from subjects with KIR/HLA combinations associated with potent NK cell anti-HIV activity secreted more chemokines than those from subjects with KIR/HLA combinations associated with weak anti-HIV NK cell activity.

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P, Author Summary RNA viruses are the fastest evolving human pathogens, making their treatment and control difficult. Compared to DNA viruses, RNA viruses replicate with much lower fidelity, which can explain why RNA viruses evolve significantly faster than most DNA viruses. However, there is tremendous variation among the evolutionary rates of different RNA viruses, which is not explained by variation in mutation rates. Here we present a survey of mammalian RNA virus rates of evolution, and a comprehensive comparison of these rates to different properties of virus genomic architecture and ecology. We found that cell tropism is the most significant predictor of long-term rates of mammalian RNA virus evolution. For instance, viruses targeting epithelial cells evolve significantly faster than viruses that target neurons. Our results provide mechanistic insight into why viruses that infect respiratory and gastrointestinal epithelia have been difficult to control.

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5′-triphosphate (5′-ppp) RNA and mediate IFN production. Cytosolic 5′-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-β independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5′-ppp dsRNA and induces IFN-β through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-β induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-β production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity., Author Summary Type I interferons (IFN) are critical for mounting effective antiviral responses by the host cells. For RNA viruses, it is believed that IFN is triggered exclusively by viral double-stranded RNA (dsRNA) or RNA containing a 5′-triphosphate (5′-ppp) that is produced during viral genome replication or transcription driven by viral replicases. Here, we provide strong evidence suggesting that the viral replicase also generates 5′-ppp dsRNA using cellular RNA templates, which trigger IFN. This finding indicates that viral replicase is capable of activating the host innate immune response, deviating from the paradigm that viral nucleic acid replication or transcription must be initiated in the host cell to trigger IFN production. Using Semliki Forest virus (SFV) as a model, we show that the magnitude of innate immune response activation by the viral replicase plays a decisive role in establishing viral infection. We demonstrate that in contrast to the wild-type SFV replicase, a non-pathogenic mutant replicase triggers increased IFN production, which leads to a shutdown of virus replication. Consequently, excessive IFN induction by the viral replicase can be dangerous for an RNA virus. Thus, we delineate a novel mechanism by which an RNA virus triggers the host cell immune response leading to RNA virus replication shutdown.

This work was funded by the Fralin Life Science Institute at Virginia Tech, as well as the College of Agriculture and Life Sciences and the Department of Entomology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus., Author Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing agent in immunocompromised patients that establishes long-lasting infections in its hosts. Initially described in 1994 and extensively studied ever since, KSHV molecular biology is understood in broad outline, but many detailed questions are still to be resolved. After almost two decades, specific aspects pertaining to the organization of the KSHV genome as well as the fate of the viral transcripts during the productive stages of infection remain unexplored. Here we use a systematic genome-wide approach to investigate changes in gene and protein expression during the productive stage of infection known as the lytic cycle. We found that the viral genome has a large coding capacity, capable of generating at least 45% more products than initially anticipated by bioinformatic analyses alone, and that it uses multiple strategies to expand its coding capacity well beyond what is determined solely by the DNA sequence of its genome. We also provide an expanded and highly detailed annotation of known and new genomic features in KSHV. We have termed this new architectural and functional annotation KSHV 2.0. Our results indicate that viral genomes are more complex than anticipated, and that they are subject to tight mechanisms of regulation to ensure correct gene expression.

