Indirect CD4+ T cell protection against mouse gamma-herpesvirus infection via interferon gamma.

CD4+ T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4+ T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4+ T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4+ T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c+ cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII+ CD11c+ antigen-presenting cells is key to the CD4+ T cell/NK cell protection axis. By contrast, CD8+ T cell control of AEC1 infection appeared to operate independently. IMPORTANCE: CD4+ T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4+ T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c+ dendritic cells. This mechanism of CD4+ T cell control releases the need for the direct engagement of CD4+ T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Open reading frames M12/M13 jointly contribute to MHV-68 latency.

Murine gammaherpesvirus 68 (MHV-68), a widely used small-animal model for the analysis of gammaherpesvirus pathogenesis, encodes the MHV-68-specific ORFs M12 and M13. The function of M12 and M13 has not been investigated so far. Therefore, we constructed and analysed recombinant MHV-68 with mutations in either M12, M13 or M12/M13. Both the M12 and M13 mutants did not display any phenotype in vitro or in vivo. However, although the M12/13 double mutant showed similar lytic growth in fibroblasts in vitro and in the lungs of infected mice as wild-type MHV-68, it was significantly attenuated in vivo during latency. This phenotype was completely restored in a revertant of the M12/13 double mutant. Thus, it appears that M12 and M13 might have redundant functions that are only revealed if both genes are lacking. The observation that M12/13 have a function during latency not only contributes to the further understanding of the pathogenesis of MHV-68 infection but might also be of interest considering that M12/13 are located at a genomic position similar to that of LMP2A and K15. The latter are important proteins of their respective human gammaherpesviruses EBV and KSHV that contribute to cellular survival, cell activation and proliferation, which was deduced from in vitro studies.

Amino acids 1811-1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection.

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

Rewiring of the Host Cell Metabolome and Lipidome during Lytic Gammaherpesvirus Infection Is Essential for Infectious-Virus Production.

Oncogenic virus infections are estimated to cause ~15% of all cancers. Two prevalent human oncogenic viruses are members of the gammaherpesvirus family: Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV). We use murine herpesvirus 68 (MHV-68), which shares significant homology with KSHV and EBV, as a model system to study gammaherpesvirus lytic replication. Viruses implement distinct metabolic programs to support their life cycle, such as increasing the supply of lipids, amino acids, and nucleotide materials necessary to replicate. Our data define the global changes in the host cell metabolome and lipidome during gammaherpesvirus lytic replication. Our metabolomics analysis found that MHV-68 lytic infection induces glycolysis, glutaminolysis, lipid metabolism, and nucleotide metabolism. We additionally observed an increase in glutamine consumption and glutamine dehydrogenase protein expression. While both glucose and glutamine starvation of host cells decreased viral titers, glutamine starvation led to a greater loss in virion production. Our lipidomics analysis revealed a peak in triacylglycerides early during infection and an increase in free fatty acids and diacylglyceride later in the viral life cycle. Furthermore, we observed an increase in the protein expression of multiple lipogenic enzymes during infection. Interestingly, pharmacological inhibitors of glycolysis or lipogenesis resulted in decreased infectious virus production. Taken together, these results illustrate the global alterations in host cell metabolism during lytic gammaherpesvirus infection, establish essential pathways for viral production, and recommend targeted mechanisms to block viral spread and treat viral induced tumors. IMPORTANCE Viruses are intracellular parasites which lack their own metabolism, so they must hijack host cell metabolic machinery in order to increase the production of energy, proteins, fats, and genetic material necessary to replicate. Using murine herpesvirus 68 (MHV-68) as a model system to understand how similar human gammaherpesviruses cause cancer, we profiled the metabolic changes that occur during lytic MHV-68 infection and replication. We found that MHV-68 infection of host cells increases glucose, glutamine, lipid, and nucleotide metabolic pathways. We also showed inhibition or starvation of glucose, glutamine, or lipid metabolic pathways results in an inhibition of virus production. Ultimately, targeting changes in host cell metabolism due to viral infection can be used to treat gammaherpesvirus-induced cancers and infections in humans.

Murine herpesvirus-68-related growth factors treatment correlates with decrease of p53 and HIF-1α protein levels.

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.

Dampening type 2 properties of group 2 innate lymphoid cells by a gammaherpesvirus infection reprograms alveolar macrophages.

