Targeting the MR1-MAIT cell axis improves vaccine efficacy and affords protection against viral pathogens.

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. Additionally, MAIT cells sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unclear. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster virus-specific CD8+ T cell responses, and potentiate heterosubtypic anti-influenza protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency and Toll-like receptor 3 and type I interferon receptor signaling. The observed phenomenon was reproducible in female and male mice, and in both young and old animals. It could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

Correction: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

[This corrects the article DOI: 10.1371/journal.ppat.1010092.].

A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.

Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

BACKGROUND: Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. RESULTS: Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-ß, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. CONCLUSIONS: Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype.

Novel gammaherpesviruses in North American domestic cats, bobcats, and pumas: identification, prevalence, and risk factors.

UNLABELLED: Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus), and puma (Puma concolor) blood cell DNA samples from California, Colorado, and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4-kb region of each putative virus species, including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology. IMPORTANCE: Gammaherpesviruses (GHVs) establish lifelong infection in many animal species and can cause cancer and other diseases in humans and animals. In this study, we identified the DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus), and pumas (Puma concolor; also known as mountain lions, cougars, and panthers). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified to be native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the United States. In contrast, puma virus was found only in a specific region of Southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to the route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Detection of Felis catus Gammaherpesvirus 1 in Domestic Cat Saliva: Prevalence, Risk Factors, and Attempted Virus Isolation.

Felis catus gammaherpesvirus 1 (FcaGHV1) infects domestic cats worldwide, yet it has not been successfully propagated in cell culture, and little is known about how it is shed and transmitted. To investigate the salivary shedding of FcaGHV1, we quantified FcaGHV1 DNA in feline saliva by qPCR. For FcaGHV1-positive saliva, we sequenced a portion of the viral glycoprotein B (gB) gene and attempted to isolate the infectious virus by passage in several felid and non-felid cell lines. We detected FcaGHV1 DNA in 45/227 (19.8%) saliva samples with variable viral DNA loads from less than 100 to greater than 3 million copies/mL (median 4884 copies/mL). Multiple saliva samples collected from an infected cat over a two-month period were consistently positive, indicating that chronic shedding can occur for at least two months. Cat age, sex, and health status were not associated with shedding prevalence or viral DNA load in saliva. Feral status was also not associated with shedding prevalence. However, feral cats had significantly higher FcaGHV1 DNA load than non-feral cats. Sequencing of FcaGHV1 gB showed low sequence diversity and >99.5% nucleotide identity to the worldwide consensus FcaGHV1 gB sequence. We did not detect virus replication during the passage of FcaGHV1-positive saliva in cell culture, as indicated by consistently negative qPCR on cell lysate and supernatant. To our knowledge, these data show for the first time that cats in Canada are infected with FcaGHV1. The data further suggest that shedding of FcaGHV1 in saliva is common, can occur chronically over an extended period of time, and may occur at higher levels in feral compared to non-feral cats.

Monovalent and trivalent VSV-based COVID-19 vaccines elicit neutralizing antibodies and CD8+ T cells against SARS-CoV-2 variants.

Recombinant vesicular stomatitis virus (rVSV) vaccines expressing spike proteins of Wuhan, Beta, and/or Delta variants of SARS-CoV-2 were generated and tested for induction of antibody and T cell immune responses following intramuscular delivery to mice. rVSV-Wuhan and rVSV-Delta vaccines and an rVSV-Trivalent (mixed rVSV-Wuhan, -Beta, -Delta) vaccine elicited potent neutralizing antibodies (nAbs) against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) viruses. Prime-boost vaccination with rVSV-Beta was less effective in this capacity. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with the rVSV-Trivalent vaccine consistently effective in inducing nAbs against all the SARS-CoV-2 variants tested. All vaccines, including rVSV-Beta, elicited a spike-specific immunodominant CD8+ T cell response. Collectively, rVSV vaccines targeting SARS-CoV-2 variants of concern may be considered in the global fight against COVID-19.

Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.

Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.

Immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of Burkholderia pseudomallei infection.