Viruses are usually thought to form parasitic associations with hosts, but all members of the family Polydnaviridae are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from Microplitis demolitor bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (lef-4, lef-9), capsid assembly (vp39, vlf-1), and envelope formation (p74, pif-1) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions. Additional experiments suggested that vlf-1 together with the nudivirus-like gene int-1 also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation., Author Summary Microorganisms form symbiotic associations with animals and plants that range from parasitic (pathogens) to beneficial (mutualists). Although numerous examples of obligate, mutualistic bacteria, fungi, and protozoans exist, viruses are almost always considered to be pathogens. An exception is the family Polydnaviridae, which consists of large DNA viruses that are obligate mutualists of insects called parasitoid wasps. Prior studies show that polydnaviruses in the genus Bracovirus evolved approximately 100 million years ago from a group of viruses called nudiviruses, which are themselves closely related to a large family of insect pathogens called baculoviruses. Polydnaviruses are thus of fundamental interest for understanding the processes by which viruses can evolve into mutualists. In this study we characterized the composition of virus particles from Microplitis demolitor bracovirus (MdBV) and conducted functional experiments to assess whether BV genes share similar functions with related essential baculovirus replication genes. Our results indicate that several genes in MdBV retain ancestral functions, but select other genes have novel functions unknown from baculoviruses. Our results also provide the first experimental data on the function of polydnavirus replication genes and enhance understanding of the similarities between these viruses and their pathogenic ancestors.

Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients., Author Summary Genetic recombination is the alternative joining of nucleic acids leading to novel combinations of genetic information. While DNA recombination in cells is of importance for evolution and adaptive immunity, RNA recombination often has only transient effects. However, RNA viruses are rapidly evolving and recombination can be an important evolutionary step in addition to mutations introduced by the viral polymerase. Recombination can allow escape from the host immune system and from antiviral treatment, and recombination of live attenuated viral vaccines has led to re-emergence of disease. Hepatitis C virus (HCV) is an important human pathogen that chronically infects more than 130 million worldwide and leads to serious liver disease. For HCV, naturally occurring recombinants are rare but clinically important. HCV recombination constitutes a challenge to antiviral treatment and can potentially provide an escape mechanism for the virus. In this study, we established an assay for HCV RNA recombination and characterized the emerging homologous and non-homologous recombinant viruses. Interestingly, recombination did not depend on viral replication, occurred most efficiently between isolates of the same genotype and did not occur with strong site-specificity. Better diagnosis of clinically important recombinants and an increased knowledge on viral recombination could strengthen antiviral and vaccine development.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine., Author Summary Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.

Oncolytic viruses aim to specifically kill tumor cells. A major challenge is the effective targeting of disseminated tumors in vivo. We retargeted herpes simplex virus (HSV) tropism to HER-2 oncoprotein p185, overexpressed in ovary and breast cancers. The HER-2-retargeted R-LM249 exclusively infects and kills tumor cells expressing high levels of human HER-2. Here, we assessed the efficacy of systemically i.p. delivered R-LM249 against disseminated tumors in mouse models that recapitulate tumor spread to the peritoneum in women. The human ovarian carcinoma SK-OV-3 cells implanted intraperitoneally (i.p.) in immunodeficient Rag2−/−;Il2rg−/− mice gave rise to a progressive peritoneal carcinomatosis which mimics the fatal condition in advanced human patients. I.p. administration of R-LM249 strongly inhibited carcinomatosis, resulting in 60% of mice free from peritoneal diffusion, and 95% reduction in the total weight of neoplastic nodules. Intraperitoneal metastases are a common outcome in breast cancer: i.p. administration of R-LM249 strongly inhibited the growth of ovarian metastases of HER-2+ MDA-MB-453 breast cells. Brain metastases were also reduced. Cumulatively, upon i.p. administration the HER-2-redirected oncolytic HSV effectively reduced the growth of ovarian and breast carcinoma disseminated to the peritoneal cavity., Author Summary In the genome of human herpes simplex virus (HSV) we have replaced part of the sequences encoding the receptor-binding glycoprotein with antibody fragments directed against the oncoprotein HER-2, overexpressed in human breast and ovarian cancers. The retargeted HSV only infects and kills human cancer cells expressing high levels of HER-2. Earlier experiments showed that the retargeted HSV injected inside localized tumors inhibits human tumor growth in immunodeficient mice. As tumor dissemination is the major cause of cancer mortality, we have now used the HER-2-retargeted HSV to treat disseminated HER-2+ ovarian and breast cancer in immunodeficient mice. Intraperitoneal treatments significantly inhibited the peritoneal spread (carcinomatosis and ascites formation) of ovarian cancer cells and the metastatic growth of breast cancer cells in the ovaries. Brain metastases were also inhibited. Our results showed that a HER-2-redirected oncolytic HSV is an effective therapeutic agent against metastatic HER-2+ cancers and, more generally, provide the first demonstration of the efficacy of a systemically-administered, retargeted oncolytic HSV.

