Serological survey of Neospora caninum and Toxoplasma gondii in shelter-housed cats infected with feline immunodeficiency virus, Brazil / Inquérito sorológico para Neospora caninum e Toxoplasma gondii em um abrigo de gatos infectados com o vírus da imunodeficiência felina, Brasil

Felines play a leading role in the epidemiology of Toxoplasma gondii infection, but there is scarce information about the epidemiology of Neospora caninum, particularly in feline immunodeficiency virus (FIV)-infected cats. Cats seropositive to T. gondii do not usually show symptoms unless they are immunosuppressed, such as FIV-infected cats. The same relationship remains poorly known for N. caninum, although it has been associated with neurological disorders in HIV-infected people. Since FIV-infected cats are prone to develop encephalitis of unknown etiology, this study aimed to evaluate the presence of specific antibodies to T. gondii and N. caninum in a shelter for stray cats naturally infected with FIV. A total of 104 serum samples from cats living in a shelter, located in São Paulo city (Brazil), was assessed for T. gondii and N. caninum specific antibody by indirect fluorescent-antibody test (IFAT). Of the 104 cats, 25 (24%) were infected with FIV and, aside from these, 8 (32%) had antibodies against T. gondii (titers from 16 to 128). Only 1 (4%) of the FIV-infected cats had antibodies against N. caninum, which was the first record of coinfection. Among the FIV-naïve cats, 11 (14%) were positive for T. gondii(titers from 16 to 256) and only 1 (1.2%) had antibodies against N. caninum. Serologically positive reactions to T. gondii and N. caninum were not correlated with age or sex (p>0.05), and there was no correlation between FIV and the occurrence of anti-T. gondii or anti-N. caninum antibodies (p>0.05). Further studies encompassing larger cat populations from different origins and locations are essential to clarify the prevalence of T. gondii and N. caninum antibodies in FIV-positive cats.(AU)

Os felinos têm um papel importante na epidemiologia da infecção por Toxoplasma gondii, mas pouco se sabe sobre a epidemiologia da infecção por Neospora caninum em gatos, particularmente em gatos infectados com o vírus da imunodeficiência felina (FIV). Gatos soropositivos para Toxoplasma gondii geralmente não apresentam sintomas a não ser que estejam imunossuprimidos, como gatos infectados com FIV. A mesma relação ainda é pouco conhecida para N. caninum, embora tenha sido associada a distúrbios neurológicos em pessoas infectadas pelo HIV. Considerando que gatos infectados com FIV são propensos a desenvolver encefalite de etiologia desconhecida, o presente estudo teve como objetivo avaliar a presença de anticorpos específicos para T. gondii e N. caninum em gatos infectados com FIV. Um total de 104 amostras de soro de gatos residentes em um abrigo na cidade de São Paulo, Brasil, foram avaliadas para a presença de anticorpos contra T. gondii e N. caninum pelo teste de imunofluorescência indireta (RIFI). Dos 104 gatos, 25 (24%) estavam infectados com FIV e destes 8, (32%) tinham anticorpos contra T. gondii (titulação entre 16 e 128). Apenas 1 (4%) dos gatos infectados com FIV apresentava anticorpos contra N. caninum, sendo este o primeiro registro dessa coinfecção. Entre os gatos não infectados com FIV, 11 (14%) foram positivos para T. gondii (titulação entre 16 e 256) e apenas 1 (1,2%) tinha anticorpos contra N. caninum. A reação sorologicamente positiva para T. gondii e N. caninum não foi correlacionada com a idade ou sexo (p> 0,05), nem houve correlação entre FIV e ocorrência de anticorpos para T. gondii ou N. caninum(p> 0,05). Estudos subsequentes abrangendo populações maiores de gatos de diferentes origens e locais são essenciais para esclarecer a prevalência de anticorpos contra T. gondii e N. caninum em animais acometidos por FIV.(AU)