Immunological dysregulation in asthma is associated with changes in exposure to microorganisms early in life. Gammaherpesviruses (γHVs), such as Epstein-Barr virus, are widespread human viruses that establish lifelong infection and profoundly shape host immunity. Using murid herpesvirus 4 (MuHV-4), a mouse γHV, we show that after infection, lung-resident and recruited group 2 innate lymphoid cells (ILC2s) exhibit a reduced ability to expand and produce type 2 cytokines in response to house dust mites, thereby contributing to protection against asthma. In contrast, MuHV-4 infection triggers GM-CSF production by those lung ILC2s, which orders the differentiation of monocytes (Mos) into alveolar macrophages (AMs) without promoting their type 2 functions. In the context of γHV infection, ILC2s are therefore essential cells within the pulmonary niche that imprint the tissue-specific identity of Mo-derived AMs and shape their function well beyond the initial acute infection.

Potential outbreak by herpesvirus in equines: detection, clinical, and genetic analysis of equid gammaherpesvirus 2 (EHV-2).

BACKGROUND: Equid herpesvirus (EHV) commonly affects horses causing neurologic and respiratory symptoms beside spontaneous abortions, meaning huge economic losses for equine industry worldwide. In foals, the virus can facilitate secondary infections by Rhodococcus equi, important in morbidity and mortality in equines. A total of five genotypes of EHV were previously described in Brazil including EHV-1, EHV-2, EHV-3, EHV-4, and EHV-5. EHV-2 genotype had only been previously described in Brazil in asymptomatic animals. We report the investigation of the dead of 11 foals in Middle-west region of Brazil showing respiratory and neurological symptoms, as well as several abortions in mares from the same farm. METHODS: Clinical and laboratory exams were performed in this case study. Lung, whole blood, serum, and plasma samples were analyzed by necroscopic and histopathologic techniques followed by molecular assays (conventional and qPCR and Sanger sequencing). RESULTS AND CONCLUSION: Laboratory exams revealed neutrophilia leukocytosis. Necroscopic and histopathologic findings were suppurative bronchopneumonia and ulcerative enteritis. Molecular assays point to the absence of the bacteria Rhodococcus equi and other viruses (including other EHV). The presence of EHV-2 DNA was confirmed by sequencing in serum sample from one foal. This is the first confirmed outbreak of EHV-2 causing disease in Brazilian horses with confirmed presence of the virus, and which highlight the important role of EHV-2 in equine respiratory disease and spontaneous abortions in equid in Brazil.

Horizontal transmission of endemic viruses among rhesus macaques (Macaca mulatta): Implications for human cytomegalovirus vaccine/challenge design.

INTRODUCTION: Rhesus macaques are natural hosts to multiple viruses including rhesus cytomegalovirus (RhCMV), rhesus rhadinovirus (RRV), and Simian Foamy Virus (SFV). While viral infections are ubiquitous, viral transmissions to uninfected animals are incompletely defined. Management procedures of macaque colonies include cohorts that are Specific Pathogen Free (SPF). Greater understanding of viral transmission would augment SPF protocols. Moreover, vaccine/challenge studies of human viruses would be enhanced by leveraging transmission of macaque viruses to recapitulate expected challenges of human vaccine trials. MATERIALS AND METHODS: This study characterizes viral transmissions to uninfected animals following inadvertent introduction of RhCMV/RRV/SFV-infected adults to a cohort of uninfected juveniles. Following co-housing with virus-positive adults, juveniles were serially evaluated for viral infection. RESULTS: Horizontal viral transmission was rapid and absolute, reaching 100% penetrance between 19 and 78 weeks. CONCLUSIONS: This study provides insights into viral natural histories with implications for colony management and modeling vaccine-mediated immune protection studies.

The Interaction between Tegument Proteins ORF33 and ORF45 Plays an Essential Role in Cytoplasmic Virion Maturation of a Gammaherpesvirus.

Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.

Molecular detection and assessment of the epidemiological risk factors associated with equine herpesvirus 2 and 5 in working equids in central Ethiopia.