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets., Author Summary Hepatitis C virus (HCV) chronically infects about 170 million people worldwide and can ultimately lead to liver failure and liver cancer. HCV, like other RNA viruses, exploits cellular proteins and membranes to promote their own replication and virion production. In particular, HCV replication occurs at membranes derived from the endoplasmic reticulum, while HCV virion assembly is believed to occur at or near cellular lipid droplets. In this work, we report that Rab18, a lipid droplet-associated cellular protein, binds to the viral protein NS5A, and that the silencing of Rab18 reduces the association of other HCV replication complex components with lipid droplets. These data are consistent with a model in which Rab18 promotes the physical interaction between sites of viral replication to lipid droplets. We also speculate that Rab18 may help to link sites of viral replication to sites of virion assembly. Understanding how viruses exploit cellular proteins may result in new methods of disrupting viral infection.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV., Author Summary Schmallenberg virus (SBV) was discovered in Germany (near the town of Schmallenberg) in November 2011 and since then has been found to be the cause of malformations and stillbirths in ruminants. SBV has spread very rapidly to many European countries including the Netherlands, Belgium, France and the United Kingdom. Very little is known about the biological properties of this virus and there is no vaccine available. In this study (i) we developed an approach (called reverse genetics) that allows the recovery of “synthetic” SBV under laboratory conditions; (ii) we developed a mouse model of infection for SBV; (iii) we showed that SBV replicates in neurons of experimentally infected mice similar to naturally infected lambs and calves; (iv) we developed viral mutants that are not as pathogenic as the original virus due to the inability to counteract the host cell defenses; and v) we identified mutations that are associated with increased virulence. This work provides the experimental tools to understand how this newly emerged virus causes disease in ruminants. In addition, it will now be possible to manipulate the SBV genome in order to develop highly effective vaccines.

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14., Author Summary Enveloped viruses must develop strategies to ensure that a sufficient quantity of their receptor-binding envelope proteins are incorporated onto the surface of viruses as they form. The HIV envelope glycoprotein is specifically incorporated onto assembling virions in relevant cells such as T lymphocytes in a manner that requires its long cytoplasmic tail. The mechanism underlying this specific incorporation has remained unknown. Here, we identify a cellular trafficking pathway that is required for the incorporation of HIV envelope onto virions. A combination of the adaptor protein Rab11-FIP1C and Rab14 directs the envelope protein onto assembling virions, and loss of either of these host factors results in the production of virus particles lacking envelope. We also found that FIP1C was required for replication in T cell lines. This study identifies a trafficking complex required for HIV envelope incorporation and for the formation of infectious HIV particles.

Abel Viejo-Borbolla...et al., Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses., This work was funded by grants from the Spanish Ministry of Science and Innovation (SAF2009-07857), Wellcome Trust (051087/Z/97/Z) and Red Española de Esclerosis Múltiple (Instituto de Salud Carlos III, RD07/0060/0014). A.V.-B. has been supported by postdoctoral fellowships from the Instituto de Salud Carlos III and Consejo Superior de Investigaciones Científicas (Spanish Ministry of Science and Innovation). N.M.-M. is funded by a graduate fellowship from the Consejo Superior de Investigaciones Científicas (Spanish Ministry of Science and Innovation). PGF is supported by Science Foundation Ireland (07/IN.1/B902).

