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.

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.

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.

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.

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.

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 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.

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.

Transcriptome Analysis and In Situ Hybridization for FcaGHV1 in Feline Lymphoma.

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenic potential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats, is unknown. In other species, including humans, cellular transformation by gammaherpesviruses is typically mediated by viral genes expressed during latency. We analysed tumour RNA, from diffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and feline immunodeficiency virus (FIV) (n = 10), by high throughput transcriptome sequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identified in five tumors, including homologs of oncogenic latency-associated transcripts, latency-associated nuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORF unique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplant lymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single case in which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcripts can be detected in some FIV-associated lymphomas, but at low copy number, precluding assessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1 transcriptome in clinical samples might employ viral enrichment and greater sequencing depth to enhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-cell tumors, large granular lymphocyte lymphoma, for study.

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.

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.

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.

Bovine herpesvirus 4 DNA is not detected in free-ranging domestic cats from California, Colorado or Florida.

Objectives Several studies have reported that domestic cats can be naturally infected with bovine herpesvirus 4 (BHV4). Cats experimentally inoculated with BHV4 developed clinical signs involving the urinary tract, leading to the hypothesis that natural infection with BHV4 may be associated with feline lower urinary tract diseases. However, the question of whether BHV4 infection is common in cats remains equivocal. In this study, we sought to determine whether BHV4 is a common natural infection of domestic cats in the USA. Methods We used a sensitive nested PCR protocol specific to the BHV4 thymidine kinase gene to screen free-ranging domestic cat blood DNA samples (n = 101) collected from California, Colorado and Florida. Results Cats within this cohort were positive for seven other common pathogens of domestic cats, demonstrating the relatively high exposure of this population to endemic feline infections. In contrast, all domestic cat blood samples were negative for BHV4, while BHV4-containing tissue culture extracts were strongly positive. Conclusions and relevance BHV4 has been detected in tissues of latently infected cattle, though viral DNA is typically also detected in peripheral blood cells throughout infection. Our results suggest that persistent presence of BHV4 DNA in the blood of domestic cats is either rare or non-existent. We thus conclude that BHV4 is unlikely to be a major pathogen of cats.

Domestic cats seropositive for Felis catus gammaherpesvirus 1 are often qPCR negative.

Felis catus gammaherpesvirus 1 (FcaGHV1) is a newly described virus that infects domestic cats. To identify FcaGHV1 antigens, we developed an immunofluorescent antibody assay by expressing FcaGHV1 open reading frames (ORFs) in feline cells and incubating fixed cells with sera from FcaGHV1-positive cats. Of the seven ORFs tested, ORF52 and ORF38 had the strongest, most consistent antibody responses. We used recombinant ORF52 and ORF38 proteins to develop two FcaGHV1 ELISAs. These assays were used to detect reactivity in cats previously tested by qPCR for FcaGHV1 in blood cell DNA. Results indicated 32%FcaGHV1seroprevalence, compared to 15%qPCR-evaluated prevalence (n=133);all but one qPCR positive animal was seropositive. ELISA results confirmed infection risk factors previously identified by qPCR: geographic location, male sex, and adult age. These data suggest that FcaGHV1is a common infection of domestic cats that has a seropositive but often qPCR negative state characteristic of herpesviral latency.

Closing the gap on causal processes of infection risk from cross-sectional data: structural equation models to understand infection and co-infection.

BACKGROUND: Epidemiological studies of disease exposure risk are frequently based on observational, cross-sectional data, and use statistical approaches as crucial tools for formalising causal processes and making predictions of exposure risks. However, an acknowledged limitation of traditional models is that the inferred relationships are correlational, cannot easily distinguish direct from indirect determinants of disease risk, and are often considerable simplifications of complex interrelationships. This may be particularly important when attempting to infer causality in patterns of co-infection through pathogen-facilitation. METHODS: We describe analyses of cross-sectional data using structural equation models (SEMs), a contemporary advancement on traditional regression approaches, based on our study system of feline gammaherpesvirus (FcaGHV1) in domestic cats. RESULTS: SEMs strongly supported a latent (host phenotype) variable associated with FcaGHV1 exposure and co-infection risk, suggesting these individuals are simply more likely to become infected with multiple pathogens. However, indications of pathogen-covariance (potential facilitation) were also variably detected: potentially among FcaGHV1, Bartonella spp and Mycoplasma spp. CONCLUSIONS: Our models suggest multiple exposures are primarily driven by host phenotypic traits, such as aggressive male phenotypes, and secondarily by pathogen-pathogen interactions. The results of this study demonstrate the application of SEMs to understanding epidemiological processes using observational data, and could be used more widely as a complementary tool to understand complex cross-sectional information in a wide variety of disciplines.

First Complete Genome Sequence of Felis catus Gammaherpesvirus 1.

We sequenced the complete genome of Felis catus gammaherpesvirus 1 (FcaGHV1) from lymph node DNA of an infected cat. The genome includes a 121,556-nucleotide unique region with 87 predicted open reading frames (61 gammaherpesvirus conserved and 26 unique) flanked by multiple copies of a 966-nucleotide terminal repeat.

Identification of Novel Gammaherpesviruses in Ocelots (Leopardus pardalis) and Bobcats (Lynx rufus) in Panama and Colorado, USA.

Gammaherpesviruses (GHVs) have been identified in many species and are often associated with disease. Recently, we characterized three novel felid GHVs in domestic cats (Felis catus), bobcats (Lynx rufus), and pumas (Puma concolor). We investigated whether free-ranging ocelots (Leopardus pardalis) and bobcats are infected with additional GHVs. We screened DNA samples from ocelots on Barro Colorado Island, Panama, and bobcats in western Colorado, US, by using a degenerate nested PCR that targets the GHV glycoprotein B gene. We identified a novel GHV glycoprotein B sequence in two ocelots and a second novel sequence in a bobcat, which is distinct from the previously characterized bobcat GHV (Lynx rufus GHV 1). Utilizing additional degenerate and virus-specific PCRs, we extended these sequences to include 3.4 kilobases of the GHV glycoprotein B and DNA polymerase genes. These sequences identify the first GHV detected in ocelots and the second GHV in bobcats. These viruses were provisionally named L. pardalis GHV 1 and Lynx rufus GHV 2, respectively. The viruses are most closely related to recently identified GHVs of the Percavirus genus found in domestic cats (F. catus GHV 1) and bobcats (L. rufus GHV 1), suggesting that a cluster of felid GHVs exists within the Percavirus genus.

Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.