Burkholderia pseudomallei is a soil bacterium that is endemic in southeast Asia and northern Australia and that can cause both acutely lethal pneumonia and chronic systemic infections in humans. The effective treatment of infection with B. pseudomallei requires rapid diagnosis and prolonged treatment with high doses of antimicrobials, and even with appropriate antibiotic therapy, patient relapses are common. Thus, new approaches to the treatment of B. pseudomallei infections are needed. In the present study, we asked whether active immunotherapy with gamma interferon (IFN-gamma), a key cytokine regulating the intracellular replication of B. pseudomallei, could increase the effectiveness of conventional antimicrobial therapy for B. pseudomallei infection. Macrophage infection assays and in vivo pulmonary challenge models were used to assess the inhibitory effects of combined treatment with IFN-gamma and ceftazidime on B. pseudomallei infection. We found that treatment with even very low doses of IFN-gamma and ceftazidime elicited strong synergistic inhibition of B. pseudomallei growth within infected macrophages. In vivo, active immunotherapy markedly potentiated the effectiveness of low-dose ceftazidime therapy for the treatment of infected mice in a pulmonary challenge model of B. pseudomallei. Combined treatment was associated with a significant reduction in the bacterial burden and a significant lessening of bacterial dissemination. We concluded, therefore, that immunotherapy with either endogenous or exogenous IFN-gamma could significantly increase the effectiveness of conventional antimicrobial therapy for the treatment of acute B. pseudomallei infection.

Calculating HIV-1 infectious titre using a virtual TCID(50) method.

Studies of HIV-1 replication kinetics and fitness require an accurate determination of the level of infectious HIV-1 present in virus stocks. The standard technique for measuring the level of replication-competent infectious virus in culture supernatants or patient samples is the tissue culture dose for 50% infectivity (TCID(50)), which provides an accurate assessment of the level of infectious HIV-1. However, it is a time-consuming technique which typically takes two or more weeks to complete and requires PHA-stimulated PBMC from HIV-1 seronegative donors or an appropriate cell line. Thus rapid, cell-free surrogate measures for TCID(50) are desirable. Here, we introduce the virtual TCID(50) technique: a new cell-free method estimating a surrogate of infectious titer by comparing the reverse transcriptase activity in virus stock to that of reference viruses with a known TCID(50) value. We have demonstrated that the virtual TCID(50) obtained through this technique is comparable to the actual infectious TCID(50). This method greatly simplifies the process of accurate HIV-1 titration and is particularly beneficial for studies which require titration of large number of HIV-1 isolates.

Expression of APOBEC3 Lentiviral Restriction Factors in Cats.

Feline immunodeficiency virus (FIV) is a naturally occurring T-cell tropic lentiviral disease of felids with many similarities to HIV/AIDS in humans. Similar to primate lentiviral-host interactions, feline APOBEC3 (A3) has been shown to inhibit FIV infection in a host-specific manner and feline A3 degradation is mediated by FIV Vif. Further, infection of felids with non-native FIV strains results in restricted viral replication in both experimental and naturally occurring infections. However, the link between molecular A3-Vif interactions and A3 biological activity during FIV infection has not been well characterized. We thus examined expression of the feline A3 genes A3Z2, A3Z3 and A3Z2-Z3 during experimental infection of domestic cats with host-adapted domestic cat FIV (referred to as FIV) and non-adapted Puma concolor FIV (referred to as puma lentivirus, PLV). We determined A3 expression in different tissues and blood cells from uninfected, FIV-infected, PLV-infected and FIV/PLV co-infected cats; and in purified blood cell subpopulations from FIV-infected and uninfected cats. Additionally, we evaluated regulation of A3 expression by cytokines, mitogens, and FIV infection in cultured cells. In all feline cells and tissues studied, there was a striking difference in expression between the A3 genes which encode FIV inhibitors, with A3Z3 mRNA abundance exceeding that of A3Z2-Z3 by 300-fold or more. Interferon-alpha treatment of cat T cells resulted in upregulation of A3 expression, while treatment with interferon-gamma enhanced expression in cat cell lines. In cats, secondary lymphoid organs and peripheral blood mononuclear cells (PBMC) had the highest basal A3 expression levels and A3 genes were differentially expressed among blood T cells, B cells, and monocytes. Acute FIV and PLV infection of cats, and FIV infection of primary PBMC resulted in no detectable change in A3 expression with the exception of significantly elevated A3 expression in the thymus, the site of highest FIV replication. We conclude that cat A3 expression is regulated by cytokine treatment but, by and large, lentiviral infection did not appear to alter expression. Differences in A3 expression in different blood cell subsets did not appear to impact FIV viral replication kinetics within these cells. Furthermore, the relative abundance of A3Z3 mRNA compared to A3Z2-Z3 suggests that A3Z3 may be the major active anti-lentiviral APOBEC3 gene product in domestic cats.