BACKGROUND: Respiratory disease is the most common presenting complaint at veterinary clinics and a priority concern for equid owners and veterinary practitioners in Ethiopia. OBJECTIVES: This study aimed to report the molecular detection of EHV-2 and EHV-5 and to assess the risk factors associated with infection in working equids in central Ethiopia. METHODS: Nasopharyngeal swabs were collected from 58 horses and donkeys to detect EHV-2 and EHV-5 using PCR targeting the conserved region of glycoprotein B (gB) genes. RESULTS: From 58 equids, EHV-5 and EHV-2 were detected in 20 (34.5%) and 19 (32.8%) equids, respectively. Concurrent infection with EHV-2 and EHV-5 was found in 6 (10.3%) equids who exhibited respiratory clinical signs. EHV-2 was detected in a significantly higher (p = 0.002) proportion of horses (54.5%; n = 18) than donkeys (4%; n = 1). In contrast, EHV-5 was detected in a significantly higher (p = 0.004) proportion of donkeys (56%; n = 14) compared to horses (18.2% n = 6). EHV-2-positive equids were seven times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (Odds ratio (OR) = 6.9; 95%CI: 1.72-27.60). However, statistically significant (p = 0.832) difference was not observed for EHV-5. EHV-2 was detected in a significantly higher (p = 0.004) proportion of female (50%; n = 16) compared to male equids (11.5%; n = 3). CONCLUSIONS: This study revealed the molecular detection of EHV-2 and EHV-5 in horses and donkeys residing in central Ethiopia. The association between EHV-2-test-positive equids and displaying of clinical signs of respiratory disease was observed, which suggests EHV-2 involvement in the development of respiratory disease; however, it deserves further investigation.

The Antagonism between the Murine Gammaherpesvirus Protein Kinase and Global Interferon Regulatory Factor 1 Expression Shapes the Establishment of Chronic Infection.

Gammaherpesviruses infect most vertebrate species and are associated with B cell lymphomas. Manipulation of B cell differentiation is critical for natural infection and lymphomagenesis driven by gammaherpesviruses. Specifically, human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) drive differentiation of infected naive B cells into the germinal center to achieve exponential increase in the latent viral reservoir during the establishment of chronic infection. Infected germinal center B cells are also the target of viral lymphomagenesis, as most EBV-positive B cell lymphomas bear the signature of the germinal center response. All gammaherpesviruses encode a protein kinase, which, in the case of Kaposi's sarcoma-associated herpesvirus (KSHV) and MHV68, is sufficient and necessary, respectively, to drive B cell differentiation in vivo. In this study, we used the highly tractable MHV68 model of chronic gammaherpesvirus infection to unveil an antagonistic relationship between MHV68 protein kinase and interferon regulatory factor 1 (IRF-1). IRF-1 deficiency had minimal effect on the attenuated lytic replication of the kinase-null MHV68 in vivo. In contrast, the attenuated latent reservoir of the kinase-null MHV68 was partially to fully rescued in IRF-1-/- mice, along with complete rescue of the MHV68-driven germinal center response. Thus, the novel viral protein kinase-IRF-1 antagonism was largely limited to chronic infection dominated by viral latency and was less relevant for lytic replication during acute infection and in vitro. Given the conserved nature of the viral and host protein, the antagonism between the two, as defined in this study, may regulate gammaherpesvirus infection across species. IMPORTANCE Gammaherpesviruses are prevalent pathogens that manipulate physiological B cell differentiation to establish lifelong infection. This manipulation is also involved in gammaherpesvirus-driven B cell lymphomas, as differentiation of latently infected B cells through the germinal center response targets these for transformation. In this study, we define a novel antagonistic interaction between a conserved gammaherpesvirus protein kinase and a host antiviral and tumor suppressor transcription factor. The virus-host antagonism unveiled in this study was critically important to shape the magnitude of gammaherpesvirus-driven germinal center response. In contrast, the virus-host antagonism was far less relevant for lytic viral replication in vitro and during acute infection in vivo, highlighting the emerging concept that nonoverlapping mechanisms shape the parameters of acute and chronic gammaherpesvirus infection.

T Cell-Specific STAT1 Expression Promotes Lytic Replication and Supports the Establishment of Gammaherpesvirus Latent Reservoir in Splenic B Cells.