Detecção do vírus da imunodeficiência felina em gatos domésticos (Felis catus) pela técnica de reação em cadeia da polimerase / Detection of feline immunodeficiency virus in domestic cats (Felis catus) by polymerase chain reaction technique

O objetivo deste trabalho foi detectar a presença de DNA do Vírus da Imunodeficiência Felina (FIV) em gatos domesticos (Feliz catus) assintomáticos. Foi realizada a tecnica de reação em cadeia da polimerase (PCR) em 50 animais. Para tal, foram coletadas amostras de sangue, por venopunção da jugular, de forma asséptica para armazenamento de 1-2 mL de sangue total. Os animais que participaram do estudo fizeram parte do projeto de castração "Vida digna" da Universidade Federal Rural da Amazônia. E a escolha dos animais foi realizada de maneira aleatória, sem distinção por sexo ou idade, resultando em 29 foram fêmeas e 21 machos. Para o diagnóstico, foi realizada a extração do DNA, em seguida as amostras foram testadas em duas reações de PCR utilizando- se dois conjuntos de primers do Gene gag de FIV. Achou-se uma prevalência de 2% (1/50), confirmando assim a presença do vírus na cidade de Belém. Assim, evidenciando a importância de testar os felinos mesmo sendo assintomáticos. Desta forma, faz-se necessário a realização de trabalhos futuros que amplie o número amostral dos animais testados para assim elucidar o perfil epidemiológico da doença na região de Belém do Pará, considerando a relevância clínica desta infecção e a correta conduta médica veterinária para evitar novas infecções.

The objective of this work was to detect the presence of Feline Immunodeficiency Virus (FIV) proviral DNA in asymptomatic domestic cats (Feliz catus). The polymerase chain reaction technique was performed from 50 animals. For this, blood samples were collected by jugular venipuncture, aseptically for storage of 1-2 mL of whole blood. The animals that participated in the study were part of the castration project "Vida digna" at the Universidade Federal Rural da Amazônia. And the choice of animals was performed randomly, without distinction by sex or age, resulting in 29 females and 21 males. For diagnosis, DNA extraction was performed, then the samples were tested in two PCR reactions using two sets of FIV gag gene primers. A prevalence of 2% (1/50) was observed, thus confirming the presence of the virus in the city of Belém. Thus, highlighting the importance of testing the felines even if they are asymptomatic. Therefore, it is necessary to carry out future work that expands the sample number of animals tested in order to elucidate the epidemiological profile of the disease in the region of Belém do Pará, considering the clinical relevance of this infection and the correct veterinary medical conduct to avoid new infections.

Lack of protection against feline immunodeficiency virus infection among domestic cats in New Zealand vaccinated with the Fel-O-Vax® FIV vaccine.

Infections with feline immunodeficiency virus (FIV) are common in New Zealand, although the impact of those infections on the health status of the cats remains unclear. Although many cats are vaccinated yearly with a commercial FIV vaccine containing FIV subtypes A and D, the effectiveness of this vaccine in protection against infection with field FIVs is unclear, as a high proportion of New Zealand viruses belong to subtype C. The objective of the study was to compare the frequency of FIV infection among adult FIV-vaccinated and FIV-unvaccinated domestic cats with access to outdoors. Buccal swabs were collected by the participating veterinarians and tested for the presence of FIV provirus by quantitative PCR. Overall, 26/185 (14.0 %) samples were positive for FIV, including 7/82 (8.5 %) samples from FIV-unvaccinated and 19/103 (18.4 %) from FIV-vaccinated cats. There was no protective effect of vaccination on FIV infection among sampled cats (p = 0.05). Partial sequences of the FIV envelope gene from five New Zealand viruses were analysed by the maximum likelihood method. All clustered with other New Zealand FIV sequences from subtypes A (n = 2), C (n = 2) or putative recombinant viruses (n = 1). While the FIV vaccination did not prevent FIV infection among sampled cats, it may have had an impact on transmissibility of the virus or on disease progression. As neither was addressed in the current study, further research is needed to fully assess the potential benefits of FIV vaccination. Considering the frequency of FIV infection in FIV-vaccinated cats, FIV infection status should be monitored not only before the first vaccination, but before each yearly booster.