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication., Author Summary Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone in prokaryotes and eukaryotes, and regulates diverse cellular processes through ensuring the correct folding of numerous client proteins. However, there are no reports of direct interactions between Hsp90 with viral RNA. Here, we report that a new member of the Hsp90 proteins of Nicotiana benthamiana, NbHsp90, specifically interacts with the 3′ UTR of Bamboo mosaic virus (BaMV) genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA or that of other viruses. We further demonstrate that NbHsp90 specifically involves in the immediately early stage of BaMV RNA replication. NbHsp90 directly interacts with the BaMV 3′ UTR through the domain E, the key structural differences that distinguishes the BaMV 3′ UTR from the satBaMV 3′ UTR, which might contribute to NbHsp90's differential requirement for BaMV and satBaMV replication. Our work revealed a new role of Hsp90 in the interaction with RNA molecules, and demonstrated the differential requirement of Hsp90 in the replication of BaMV and satBaMV RNAs, which provide additional leverage for understanding the complex interactions between host, virus and its associated satellite RNA.

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ∼50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack., Author Summary There are many CNS diseases, including multiple sclerosis and amyotrophic lateral sclerosis, which have an inflammatory component, though no direct link has been established between incidence and a CNS-resident infectious agent. We reasoned that peripheral immunogens could play a role in CNS disease by inducing an immune response that is “mis-targeted” to the brain. This hypothesis was based on the immunological principle that, while education and activation of naïve cells is an antigen-driven process, recruitment is primarily antigen-independent. We developed a viral co-infection model using measles virus (MV) as a CNS activator and recruiting signal and lymphocytic choriomeningitis (LCMV) as a peripheral immune response initiator. Co-infection with both viruses resulted in significant morbidity and mortality, coincident with LCMV-specific CD8+ T cell trafficking to the brain. Death occurred due to edema, despite an apparent absence of LCMV antigens within the brain, and pathogenesis was CD8+ T cell-dependent, but perforin-independent. Thus, recruitment of peripherally activated CD8+ T cells to the CNS can potentiate neuroinflammation. This work raises the possibility that concomitant immune challenges may be an important cause of the neuroinflammation of some human CNS diseases, perhaps accounting for the inability to identify a discrete pathogenic trigger within affected brain tissues.

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog - gp180 - contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target., Author Summary Herpesvirus transmission between immune hosts implies some kind of antibody evasion. However, the underlying molecular mechanisms remain largely unknown. All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus (EBV) gp350. Gp350 binds EBV to B cells and provides a neutralization target. However, despite its immunogenicity, EBV carriers remain infectious. Here we show that the gp350 homolog of the related Bovine Herpesvirus 4 (BoHV-4), gp180, and its O-glycans, shield some otherwise vulnerable viral epitopes. Extensive O-glycosylation is common to all gammaherpesvirus gp350 homologs, suggesting that this evasion mechanism is also widespread.

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1 -/- mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1 -/- cells and mice. IRF-1 -/- mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1 -/- macrophages supported enhanced WNV replication but infection was unaltered in IRF-1 -/- fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8+ T cell expansion. Although markedly fewer CD8+ T cells were observed in naïve animals as described previously, remarkably, IRF-1 -/- mice rapidly expanded their pool of WNV-specific cytolytic CD8+ T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8+ T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8+ T cell response., Author Summary Interferon regulatory factor (IRF)-1 is a transcription factor that has been implicated in immune regulation and induction of type I IFN gene expression. To better understand the contribution of IRF-1 to antiviral immunity, we infected cells and mice lacking IRF-1 with West Nile virus (WNV), an encephalitic flavivirus. IRF-1 -/- mice were uniformly vulnerable to WNV infection with enhanced viral replication and rapid dissemination into the brain and spinal cord. Studies in cell culture revealed a cell-type specific antiviral role as IRF-1 -/- macrophages but not fibroblasts supported enhanced WNV replication. IRF-1 also had an independent effect on CD8+ T cell responses. Although fewer CD8+ T cells were observed in naïve animals, WNV-specific CD8+ T cells rapidly expanded in IRF-1 -/- mice and retained the capacity to clear infection. Collectively, our studies define independent roles for IRF-1 in restricting WNV pathogenesis and modulating the protective CD8+ T cell response.

Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans., Author Summary Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is associated with several cancers. Presently there is no licensed vaccine to prevent EBV diseases. Two types of candidate vaccines are under development; one involves immunization with the major glycoprotein (gp350) on the outside of the virus, while the other involves vaccination with EBV proteins expressed during latency. We compared these two types of candidate vaccines in a rhesus monkey model of EBV and found that the gp350 vaccine induced better protection from infection. In addition, animals that received the rhesus EBV glycoprotein and became infected had a lower level of rhesus EBV DNA in the blood at 23 months after challenge than animals that received the rhesus EBV latency protein vaccine that subsequently were infected. Since levels of EBV DNA in the blood have been predictive for EBV lymphomas in transplant patients, the ability of rhesus EBV gp350 to reduce levels of rhesus EBV in the blood after infection suggests the EBV gp350 could have a role in reducing certain EBV-associated cancers. This is the first test of candidate vaccines in the rhesus monkey model of EBV and shows that this model should be useful in further evaluation of EBV vaccines.

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses., PLoS Pathogens, 7(9), e1002215; 2011

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer., Author Summary The identification of cells that give origin to cancer is critical in order to design effective therapeutic strategies. To this end, the early stages of cancer are the most informative but they are seldom associated with clinical symptoms and therefore pass unnoticed in human patients. Studies on animal tumors are invaluable to this research area. In this study, we determined the cells originating an infectious lung cancer of sheep (ovine pulmonary adenocarcinoma, OPA) that is similar to some forms of human pulmonary adenocarcinoma. OPA is caused by a virus known as Jaagsiekte sheep retrovirus (JSRV). We show that OPA is caused by JSRV infection of proliferating type 2 pneumocytes (lung alveolar proliferating cells, LAPCs). We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection while healthy adult sheep exhibit a relatively low number of LAPCs and are resistant to OPA induction. However, adult sheep were susceptible to JSRV infection when the presence of LAPCs was stimulated by induction of a mild injury to the respiratory epithelium. Thus, our study identifies the cells originating lung adenocarcinoma in OPA and shows that inflammation to the respiratory epithelium can predispose to retrovirus infection and cancer.

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus., Author Summary Influenza virus is classified into types A, B and C. Influenza A virus is further divided into many subtypes, all of which exist in animals, indicating pandemic potential. By contrast, influenza B virus circulates almost exclusively in humans and, as there is no evidence for reassortment with influenza A virus, there is no indication of pandemic potential. Hence, there is far less accumulated research information regarding influenza B virus than influenza A virus. Influenza B virus, which is classified into two phylogenetic lineages, does, however, cause annual epidemics in humans and is therefore as essential to control as influenza A virus. Recently, the development of a universal vaccine and therapeutic strategies using human monoclonal antibodies (HuMAbs) has been gathering great interest. The present study reports a HuMAb neutralizing a wide range of influenza B viruses of both lineages. This HuMAb recognizes the conserved region of hemagglutinin. Moreover, therapeutic efficacy of this HuMAb was also confirmed by in vivo animal experiments. Thus, this study provides insight for the development of broad-spectrum therapeutics and a universal prophylactic vaccine against influenza B virus.