Gammaherpesviruses establish lifelong infections in most vertebrate species, including humans and rodents, and are associated with cancers, including B cell lymphomas. While type I and II interferon (IFN) systems of the host are critical for the control of acute and chronic gammaherpesvirus infection, the cell type-specific role(s) of IFN signaling during infection is poorly understood and is often masked by the profoundly altered viral pathogenesis in the hosts with global IFN deficiencies. STAT1 is a critical effector of all classical IFN responses along with its involvement in other cytokine signaling pathways. In this study, we defined the effect of T cell-specific STAT1 deficiency on the viral and host parameters of infection with murine gammaherpesvirus 68 (MHV68). MHV68 is a natural rodent pathogen that, similar to human gammaherpesviruses, manipulates and usurps B cell differentiation to establish a lifelong latent reservoir in B cells. Specifically, germinal center B cells host the majority of latent MHV68 reservoir in the lymphoid organs, particularly at the peak of viral latency. Unexpectedly, T cell-specific STAT1 expression, while limiting the overall expansion of the germinal center B cell population during chronic infection, rendered these B cells more effective at hosting the latent virus reservoir. Further, T cell-specific STAT1 expression in a wild type host limited circulating levels of IFNγ, with corresponding increases in lytic MHV68 replication and viral reactivation. Thus, our study unveils an unexpected proviral role of T cell-specific STAT1 expression during gammaherpesvirus infection of a natural intact host. IMPORTANCE Interferons (IFNs) represent a major antiviral host network vital to the control of multiple infections, including acute and chronic gammaherpesvirus infections. Ubiquitously expressed STAT1 plays a critical effector role in all classical IFN responses. This study utilized a mouse model of T cell-specific STAT1 deficiency to define cell type-intrinsic role of STAT1 during natural gammaherpesvirus infection. Unexpectedly, T cell-specific loss of STAT1 led to better control of acute and persistent gammaherpesvirus replication and decreased establishment of latent viral reservoir in B cells, revealing a surprisingly diverse proviral role of T cell-intrinsic STAT1.

Frequency of Detection of Respiratory Pathogens in Nasal Secretions From Healthy Sport Horses Attending a Spring Show in California.

The objective of this study was to determine detection frequency of respiratory viruses (equine influenza virus [EIV], equine herpesvirus-1 [EHV-1], EHV-2, EHV-4, EHV-5, equine rhinitis A virus [ERAV], ERBV) and bacteria (Streptococcus equi ss. equi[S. equi], S. equi ss. zooepidemicus[S. zooepidemicus]) in 162 nasal secretions and 149 stall swabs from healthy sport horses attending a spring show in California. Nasal and stall swabs were collected at a single time point and analyzed using qPCR. The detection frequency of respiratory pathogens in nasal secretions was 38.9% for EHV-2, 36.4% for EHV-5, 19.7% for S. zooepidemicus, 1.2% for ERBV, 0.6% for S. equi and 0% for EIV, EHV-1, EHV-4 and ERAV. The detection frequency of respiratory pathogens in stall swabs was 65.8% for S. zooepidemicus, 33.5% for EHV-2, 27.5% for EHV-5, 3.3% for EHV-1, 1.3% for EHV-4 and 0% for EIV, ERAV, ERBV and S. equi. Commensal viruses and bacteria were frequently detected in nasal secretions and stall swabs from healthy sport horses. This was in sharp contrast to the subclinical shedding of well-characterized respiratory pathogens. Of interest was the clustering of five EHV-1 qPCR-positive stalls from apparently healthy horses with no evidence of clinical spread. The results highlight the role of subclinical shedders in introducing respiratory pathogens to shows and their role in environmental contamination. The results also highlight the need to improve cleanliness and disinfection of stalls utilized by performance horses during show events.

A Polymorphism in the Epstein-Barr Virus EBER2 Noncoding RNA Drives In Vivo Expansion of Latently Infected B Cells.