Molecular and serological investigation of cat viral infectious diseases in China from 2016 to 2019.

In order to analyse the prevalence of cat viral diseases in China, including feline parvovirus (FPV), feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV) and feline infectious peritonitis virus (FIPV), a total of 1,326 samples of cats from 16 cities were investigated from 2016 to 2019. Collectively, 1,060 (79.9%) cats were tested positive for at least one virus in nucleotide detection, and the positive rates of cat exposure to FeLV, FPV, FHV-1, FCV, FIV and FIPV were 59.6%, 19.2%, 16.3%, 14.2%, 1.5% and 0.5%, respectively. The prevalence of FHV-1 and FPV was dominant in winter and spring. Cats from north China showed a higher positive rate of viral infection than that of cats from south China. The virus infection is not highly correlated with age, except that FPV is prone to occur within the age of 12 months. In the serological survey, the seroprevalences of 267 vaccinated cats to FPV, FCV and FHV-1 were 83.9%, 58.3% and 44.0%, respectively. Meanwhile, the seroprevalences of 39 unvaccinated cats to FPV, FCV and FHV-1 were 76.9% (30/39), 82.4% (28/34) and 58.6% (17/29), respectively. This study demonstrated that a high prevalence of the six viral diseases in China and the insufficient serological potency of FCV and FHV-1 remind the urgency for more effective vaccines.

Serological and molecular investigation of Leishmania spp. infection in cats from an area endemic for canine and human leishmaniasis in Northeast Brazil / Investigação sorológica e molecular da infecção por Leishmania spp. em gatos provenientes de uma área endêmica para leishmaniose canina e humana no nordeste brasileiro

Abstract The aim of this study was to investigate the occurrence of Leishmania spp. antibodies, and its association with feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), in domestic cats from an area endemic for canine and human leishmaniasis in Rio Grande do Norte State, Brazil. Ninety-one cats were subjected to a complete clinical exam, and blood samples were collected. An epidemiological questionnaire was used to investigate the risk factors. IgG anti-Leishmania spp. antibodies were detected by immunofluorescence antibody test (IFAT), with a cut-off value of 1:40. Polymerase chain reaction (PCR) was performed to detect genetic material of Leishmania spp. in the blood samples. The presence of antibodies against FIV and antigens of FeLV was evaluated using an immunochromatographic test. Seropositivity for Leishmania spp., FIV, and FeLV was observed in 14/91 (15.38%), 26/91 (28.57%), and 3/91 (3.29%) cats, respectively. All samples gave negative results on PCR analysis. Based on these data, no significant statistical association was observed between seropositivity for Leishmania spp., and sex, age, presence of clinical signs, evaluated risk factors, and positivity for retroviruses. These findings demonstrated for the first time that cats from Mossoró, Rio Grande do Norte, are being exposed to this zoonosis and might be part of the epidemiological chain of transmission of visceral leishmaniasis.

Resumo O objetivo do presente estudo foi investigar a ocorrência de anticorpos contra Leishmania spp., e sua associação com o vírus da imunodeficiência felina (FIV) e o vírus da leucemia felina (FeLV), em felinos domésticos provenientes de uma área endêmica no estado do Rio Grande do Norte, para a leishmaniose visceral canina e humana. Noventa e um gatos foram submetidos a exame clínico completo e amostras de sangue foram coletadas. Um questionário epidemiológico foi feito para investigar fatores de risco. Anticorpos IgG anti-Leishmania spp. foram identificados por meio da imunofluorescência indireta (RIFI), adotando-se como ponto de corte a diluição de 1:40. A reação em cadeia da polimerase (PCR) foi executada visando detectar o material genético de Leishmania spp. a partir de amostras de sangue total. Para avaliar a presença de anticorpos contra o FIV e antígenos do FeLV foi utilizado um teste imunocromatográfico. Observou-se soropositividade em 14/91 (15,38%), 26/91 (28,57%) e 3/91 (3,29%) animais para Leishmania spp., FIV e FeLV, respectivamente. Nenhuma amostra foi positiva na PCR. Baseado nestes dados, não foi observada nenhuma associação estatística significativa entre a soropositividade para Leishmania spp. e gênero, idade, presença de sinais clínicos, fatores de risco avaliados e positividade para as retroviroses. Esses achados demonstram pela primeira vez que felinos da cidade Mossoró, Rio Grande do Norte, estão sendo expostos a esta zoonose, sugerindo que os mesmos podem estar participando da cadeia epidemiológica de transmissão da leishmaniose visceral.