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution., Author Summary Influenza C viruses infect most humans during childhood. Unlike influenza A viruses, influenza C viruses exhibit little genetic variability and evolve at a comparably slower rate. Influenza A viruses exist as multiple subtypes and cause disease in numerous mammals. In contrast, influenza C viruses are comprised of a single subtype in its primary human host. Here we characterize a novel swine influenza virus, C/swine/Oklahoma/1334/2011 (C/OK), having only modest genetic similarity to human influenza C viruses. No cross-reaction was observed between C/OK and human influenza C viruses. Antibodies that cross react with C/OK were identified in a significant number of swine but not human sera samples, suggesting that C/OK circulates in pigs. Additionally, we show that C/OK is capable of infecting and transmitting by direct contact in both pigs and ferrets. These results suggest that C/OK represents a new subtype of influenza C viruses. This is significant, as co-circulation of multiple subtypes of influenza allows for rapid viral evolution through antigenic shift, a property previously only shown for influenza A viruses. The ability of C/OK to infect ferrets along with the absence of antibodies to C/OK in humans, suggests that such viruses may become a potential threat to human health.

Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses., Author Summary Hantaviruses are important human pathogens, occasionally emerging from animal reservoirs. However, both the biodiversity of hantaviruses in nature, as well as the frequency with which they have jumped species barriers in the past, are unclear. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) that were sampled from bats and shrews in China. These viruses are different from known hantaviruses, with each representing a novel species. An evolutionary analysis of all known hantaviruses including the novel viruses described here reveals the existence of four distinct phylogenetic groups of viruses that infect a range of mammalian hosts, and which have sometimes exchanged genes through segment reassortment. Our analysis also suggests that hantaviruses might have first appeared in bats or insectivores, before spreading to rodents, even though rodents are currently the best documented hosts of hantaviruses. Because the phylogenetic trees of the hantaviruses do not always match those of their mammalian hosts, we conclude that both host-jumping and co-divergence have played important roles in hantavirus evolution. Overall, our study shows that bats are likely to be important natural reservoir hosts of hantaviruses from which novel hantaviruses may emerge in the future.

Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5′ and 3′ untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5′-UTRs lack internal ribosomal entry site function. However, the 5′-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5′-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5′-UTR. The L 5′-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5′-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a “weak” Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10–100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target., Author Summary Filoviruses (Ebola and Marburg viruses) are emerging zoonotic pathogens that cause lethal hemorrhagic fever in humans and have the potential to be employed as bioterrorism agents. Currently, approved therapeutics to treat filovirus infections are not available and new treatment strategies could be facilitated by improved mechanistic insight into the virus replication cycle. Compared to other related viruses, filovirus messenger RNAs have unusually long 5′ untranslated regions (UTRs) with undefined functions. In the Zaire ebolavirus (EBOV) genome, four of its seven messenger RNAs have 5′-UTRs with a small upstream open reading frame (uORF). We found that a uORF present in the EBOV polymerase (L) 5′-UTR suppresses L protein production and established a reporter assay to demonstrate that this uORF maintains L translation following the induction of an innate immune response; a phenomenon observed with several uORF-containing cellular messenger RNAs. The presence of the uORF is important for optimal virus replication, because a mutant virus lacking the upstream reading frame replicates less efficiently than a wildtype virus, an attenuation which is more pronounced following the induction of cellular stress. These studies define a novel mechanism by which filovirus upstream open reading frames modulate virus protein translation in the face of an innate immune response and highlight their importance in filovirus replication.

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production., Author Summary Influenza viruses cause annual epidemics and occasionally, major global pandemics. To establish productive infection these viruses have mechanisms to evade host immune responses, including the type-I interferon (IFN) response. An important component of the IFN system is the helicase RIG-I that recognizes viral RNA, and is subsequently ubiquitinated by TRIM25 ubiquitin E3 ligase to induce downstream signaling resulting in IFN-α/β production. The NS1 protein of influenza A viruses binds to human TRIM25 and inhibits TRIM25-dependent RIG-I ubiquitination and downstream RIG-I signaling. An important unresolved question is how viruses can inhibit the RIG-I pathway when infecting new hosts. Here we show that while human TRIM25 is able to bind to different NS1 proteins, chicken TRIM25 binds preferentially to the NS1 from an avian virus. Strikingly, mouse TRIM25 was unable to bind NS1. We found that NS1 blocks RIG-I signaling in mouse and human cells by different mechanisms. While NS1 inhibits human TRIM25-mediated RIG-I ubiquitination, in mouse cells NS1 suppresses RIG-I signaling by binding to and inhibiting the ubiquitin E3 ligase Riplet. These results help understand the immune evasion strategies used by influenza virus in different species, and may partly explain the ability of this virus to adapt to different host species.