Identification of a Novel Gammaherpesvirus in Canada lynx (Lynx canadensis).

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22-53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8-31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study.

Feline Foamy Virus Infection: Characterization of Experimental Infection and Prevalence of Natural Infection in Domestic Cats with and without Chronic Kidney Disease.

Foamy viruses (FVs) are globally prevalent retroviruses that establish apparently apathogenic lifelong infections. Feline FV (FFV) has been isolated from domestic cats with concurrent diseases, including urinary syndromes. We experimentally infected five cats with FFV to study viral kinetics and tropism, peripheral blood mononuclear cell (PBMC) phenotype, urinary parameters, and histopathology. A persistent infection of primarily lymphoid tropism was detected with no evidence of immunological or hematologic perturbations. One cat with a significant negative correlation between lymphocytes and PBMC proviral load displayed an expanded FFV tissue tropism. Significantly increased blood urea nitrogen and ultrastructural kidney changes were noted in all experimentally infected cats, though chemistry parameters were not outside of normal ranges. Histopathological changes were observed in the brain, large intestine, and other tissues. In order to determine if there is an association of FFV with Chronic Kidney Disease, we additionally screened 125 Australian pet cats with and without CKD for FFV infection and found that FFV is highly prevalent in older cats, particularly in males with CKD, though this difference was not statistically significant compared to controls. Acute FFV infection was clinically silent, and while some measures indicated mild changes, there was no overt association of FFV infection with renal disease.

Identification of gammaherpesvirus infection in free-ranging black bears (Ursus americanus).

Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.

In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture.

A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested.

A radiolabeled oligonucleotide ligation assay demonstrates the high frequency of nevirapine resistance mutations in HIV type 1 quasispecies of NVP-treated and untreated mother-infant pairs from Uganda.

This study explores the levels of NVP and AZT resistance mutations in untreated, NVP- or AZT-treated mother-infant pairs in Uganda. PCR-amplified reverse transcriptase (RT) gene fragments derived from PBMC samples of 85 mothers (10 AZT treated, 35 NVP treated, and 40 untreated) and their 52 infected infants (5 AZT, 9 NVP, and 38 untreated) were classified as subtype A (59%), D (29%), C (3%), and recombinant forms (9%) by population sequencing. Only 16% of the NVP-treated infected mothers and infants harbored either the K103N or the Y181C at 6 weeks postdelivery. The majority of these samples (n = 107) were then analyzed using a radiolabeled oligonucleotide ligation assay (OLA) specific for K70R, K103N, and Y181C, using nonstandard bases to accommodate sequence heterogeneity. By OLA, 43% of the NVP-treated group had K103N and/or Y181C mutations in their HIV-1 population, using >0.6% cutoff based on a comparative clonal analysis of clinical isolates. Surprisingly, an equal fraction of the untreated and NVP-treated mother-infant group had the K103N mutation in their HIV-1 population in the range of 0.6-5%. These findings suggest a relatively high frequency of K103N mutation in the drug-naive, subtype A and D infected Ugandan population as compared to the very low frequency of the Y181C and K70R mutation (<0.6%). The prevalence of the K103N mutations may be related to its low fitness cost and high genetic stability. The persistence of these mutations may reduce the effectiveness of subsequent NVP use in treatment or prevention of perinatal transmission.