Serological and molecular investigation of Leishmania spp. infection in cats from an area endemic for canine and human leishmaniasis in Northeast Brazil.

The aim of this study was to investigate the occurrence of Leishmania spp. antibodies, and its association with feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), in domestic cats from an area endemic for canine and human leishmaniasis in Rio Grande do Norte State, Brazil. Ninety-one cats were subjected to a complete clinical exam, and blood samples were collected. An epidemiological questionnaire was used to investigate the risk factors. IgG anti-Leishmania spp. antibodies were detected by immunofluorescence antibody test (IFAT), with a cut-off value of 1:40. Polymerase chain reaction (PCR) was performed to detect genetic material of Leishmania spp. in the blood samples. The presence of antibodies against FIV and antigens of FeLV was evaluated using an immunochromatographic test. Seropositivity for Leishmania spp., FIV, and FeLV was observed in 14/91 (15.38%), 26/91 (28.57%), and 3/91 (3.29%) cats, respectively. All samples gave negative results on PCR analysis. Based on these data, no significant statistical association was observed between seropositivity for Leishmania spp., and sex, age, presence of clinical signs, evaluated risk factors, and positivity for retroviruses. These findings demonstrated for the first time that cats from Mossoró, Rio Grande do Norte, are being exposed to this zoonosis and might be part of the epidemiological chain of transmission of visceral leishmaniasis.

Effects of Regulatory T Cell Depletion on NK Cell Responses against Listeria monocytogenes in Feline Immunodeficiency Virus Infected Cats.

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance, preventing autoimmune diseases and restraining chronic inflammatory diseases. Evidence suggests Treg cells and NK cells have important roles in feline immunodeficiency virus (FIV) pathogenesis; however, in vivo studies investigating the interplay between these two cell populations are lacking. We previously described innate immune defects in FIV-infected cats characterized by cytokine deficits and impaired natural killer cell (NK) and NK T cell (NKT) functions. In this study, we investigated whether in vivo Treg depletion by treatment with an anti-feline CD25 monoclonal antibody would improve the innate immune response against subcutaneous challenge with Listeria monocytogenes (Lm). Treg depletion resulted in an increased overall number of cells in Lm-draining lymph nodes and increased proliferation of NK and NKT cells in FIV-infected cats. Treg depletion did not normalize expression of perforin or granzyme A by NK and NKT cells, nor did Treg depletion result in improved clearance of Lm. Thus, despite the quantitative improvements in the NK and NKT cell responses to Lm, there was no functional improvement in the early control of Lm. CD1a+ dendritic cell percentages in the lymph nodes of FIV-infected cats were lower than in specific-pathogen-free control cats and failed to upregulate CD80 even when Treg were depleted. Taken together, Treg depletion failed to improve the innate immune response of FIV-infected cats against Lm and this may be due to dendritic cell dysfunction.

Molecular and serological survey of carnivore pathogens in free-roaming domestic cats of rural communities in southern Chile.