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study., Author Summary The ability of arboviruses to perpetuate in nature given that they must infect two disparate hosts (the mosquito vector and the vertebrate host) remains a mystery. We studied how viral genetic diversity is impacted by the dual host transmission cycle. Our studies of an enzootic cycle using Venezuelan equine encephalitis virus (VEEV) and its natural mosquito, Culex taeniopus, revealed the stages of infection that result in a viral population bottleneck. Using a set of marked VEEV clones and repeated sampling at various time points following C. taeniopus infection, we determined the number of clones in various mosquito tissues culminating in transmission. Bottlenecks were identified but the stage of occurrence was dependent on the dose that initiated infection. Understanding the points at which mosquito-borne viruses are constrained will shed light on the ways in which virus diversity varies, leading to selection of mutants that may result in host range changes or alterations in virulence.

Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases., Author Summary Influenza A virus, a negative stranded RNA, can cause severe illness in humans and animals and stimulates many receptors including Toll like receptors 7 (TLR7). TLR signaling induces maturation of dendritic cells and the production of a variety of inflammatory cytokines that are crucial for both innate and adaptive immunity. TLR7 is an intracellular receptor, which resides in endosomes and senses viruses to trigger host defence. Previous data have shown that TLR9 requires proteolysis to be functional but it is unclear whether other intracellular TLRs (TLR3 and TLR7) are also subject to degradation. Here, we used a protease deficient mouse model to show the in vivo importance of TLR7 processing in influenza infection. Inflammation monitored by cytokine release and adaptive immunity measured by cross priming of CD8+ T cells was significantly reduced in infected protease-deficient animals in comparison to control animals. We showed that TLR7 requires a proteolytic cleavage by a cysteine endopeptidase in order to be functional. Our findings indicate that TLR7 processing mediated by a protease, asparagynil endopeptidase, is critical for inducing robust anti-influenza immune responses. Given our results, targeting TLR7 response in the lungs through proteases may offer new therapeutic potential in pulmonary infection.

Textbooks define viruses as infectious agents with nucleic acid genomes (RNA or DNA), which replicate inside living host cells to produce particles (virions) that can transfer the genome to other cells [1], [2]. The Polydnaviridae was recognized as a family of viruses in 1995, and is currently divided into two genera named the Bracovirus and Ichnovirus [3]. Polydnavirus (PDV) virions consist of enveloped nucleocapsids and package multiple circular, double-stranded (ds) DNAs with aggregate sizes that range from 190 to more than 500 kbp [4]. PDVs are also strictly associated with insects called parasitoid wasps (Hymenoptera), which are free living nectar feeders as adults but which develop during their immature stages by feeding inside the body of another insect (the host) [3], [4]. Recent studies, however, indicate that PDVs differ from all other known viruses in ways that challenge traditional views of what viruses are and how they function.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-κB (NF-κB) transcription factors, and inhibitor κB (IκB) proteins that complex with and regulate NF-κBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IκBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IκB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IκBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IκB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and –N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IκB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and –N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-κB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IκB binding affinities, while also showing that viral mimics disable Relish processing., Author Summary Central to the study of host-pathogen interactions is understanding how the immune system of hosts responds to infection, and reciprocally how pathogens subvert host defenses. In the case of insects, understanding of how the immune system responds to infection greatly exceeds understanding of pathogen counterstrategies. Parasitoid wasps are key mortality agents of insects. Thousands of wasp species have also evolved a symbiotic relationship with large DNA viruses in the family Polydnaviridae whose primary function is to deliver immunosuppressive virulence genes to the insect hosts that wasps parasitize. The function of most PDV-encoded virulence genes, however, remains unknown. In this article, we investigated the function of two ank gene family members from Microplitis demolitor bracovirus (MdBV). Our results indicate that Ank-H4 and Ank-N5 function as mimics of IκB proteins, which regulate a family of transcription factors called NF-κBs that control many genes of the insect immune system. IκBs and NF-κBs also function as key regulators of the mammalian immune system. Our results thus suggest that viral Ank proteins subvert the immune system of host insects by targeting conserved signaling pathways used by a diversity of organisms.

