Feline foamy virus in domestic cats: use as a vaccine vector, characterization of infection, and association with other diseases

2019 Spring.Includes bibliographical references.Foamy viruses (FVs) are retroviruses from the Spumaretrovirinae subfamily. FVs are globally prevalent retroviruses with a unique molecular biology. FVs establish apparently apathogenic lifelong infections. Due to this, FVs are considered attractive vectors for vaccine and gene therapy development. Feline foamy virus (FFV) infects domestic cats and has widespread and high prevalence around the world. However, FFV has also been isolated from cats suffering from concurrent disease, including renal syndromes and other retroviral co- infections such as feline immunodeficiency virus (FIV). Much remains unknown about FFV infection and in vivo experimental infections are rare in the literature. To test FFV's use as a vaccine vector and understand the interaction between viral proteins and host antiviral restriction factors, we developed an infective chimeric vaccine containing lentiviral FIV vif replacing FFV bet. FFV Bet and FIV Vif counteract feline innate APOBEC3 (feA3) restriction factors through different mechanisms. FeA3 action on retroviral genomes lead to hypermutation and degradation of viral DNA. In vitro, we show that vif can replace bet to yield replication-competent chimeric viruses. We experimentally inoculated 12 domestic cats (n=4 per group in naïve, wild-type, and chimera-inoculated groups) with the FFV-Vif chimera and wild- type FFV in order to compare viral replication kinetics through PCR and specific antibody development through ELISA. Inoculation with the chimeric vector resulted in the development of a specific immune response against FFV Gag and Bet and FIV Vif proteins. In addition, we show that the domestic cat can be superinfected with different strains of FFV. The chimeric virus displayed attenuated infection in vivo, as provirus was not detected in PBMC for any chimera-only inoculated animals. Thus, Bet may have additional functions other than A3 antagonism required for successful in vivo infection. Our studies further exemplify how FV vaccine vectors are an attractive tool to counteract lentiviral infections and poses the possibility to induce immunity against other lentiviral antigens. In order to further characterize wild-type infection, we also collected blood, saliva, and urine over a 6-month time-period with a necropsy and tissue collection at the end of the study. Animals were monitored daily for clinical signs of disease and temperature and weight data were collected weekly. None of the cats showed clinical signs of infection and complete blood count and chemistry were unremarkable. However, we found significant differences in blood urea nitrogen, one of the markers used to assess renal function, when comparing infected versus control animals. All animals inoculated with wild-type virus showed a persistent proviremia (in PBMCs) and viral tissue tropism was primarily lymphoid with the exception of one cat that had an expanded tissue tropism to other lymphoid tissues and oral mucosa. This animal had altered viral kinetics compared to the rest of infected animals, in addition to a negative correlation between lymphocyte count and viral load. Histopathological analysis showed evidence of microscopic pathology in the kidneys, lung, and brain of infected animals. This same cat had an increase in urine protein at the time of highest PBMC proviremia. Additionally, transmission electron microscopy showed ultrastructural changes indicative of transient renal injury in the kidneys of infected animals. We additionally found electron dense structures in the cytoplasm of tubular epithelial of as of yet unknown origin. Due to the renal changes we saw in the experimental study and pathology reported in the literature, we conducted a survey of FFV in pet cats in the USA and Australia (AU) suffering from chronic kidney disease (CKD) and compared findings to age- and sex-matched controls without CKD. We found an association between CKD and FFV in males, and males in general are also at a significantly increased risk of FFV infection. We then assessed through an FFV serosurvey whether FFV was associated with FIV and causing potentiation of FIV disease in two cohorts of naturally FIV-infected cats. One of the groups consisted of cats living in 1-2 cat households that did not experience much FIV-related morbidity and mortality, while the second group of cats housed in a large multicat household suffered from severe clinical symptoms and mortality. We hypothesized the reason for this discrepancy could be an increase in FFV/FIV co-infection rate in the group of cats with higher morbidity and mortality. We found that FFV is associated with FIV in these groups and that males are also at an increased risk for FFV infection. Finally, we conducted an in vitro co- infection study to assess potentiated infection as determined by more rapid development of cytopathic effects (CPE) and higher viral titers in the supernatant. GFox cells were inoculated with FFV and FIV in single, and dual simultaneous and staggered inoculations. A p26 ELISA was used to determine amount of FIV reactivity in the cells, while a chemiluminescent β- galactosidase assay was used to detect amount of β -gal produced in FeFAB FFV reporter cells. The in vitro assays showed increased permissivity of either virus following an initial infection of the other virus, showing these two retroviruses can accelerate and potentiate a secondary infection regardless of which virus infected initially. Overall, we have demonstrated the suitability of FFV as a vaccine vector candidate. Additionally, we have documented that FFV may cause subclinical alterations that in certain cohorts of domestic cats, may contribute to disease development in chronic cases. Finally, we showed that FFV interacts with another retrovirus and could potentially affect FIV-related disease. More studies should focus on the effects of FFV in chronic infections in addition to the effect of FFV on co-morbidities in a chronic timeline