Owned, free-roaming domestic cats are abundant in the Chilean countryside, having high probability of contact with wildlife and potentially participating as reservoirs of zoonotic pathogens. In the present study, 131 cats from two remote study areas (Valdivia and Chiloe Island) in southern Chile were analyzed for infection/exposure to eight pathogens. Serum samples from 112 cats were tested for antigens against feline leukemia virus (FeLV antigen-ELISA) and antibodies against feline immunodeficiency virus (FIV-ELISA) and canine distemper virus (CDV-serum neutralization), yielded occurrence of 8.9, 1.7 and 0.8% respectively. The presence of DNA of five vector-borne pathogens, piroplasmids, Ehrlichia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp. was investigated in thirty cats. Overall observed occurrence was 6.6% (2/30) for both Anaplasma platys, and B. henselae, and 3.3% (1/30) for both Bartonella sp. and Theileria equi. Observed occurrence for all vector-borne pathogens in Valdivia area was significantly higher than in Chiloe Island (5/15 vs 0/15; P=0.04). Our results represent the first description of exposure to CDV and DNA detection of T. equi and A. platys in domestic cats in Chile. The results highlight the importance of performing pathogen screening in owned, free-roaming rural cats to evaluate their potential role as reservoirs of infection and vectors for disease transmission to wildlife.

Immunopathologic Effects of Prednisolone and Cyclosporine A on Feline Immunodeficiency Virus Replication and Persistence.

Feline immunodeficiency virus (FIV) induces opportunistic disease in chronically infected cats, and both prednisolone and cyclosporine A (CsA) are clinically used to treat complications such as lymphoma and stomatitis. However, the impact of these compounds on FIV infection are still unknown and understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective therapies. To determine the immunologic and virologic effects of prednisolone and CsA during FIV infection, FIV-positive cats were administered immunosuppressive doses of prednisolone (2 mg/kg) or CsA (5 mg/kg). Both prednisolone and CsA induced acute and transient increases in FIV DNA and RNA loads as detected by quantitative PCR. Changes in the proportion of lymphocyte immunophenotypes were also observed between FIV-infected and naïve cats treated with CsA and prednisolone, and both treatments caused acute increases in CD4+ lymphocytes that correlated with increased FIV RNA. CsA and prednisolone also produced alterations in cytokine expression that favored a shift toward a Th2 response. Pre-treatment with CsA slightly enhanced the efficacy of antiretroviral therapy but did not enhance clearance of FIV. Results highlight the potential for drug-induced perturbation of FIV infection and underscore the need for more information regarding immunopathologic consequences of therapeutic agents on concurrent viral infections.

Prevalence, Geographic Distribution, Risk Factors and Co-Infections of Feline Gammaherpesvirus Infections in Domestic Cats in Switzerland.

Recently, a gammaherpesvirus was described in domestic cats (FcaGHV1). The goal of the present study was to investigate the presence of FcaGHV1 in Swiss domestic cats and analyze potential risk factors. Blood samples from 881 cats presented to veterinarians in all Swiss cantons and from 91 stray cats and neoplastic tissue samples from 17 cats with lymphoma were evaluated. FcaGHV1 was detected by real-time PCR targeting the glycoprotein B gene, followed by sequencing. Blood samples were also tested for feline hemoplasmas, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). The molecular prevalence of FcaGHV1 was 6.0% (95% confidence interval (CI), 4.5-7.8%) in cats presented to veterinarians and 5.5% (95% CI, 1.8-12.4%) in stray cats. FcaGHV1 PCR-positive cats originated from 19/26 Swiss cantons. Factors significantly associated with FcaGHV1 detection included male sex, age >3 years, nonpedigree status and co-infection with FIV and hemoplasmas. Moreover, FeLV viremia tended to be associated with FcaGHV1 detection. High FcaGHV1 blood loads were found more frequently in FeLV-viremic cats and less frequently in hemoplasma-infected cats than in uninfected cats. Clinical information was unavailable for most of the 881 cats, but leukemia, carcinoma and cardiomyopathy were reported in FcaGHV1-positive cats. None of the tissue samples from the 17 cats with lymphoma tested positive for FcaGHV1. Sequence analyses revealed homogeneity among the Swiss isolates and >99.7% identity to published FcaGHV1 sequences. In conclusion, FcaGHV1 is present in Switzerland with a similar prevalence in cats presented to veterinarians and in stray cats. The pathogenic potential of FcaGHV1 needs further evaluation.