Rates of evolution span orders of magnitude among RNA viruses with important implications for viral transmission and emergence. Although the tempo of viral evolution is often ascribed to viral features such as mutation rates and transmission mode, these factors alone cannot explain variation among closely related viruses, where host biology might operate more strongly on viral evolution. Here, we analyzed sequence data from hundreds of rabies viruses collected from bats throughout the Americas to describe dramatic variation in the speed of rabies virus evolution when circulating in ecologically distinct reservoir species. Integration of ecological and genetic data through a comparative Bayesian analysis revealed that viral evolutionary rates were labile following historical jumps between bat species and nearly four times faster in tropical and subtropical bats compared to temperate species. The association between geography and viral evolution could not be explained by host metabolism, phylogeny or variable selection pressures, and instead appeared to be a consequence of reduced seasonality in bat activity and virus transmission associated with climate. Our results demonstrate a key role for host ecology in shaping the tempo of evolution in multi-host viruses and highlight the power of comparative phylogenetic methods to identify the host and environmental features that influence transmission dynamics., Author Summary Rapid evolution of RNA viruses is intimately linked to their success in overcoming the defenses of their hosts. Several studies have shown that rates of viral evolution can vary dramatically among distantly related viral families. Variability in the speed of evolution among closely related viruses has received less attention, but could be an important determinant of the geographic or host species origins of viral emergence if certain species or regions promote especially rapid evolution. Here, using a dataset of rabies virus sequences collected from bat species throughout the Americas, we test the role of inter-specific differences in reservoir host biology on the tempo of viral evolution. We show the annual rate of molecular evolution to be a malleable trait of viruses that is accelerated in subtropical and tropical bats compared to temperate species. The association between geography and the speed of evolution appears to reflect differences in the seasonality of rabies virus transmission in different climatic zones. Our results illustrate that the viral mechanisms that are commonly invoked to explain heterogeneous rates of evolution among viral families may be insufficient to explain evolution in multi-host viruses and indicate a role for host biology in shaping the speed of viral evolution.

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 −/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD50 values in Ifit1 −/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 −/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8+ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types., Author Summary We recently showed that a West Nile virus (WNV) mutant in NS5 (WNV-E218A) lacking 2′-O methyltransferase activity was attenuated in primary macrophages but replicated well in cells lacking type I interferon (IFN) signaling or expression of Ifit1, an IFN-stimulated gene. Here, we follow-up these studies by examining the pathogenesis in Ifit1−/− mice of WNV-E218A, the mutant virus lacking 2′-O methyltransferase activity. Because a deficiency of Ifit1 did not alter pathogenesis of wild type WNV, we conclude that the viral 2′-O methyltransferase encoded by NS5 largely overcomes Ifit1-mediated control of infection. In comparison, WNV-E218A showed increased infection in peripheral tissues of Ifit1 −/− mice after subcutaneous infection, yet this did not result in enhanced replication in the brain. However, WNV-E218A caused lethal infection when it was directly introduced into the brain. We observed cell-type specific differences in the ability of an Ifit1 deficiency to rescue replication of WNV-E218A; for example, WNV-E218A showed equivalently impaired infection in wild type and Ifit1 −/− brain endothelial cells, which potentially allow virus access into the brain. Our results suggest that virulent strains of WNV have evaded the antiviral effects of Ifit1, and mutants lacking 2′-O methylation are restricted by cell-type specific Ifit1-dependent and -independent mechanisms.