High prevalence of Lynx rufus gammaherpesvirus 1 in wild Vermont bobcats.

Gammaherpesviruses (GHVs) are host specific DNA viruses that infect a large range of mammalian species. These viruses preferentially target host lymphocyte cell populations and infection may lead to morbidity or mortality in immunocompromised, co-infected, or non-adapted hosts. In this study, we tested for the presence of Lynx rufus gammaherpesvirus 1 (LruGHV1) in a northeastern United States population of wild bobcats (L. rufus). We estimated prevalence of infection and viral load in infected individuals using quantitative real-time PCR analysis of spleen DNA from 64 Vermont bobcats. We observed an overall prevalence of 64% using this methodology. Bobcat age was significantly positively associated with GHV infection status, and we noted a trend for higher viral loads in young animals, but prevalence and viral load were similar in male and female bobcats. A single LruGHV1 variant was identified from the sequencing of the viral glycoprotein B gene of Vermont bobcats. This gene sequence was 100% similar to that reported in Florida bobcats and slightly variant from other isolates identified in the Western USA. Our work suggests broad geographic distribution and high prevalence of LruGHV1 in bobcat populations across the United States with infection attributes that suggest horizontal transmission of the agent. Geographic differences in viral genotype may reflect historical migration and expansion events among bobcat populations.

Prior Puma Lentivirus Infection Modifies Early Immune Responses and Attenuates Feline Immunodeficiency Virus Infection in Cats.

We previously showed that cats that were infected with non-pathogenic Puma lentivirus (PLV) and then infected with pathogenic feline immunodeficiency virus (FIV) (co-infection with the host adapted/pathogenic virus) had delayed FIV proviral and RNA viral loads in blood, with viral set-points that were lower than cats infected solely with FIV. This difference was associated with global CD4⁺ T cell preservation, greater interferon gamma (IFN-γ) mRNA expression, and no cytotoxic T lymphocyte responses in co-infected cats relative to cats with a single FIV infection. In this study, we reinforced previous observations that prior exposure to an apathogenic lentivirus infection can diminish the effects of acute infection with a second, more virulent, viral exposure. In addition, we investigated whether the viral load differences that were observed between PLV/FIV and FIV infected cats were associated with different immunocyte phenotypes and cytokines. We found that the immune landscape at the time of FIV infection influences the infection outcome. The novel findings in this study advance our knowledge about early immune correlates and documents an immune state that is associated with PLV/FIV co-infection that has positive outcomes for lentiviral diseases.

A Preliminary Proteomic Investigation of Circulating Exosomes and Discovery of Biomarkers Associated with the Progression of Osteosarcoma in a Clinical Model of Spontaneous Disease.

Circulating cancer exosomes are microvesicles which originate from malignant cells and other organs influenced by the disease and can be found in blood. The exosomal proteomic cargo can often be traced to the cells from which they originated, reflecting the physiological status of these cells. The similarities between cancer exosomes and the tumor cells they originate from exhibit the potential of these vesicles as an invaluable target for liquid biopsies. Exosomes were isolated from the serum of eight osteosarcoma-bearing dogs, five healthy dogs, and five dogs with traumatic fractures. We also characterized exosomes which were collected longitudinally from patients with osteosarcoma prior and 2 weeks after amputation, and eventually upon detection of lung metastasis. Exosomal proteins fraction were analyzed by label-free mass spectrometry proteomics and were validated with immunoblots of selected proteins. Ten exosomal proteins were found that collectively discriminate serum of osteosarcoma patients from serum healthy or fractured dogs with an accuracy of 85%. Additionally, serum from different disease stages could be distinguished with an accuracy of 77% based on exosomal proteomic composition. The most discriminating protein changes for both sample group comparisons were related to complement regulation, suggesting an immune evasion mechanism in early stages of osteosarcoma as well as in advanced disease.