Prevalence of and factors associated with feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) in cats of the state of Santa Catarina, Brazil.

A cross-sectional study was conducted in 274 cats for determination of FeLV antigenemia and FIV seropositivity and factors associated with those infections in cats presented at the Veterinary Hospital of the Santa Catarina State University - UDESC (Brazil). Apparent prevalence for sick cats at the hospital population was 28.41% (95%CI 21.88-34.94%) for FeLV, 7.65% (95%CI 3.71-11.50%) for FIV and 2.18% (95%CI 0.56-5.47%) for both viruses. For healthy cats, the apparent prevalence was 9.89% (95%CI 3.75-16.02%) for FeLV, 2.20% (95%CI 0.34-7.75%) for FIV by immunoassay (ELISA). Average age for FeLV- and FIV-positive individuals was 38.32 and 64.25 months, respectively. Behavior such as aggressiveness and sex (male) were both associated with increased odds of result positivity test for FeLV and FIV; older animals were also associated with FIV test results. A very small proportion of the animals were vaccinated against FeLV and none against FIV. Most of the animals were adopted from shelters or rescued from streets, living with multiple cats that had access to outdoors. The high prevalence of FeLV suggests a need for better control strategies against this disease.

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians.

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel-O-Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point-of-care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV-vaccinated cats, because of the production of vaccine-induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV-vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness™ and Anigen Rapid™) were able to accurately distinguish between FIV-vaccinated and FIV-infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer-reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV-vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV-vaccinated cats to detect 'vaccine breakthroughs'; and the potential off-label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.

Co-infection with feline retrovirus is related to changes in immunological parameters of cats with sporotrichosis.

Feline sporotrichosis due to Sporothrix brasiliensis is frequently severe and often correlated to zoonotic transmission. Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) cause immunodeficiency in cats; no association has been identified with critical cases of sporotrichosis. Moreover, the cytokine profile in Sporothrix-infected cats and a potential impact of retrovirus co-infections on their immunity is unknown. This study assessed immunological parameters in cats with sporotrichosis with and without FIV or FeLV co-infection. FeLV infection was detected by antigen ELISA and by provirus PCR. FIV infection was investigated through ELISA and Western blot. Cytokine transcription (IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α) was quantified using RT-qPCR and lymphocyte subpopulations (CD4, CD8, CD5 and CD21) were assessed by flow cytometry. Thirty cats with sporotrichosis were recruited to the study, including three FIV-positive and five FeLV-positive (progressive infection) cats. One cat with regressive FeLV infection was excluded from statistics. In comparison to retrovirus-negative cats, FIV-positive cats and FeLV-positive cats had higher IL-10 levels, FeLV-positive cats had lower IL-4 levels and FIV-positive cats had lower IL-12 levels and a lower CD4+/CD8+ ratio. Remarkably, all cats with poor general condition were FeLV (progressive infection) or FIV-positive, but the retrovirus status was not associated with the sporotrichosis treatment length or outcome. The immunological changes and the more severe clinical presentation observed in cats with retrovirus co-infections encourage future prospective studies that address the impact of these changes on prognostic determinants of feline sporotrichosis and the development of new therapy strategies that control disease spread.

Prospects in Innate Immune Responses as Potential Control Strategies against Non-Primate Lentiviruses.