Developmental Aspects of SARS-CoV-2, Potential Role of Exosomes and Their Impact on the Human Transcriptome

With over 4.8 million deaths within 2 years, time is of the essence in combating COVID-19. The infection now shows devastating impacts on the younger population, who were not previously predicted to be vulnerable, such as in the older population. COVID-19-related complications have been reported in neonates whose mothers were infected with SARS-CoV-2 during pregnancy, and in children who get infected. Hence, a deeper understanding of the pathophysiology of COVID-19 during various developmental stages and placental transmission is essential. Although a connection has not yet been established between exosomal trafficking and the placental transmission of COVID-19, reports indicate that SARS-CoV-2 components may be trafficked between cells through exosomes. As the infection spreads, the transcriptome of cells is drastically perturbed, e.g., through the severe upregulation of several immune-related genes. Consequently, a major outcome of COVID-19 is an elevated immune response and the detection of viral RNA transcripts in host tissue. In this direction, this review focuses on SARS-CoV-2 virology, its in utero transmission from infected pregnant mothers to fetuses, SARS-CoV-2 and exosomal cellular trafficking, transcriptomic impacts, and RNA-mediated therapeutics against COVID-19. Future research will establish stronger connections between the above processes to develop diagnostic and therapeutic solutions towards COVID-19 and similar viral outbreaks

Improved DNA Vaccine Delivery with Needle-Free Injection Systems

DNA vaccines have inherent advantages compared to other vaccine types, including safety, rapid design and construction, ease and speed to manufacture, and thermostability. However, a major drawback of candidate DNA vaccines delivered by needle and syringe is the poor immunogenicity associated with inefficient cellular uptake of the DNA. This uptake is essential because the target vaccine antigen is produced within cells and then presented to the immune system. Multiple techniques have been employed to boost the immunogenicity and protective efficacy of DNA vaccines, including physical delivery methods, molecular and traditional adjuvants, and genetic sequence enhancements. Needle-free injection systems (NFIS) are an attractive alternative due to the induction of potent immunogenicity, enhanced protective efficacy, and elimination of needles. These advantages led to a milestone achievement in the field with the approval for Restricted Use in Emergency Situation of a DNA vaccine against COVID-19, delivered exclusively with NFIS. In this review, we discuss physical delivery methods for DNA vaccines with an emphasis on commercially available NFIS and their resulting safety, immunogenic effectiveness, and protective efficacy. As is discussed, prophylactic DNA vaccines delivered by NFIS tend to induce non-inferior immunogenicity to electroporation and enhanced responses compared to needle and syringe

Feline Foamy Virus is Highly Prevalent in Free-Ranging <i>Puma concolor</i> from Colorado, Florida and Southern California

