Vaccination against the Koala Retrovirus (KoRV): Problems and Strategies

The koala retrovirus (KoRV) is spreading in the koala population from the north to the south of Australia and is also in the process of endogenization into the koala genome. Virus infection is associated with tumorigenesis and immunodeficiency and is contributing to the decline of the animal population. Antibody production is an excellent marker of retrovirus infection; however, animals carrying endogenous KoRV are tolerant. Therefore, the therapeutic immunization of animals carrying endogenous KoRV seems to be ineffective. Using the recombinant transmembrane (TM) envelope protein of the KoRV, we immunized goats, rats and mice, obtaining in all cases neutralizing antibodies which recognize epitopes in the fusion peptide proximal region (FPPR), and in the membrane-proximal external region (MPER). Immunizing several animal species with the corresponding TM envelope protein of the closely related porcine endogenous retrovirus (PERV), as well as the feline leukemia virus (FeLV), we also induced neutralizing antibodies with similar epitopes. Immunizing with the TM envelope protein in addition to the surface envelope proteins of all three viruses resulted in higher titers of neutralizing antibodies. Immunizing KoRV-negative koalas with our vaccine (which is composed of both envelope proteins) may protect these animals from infection, and these may be the starting points of a virus-free population.

First Description of a Multisystemic and Lethal SARS-CoV-2 Variant of Concern P.1 (Gamma) Infection in a FeLV-Positive Cat: New Concerns Regarding Viral Re-emergence and Adaption to Pets

Background: Coronaviruses are recognized for their ability to cross the species barrier and infect new hosts. The coronavirus disease 2019 (COVID-19) is caused by the new coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). It remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. Methods: In this study, we analysed the first fatal case of a SARS-CoV-2 and FeLV (Feline leukemia virus) co-infection of an eight-year-old male cat. We carried out a clinical evaluation, pathological analysis, and viral genomic analysis. Results: As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to animal’s death. The causative agent was confirmed to be SARS-CoV-2, variant of concern P.1 (Gamma). The virus presented a pattern of mutations potentially associated with feline infection. In addition, the virus was detected by RT-qPCR in the spleen, liver, heart, lungs, trachea, intestines and kidneys, indicating a multisystemic viral infection. The virus was found in a high load in the trachea, suggesting a capacity of transmitting the virus. Conclusion: Our data show that felines, such as FeLV-positive cats, are susceptible to SARS-CoV-2 infection, and may be intermediate hosts in this pandemic.

Rapid characterization of feline leukemia virus infective stages by a novel nested recombinase polymerase amplification (RPA) and reverse transcriptase-RPA

Feline leukemia virus (FeLV) is a major viral disease in cats, causing leukemia and lymphoma. The molecular detection of FeLV RNA and the DNA provirus are important for staging of the disease. However, the rapid immunochromatographic assay commonly used for antigen detection can only detect viremia at the progressive stage. In this study, nested recombinase polymerase amplification (nRPA) was developed for exogenous FeLV DNA provirus detection, and reverse transcriptase polymerase amplification (RT-RPA) was developed for the detection of FeLV RNA. The approaches were validated using 108 cats with clinicopathologic abnormalities due to FeLV infection, and from 14 healthy cats in a vaccination plan. The nRPA and RT-RPA assays could rapidly amplify the FeLV template, and produced high sensitivity and specificity. The FeLV detection rate in regression cats by nRPA was increased up to 45.8% compared to the rapid immunochromatographic assay. Hence, FeLV diagnosis using nRPA and RT-RPA are rapid and easily established in low resource settings, benefiting FeLV prognosis, prevention, and control of both horizontal and vertical transmission.

Endogenous feline leukemia virus siRNA transcription may interfere with exogenous FeLV infection

Endogenous retroviruses (ERVs) are increasingly recognized for biological impacts on host cell function and susceptibility to infectious agents, particularly in relation to interactions with exogenous retroviral progenitors (XRVs). ERVs can simultaneously promote and restrict XRV infections using mechanisms that are virus- and host-specific. The majority of endogenous-exogenous retroviral interactions have been evaluated in experimental mouse or chicken systems which are limited in their ability to extend findings to naturally infected outbred animals. Feline leukemia virus (FeLV) has a relatively well-characterized endogenous retrovirus with a coexisting virulent exogenous counterpart and is endemic worldwide in domestic cats. We have previously documented an association between endogenous FeLV LTR copy number and abrogated exogenous FeLV in naturally infected cats and experimental infections in tissue culture. Analyses described here examine limited FeLV replication in experimentally infected peripheral blood mononuclear cells which correlates with higher enFeLV transcripts in these cells compared to fibroblasts. We further examine NCBI Sequence Read Archive RNA transcripts to evaluate enFeLV transcripts and RNA interference precursors. We find that lymphoid-derived tissues, which are experimentally less permissive to exogenous FeLV infection, transcribe higher levels of enFeLV under basal conditions. Transcription of enFeLV-LTR segments is significantly greater than other enFeLV genes. We documented transcription of a 21-nt miRNA just 3’ to the enFeLV 5’-LTR in the feline miRNAome of all datasets evaluated (n=27). Our findings point to important biological functions of enFeLV transcription linked to solo LTRs distributed within the domestic cat genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. Importance Endogenous retroviruses (ERVs) are increasingly implicated in host cellular processes and susceptibility to infectious agents, specifically regarding interactions with exogenous retroviral progenitors (XRVs). Exogenous feline leukemia virus (FeLV) and its endogenous counterpart (enFeLV) represent a well characterized, naturally occurring XRV-ERV dyad. We have previously documented an abrogated FeLV infection in both naturally infected cats and experimental fibroblast infections that harbor higher enFeLV proviral loads. Using an in silico approach, we provide evidence of miRNA-transcription that are produced in tissues most important for FeLV infection, replication, and transmission. Our findings point to important biological functions of enFeLV transcription linked to solo-LTRs distributed within the feline genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. This body of work provides additional evidence of RNAi as a mechanism of viral interference and is a demonstration of ERV exaptation by the host to defend against related XRVs.

Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

Prevalence of feline coronavirus, feline leukemia virus, and feline immunodeficiency virus in client-owned cats in Croatia

This study aimed to determine prevalences for anti-FCoV antibody, FeLV antigen, FeLV proviral DNA, and anti-FIV antibody among client-owned cats from the cities of Zagreb and Varaždin in Croatia. Subjects included 106 client-owned cats tested at the Faculty of Veterinary Medicine, Vienna, Austria. Blood samples were tested with IFA for anti-FCoV antibody and IFA FCoV antibody titeres, with ELISA for FeLV p27 antigen, with PCR for FeLV proviral DNA, and with RIM for anti-FIV antibody. Prevalence of FCoV and FeLV was 41.51% and 6.60%, respectively. A coinfection with FeLV/FCoV and FIV/FCoV prevalence was 7.55% and 5.66%. No cats were coinfected with FIV and FeLV. All three viruses were detected, confirming their presence in Croatia. The seroepidemiological findings demonstrate that both feline retroviruses and feline coronavirus are important feline pathogens in Croatia.

Cytomorphological similarities between feline viral leukemia, bovine enzootic leukosis and adult T-cell leukemia/lymphoma: A review

Leukemias are malignant neoplasms of hematological origin and originating from bone marrow cells. Innumerable species can be affected by this disease, which can be originated by several causes, including infection by viruses belonging to the Retroviridae family. In felines, humans and cattle, the leukemia-inducing retroviruses are Feline Leukemia Virus (FeLV), human T-cell lymphotropic virus type 1 (HTLV-1) and Bovine Leukosis Virus (BLV), respectively. In Brazil, the number of domestic cats infected with FeLV grows progressively, when compared to the incidence of infected animals in developed countries, such as the United States. In cattle, viral leukemia or enzootic bovine leukosis (EBL), caused by BLV, although asymptomatic, leads to decreased production and economic losses. In humans, HTLV-1 was the first human retrovirus described in the 1980s. In this work, the similarities between cytomorphological changes in felines, cattle and humans affected by FeLV, BLV and HTLV-1, respectively, were analyzed. The bibliographic findings showed that the affected species addressed share the presence of atypical and/or reactive lymphocytes, smudge cells, immature cells and nuclear cell atypias in peripheral blood.

Feline cystadenomatosis affecting the ears and skin of 57 cats (2011–2019)

Objectives This study aimed to understand epidemiological factors associated with feline cystadenomatosis, including signalment and papillomavirus PCR status. Cystadenomatosis is an uncommon condition primarily involving the ceruminous and apocrine skin and ear glands. Methods This was a retrospective case series. Clinical records from 2011 to 2019 from a tertiary referral hospital in Boston, MA, USA were screened for cases, and case data were re-evaluated and analyzed. The total patient pool contained 65,385 individual cats, of which 797 were referred to the dermatology service. Medical records and biopsy specimens were reviewed; the information collected included signalment, clinical signs, physical examination and diagnostic tests, comorbidities and histopathologic findings. PCR was performed on biopsy specimens to test for papillomavirus DNA. Results The cystadenomatosis population consisted of 57 cases (7.1% of total cases referred to the dermatology service) with 105 affected ears. Twenty-seven cases (48 ears) were confirmed via histopathology; four cats (7%) exhibited clinically cystic lesions on the periocular, periorbital and perianal regions; only one cat did not have pinnal lesions. Domestic shorthair cats were most often affected. Relative risk for cystadenomatosis was 2.24 times higher in male cats. In 48 cats (84.2%), ears were bilaterally affected. Seven cats (12.3%) had malignant neoplasia, which included: inflamed adenocarcinoma (n = 5); mast cell tumor (n = 1); or squamous cell carcinoma (n = 1). PCR testing on biopsy specimens from 24 cats revealed feline papillomavirus type 2 DNA in only four cats. Conclusions and relevance Cystadenomatosis was more prevalent in senior non-purebred cats, over-represented in male cats and did not appear to be associated with papillomavirus, feline infectious peritonitis, feline immunodeficiency virus/feline leukemia virus status or other identifiable illnesses. Further studies are needed to investigate the causes of cystadenomatosis.