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are associated with numerous malignancies, including B cell lymphomas and nasopharyngeal carcinoma. These viruses employ numerous molecular strategies to colonize the host, including the expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2, respectively) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. In work here, we used chimeric MHV68 viruses in an in vivo complementation system to test whether EBV EBER2 contributes to acute and/or chronic phases of infection. Expression of EBER2 derived from EBV strain B95-8 resulted in a significant expansion of latently infected B cells in vivo, which was accompanied by a decrease in virus-infected plasma cells. EBV strains typically carry one of two variants of EBER2, which differ primarily by a 5-nucleotide core polymorphism identified initially in the EBV strain M81. Strikingly, mutation of the 5 nucleotides that define this core polymorphism resulted in the loss of the infected B cell expansion and restored plasma cell infection. This work reveals that the B95-8 variant of EBER2 promotes the expansion of the latently infected B cell pool in vivo and may do so in part through inhibition of terminal differentiation. These findings provide new insight into mechanisms by which viral ncRNAs promote in vivo colonization and further and provide further evidence of the inherent tumorigenic risks associated with gammaherpesvirus manipulation of B cell differentiation. IMPORTANCE The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68, employ numerous strategies to colonize the host, including expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs ever identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. Work here reveals that an EBV EBER2 variant highly associated with B cell lymphoma promoted a significantly increased expansion of the infected B cell pool in vivo, which coincided with altered B cell differentiation. Mutation of the 5 nucleotides that define this EBER2 variant resulted in the loss of B cell expansion and normal B cell differentiation. These findings provide new insight into the mechanisms by which EBV manipulates B cells in vivo to retain infected cells in the high-risk B cell differentiation pathway where they are poised for tumorigenesis.

T Cell-Intrinsic Interleukin 17 Receptor A Signaling Supports the Establishment of Chronic Murine Gammaherpesvirus 68 Infection.

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), are species-specific, ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a unique and robust polyclonal germinal center response to establish a long-term latent reservoir in memory B cells. The robust polyclonal germinal center response driven by gammaherpesvirus infection increases the risk for B cell transformation. Unsurprisingly, many gammaherpesvirus cancers are derived from germinal center or post-germinal center B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined. We previously showed that host interleukin 17 receptor A (IL-17RA) signaling promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we found that T cell-intrinsic IL-17RA signaling recapitulates some proviral aspects of global IL-17RA signaling during MHV68 infection. Specifically, we found that T cell-intrinsic IL-17RA signaling supports the MHV68-driven germinal center response, the establishment of latency in the spleen, and viral reactivation in the spleen and peritoneal cavity. Our study unveils an unexpected finding where the T cell-specific IL-17RA signaling supports the establishment of a latent reservoir of a B cell-tropic gammaherpesvirus. IMPORTANCE Gammaherpesviruses, such as human EBV, establish lifelong infection in >95% of adults and are associated with B cell lymphomas. Gammaherpesviruses usurp the germinal center response to establish latent infection, and the germinal center B cells are thought to be the target of viral transformation. We previously found that global expression of IL-17RA promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we showed that T cell-intrinsic IL-17RA signaling is necessary to promote the MHV68-driven germinal center response by supporting CD4+ T follicular helper cell expansion. We also found that T cell-intrinsic IL-17RA signaling contributes to but is not solely responsible for the systemic proviral role of IL-17RA signaling, highlighting the multifaceted function of IL-17RA signaling during MHV68 infection.

Kinetics of the Equid Herpesvirus 2 and 5 Infections among Mares and Foals from Three Polish National Studs.

Equid herpesvirus 2 (EHV-2) and 5 (EHV-5) are two γ-herpesviruses that are commonly detected from horses worldwide, based on several cross-sectional molecular surveys. Comparatively few studies examined the dynamics of γ-herpesvirus infection over time in a group of horses. The aim of the current study was to investigate the dynamics of EHV-2/5 infections among mares and their foals at three Polish national studs with different breeds of horses: Arabians, Thoroughbreds and Polish Konik horses. Nasal swabs were collected from each of 38 mare-foal pairs monthly for a period of 6 to 8 months. Virus-specific quantitative PCR assays were used to determine the viral load of EHV-2 and EHV-5 in each sample. All 76 horses sampled were positive for EHV-2 or EHV-5 on at least one sampling occasion. The majority (73/76, 96%) were infected with both EHV-2 and EHV-5. In general, the mean load of viral DNA was higher in samples from foals than from mares, but similar for EHV-2 and EHV-5 at most sampling occasions. There was, however, a considerable variability in the viral DNA load between samples collected at different times from the same foal, as well as between samples from different foals. The latter was more apparent for EHV-2 than for EHV-5. All foals became infected with both viruses early in life, before weaning, and remained positive on all, or most, subsequent samplings. The virus shedding by mares was more intermittent, indicating the existence of age-related differences. Overall, the data presented extend our knowledge of EHV-2/5 epidemiology among mares and foals.