Feline foamy virus (FFV) is a retrovirus that has been detected in multiple feline species, including domestic cats (Felis catus) and pumas (Puma concolor). FFV results in persistent infection but is generally thought to be apathogenic. Sero-prevalence in domestic cat populations has been documented in several countries, but the extent of viral infections in nondomestic felids has not been reported. In this study, we screened sera from 348 individual pumas from Colorado, Southern California and Florida for FFV exposure by assessing sero-reactivity using an FFV anti-Gag ELISA. We documented a sero-prevalence of 78.6% across all sampled subpopulations, representing 69.1% in Southern California, 77.3% in Colorado, and 83.5% in Florida. Age was a significant risk factor for FFV infection when analyzing the combined populations. This high prevalence in geographically distinct populations reveals widespread exposure of puma to FFV and suggests efficient shedding and transmission in wild populations

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Additional file 2. Partial genome sequences from pCF7-Vif-4 and the stop mutations of the in vitro-selected FFV-Vif variants. The Trp codon and the downstream G residue (TGGG) ~ 130 bp upstream of the vif coding sequence are in bold face letters and underlined. In pCF7-Vif W/*1 (in blue), the mutation is from TGG to TGA and for mutant W/*2 (in green) the mutation is from TGGG to TAGA, with both mutations resulting in a Trp (W) to Stop (*) mutation (W/*) as indicated. The bet nucleotide sequence is in black, the linker sequence in pink with recognition sites for NheI (in brown) and SacII (in light violet). The vif gene is marked in blue with the authentic Met start codon in bold. The BettrVif fusion protein is highlighted in yellow with the amino acids color-coded as described above for the genes. The Met residue 14 amino acids upstream of the authentic vif start codon is highlighted in bold and underlining. The C-terminal amino acid sequence of tas is highlighted in red

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Additional file 4. Titers of pCF-7, pCF7-Vif-4 and engineered pCF7-Vif W/*1 and pCF7-Vif W/*2 variants. Plasmid pCF-7, pCF7-Vif-4, pCF7-Vif W/*1, and pCF7-Vif W/*2 and their engineered M/T and M+ variants were transfected into HEK 293T cells and 2 days post-transfection, cell-free supernatants were inoculated on CrFK cells and serially passaged every A 60 and B 84 h p.i. FFV titers were determined in duplicate using FeFAB reporter cells and are shown as bar diagrams for the different passages. Error bars represent the standard deviation

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Additional file 1. Rescue of Vif-deficient FIV and Bet-deficient FFV by FIV Vif and FFV Bet. A Vif-deficient FIV plasmid DNA was co-transfected with plasmids expressing FIV Vif or FFV Bet together with different feA3 restriction factors as given in the legend (left panel). Empty vector pcDNA3.1 served as control. Two days after transfection, cell-free supernatants were used to infect FIV reporter cells and luc activity induced by FIV infection was measured two days p.i. Titers are expressed as luc values of a representative experiment. B The Bet-deficient FFV genome pCF7-BBtr was co-transfected with plasmids expressing untagged and V5-tagged FFV Bet or two different amounts of FIV Vif expression plasmid together with the major FFV-restricting feA3Z2b-HA as shown below the bar diagram (right panel). Empty vector pcDNA3.1 served as control. Two days after transfection, cell-free supernatants were titrated in triplicate using FFV reporter cells as described in the “Methods” section and are expressed as focus-forming units (FFU) per ml inoculum of a representative experiment. Error bars represent the standard deviation

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Additional file 3. Mutations in Tas generated during the analysis of the upstream ATG do not affect Tas-mediated LTR transactivation. The LTR promoter-based luc reporter construct pFeFV-LTR-luc [73] was cotransfected into HEK 293T cells together with a CMV-IE-driven FFV Tas expression construct, the empty control pcDNA3.1 and proviral genomes pCF-7, pCF7-Vif-4, pCF7-Vif W/*1, and pCF7-Vif W/*2, and their engineered M/T and M+ variants. Two days post transfection, luc activity induced by FFV Tas expression was measured in duplicates. Data from a representative experiment normalized to co-expressed β-gal are expressed in a logarithmic bar diagram