Lentiviruses are infectious agents of a number of animal species, including sheep, goats, horses, monkeys, cows, and cats, in addition to humans. As in the human case, the host immune response fails to control the establishment of chronic persistent infection that finally leads to a specific disease development. Despite intensive research on the development of lentivirus vaccines, it is still not clear which immune responses can protect against infection. Viral mutations resulting in escape from T-cell or antibody-mediated responses are the basis of the immune failure to control the infection. The innate immune response provides the first line of defense against viral infections in an antigen-independent manner. Antiviral innate responses are conducted by dendritic cells, macrophages, and natural killer cells, often targeted by lentiviruses, and intrinsic antiviral mechanisms exerted by all cells. Intrinsic responses depend on the recognition of the viral pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), and the signaling cascades leading to an antiviral state by inducing the expression of antiviral proteins, including restriction factors. This review describes the latest advances on innate immunity related to the infection by animal lentiviruses, centered on small ruminant lentiviruses (SRLV), equine infectious anemia virus (EIAV), and feline (FIV) and bovine immunodeficiency viruses (BIV), specifically focusing on the antiviral role of the major restriction factors described thus far.

Utilization of Feline ELISpot to Evaluate the Immunogenicity of a T Cell-Based FIV MAP Vaccine.

The prototype and the commercial dual-subtype feline immunodeficiency virus (FIV) vaccines conferred protection against homologous FIV strains as well as heterologous FIV strains from the vaccine subtypes with closely related envelope (Env) sequences. Such protection was mediated by the FIV neutralizing antibodies (NAbs) induced by the vaccines. Remarkably, both prototype and commercial FIV vaccines also conferred protection against heterologous FIV subtypes with highly divergent Env sequences from the vaccine strains. Such protection was not mediated by the vaccine-induced NAbs but was mediated by a potent FIV-specific T-cell immunity generated by the vaccines (Aranyos et al., Vaccine 34: 1480-1488, 2016). The protective epitopes on the FIV vaccine antigen were identified using feline interleukin-2 (IL-2) and interferon-γ (IFNγ) ELISpot assays with overlapping FIV peptide stimulation of the peripheral blood mononuclear cells (PBMC) from cats immunized with prototype FIV vaccine. Two of the protective FIV peptide epitopes were identified on FIV p24 protein and another two protective peptide epitopes were found on FIV reverse transcriptase. In the current study, the multiple antigenic peptides (MAPs) of the four protective FIV peptides were combined with an adjuvant as the FIV MAP vaccine. The laboratory cats were immunized with the MAP vaccine to evaluate whether significant levels of vaccine-specific cytokine responses can be generated to the FIV MAPs and their peptides at post-second and post-third vaccinations. The PBMC from vaccinated cats and non-vaccinated control cats were tested for IL-2, IFNγ, and IL-10 ELISpot responses to the FIV MAPs and peptides. These results were compared to the results from CD4+ and CD8+ T-cell proliferation to the FIV MAPs and peptides. Current study demonstrates that IL-2 and IFNγ ELISpot responses can be used to detect memory responses of the T cells from vaccinated cats after the second and third vaccinations.

Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential.

BACKGROUND: Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication. RESULTS: We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV. CONCLUSIONS: Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.

A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia.

High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation.

Lessons Learned in Developing a Commercial FIV Vaccine: The Immunity Required for an Effective HIV-1 Vaccine.

The feline immunodeficiency virus (FIV) vaccine called Fel-O-Vax® FIV is the first commercial FIV vaccine released worldwide for the use in domestic cats against global FIV subtypes (A⁻E). This vaccine consists of inactivated dual-subtype (A plus D) FIV-infected cells, whereas its prototype vaccine consists of inactivated dual-subtype whole viruses. Both vaccines in experimental trials conferred moderate-to-substantial protection against heterologous strains from homologous and heterologous subtypes. Importantly, a recent case-control field study of Fel-O-Vax-vaccinated cats with outdoor access and ≥3 years of annual vaccine boost, resulted in a vaccine efficacy of 56% in Australia where subtype-A viruses prevail. Remarkably, this protection rate is far better than the protection rate of 31.2% observed in the best HIV-1 vaccine (RV144) trial. Current review describes the findings from the commercial and prototype vaccine trials and compares their immune correlates of protection. The studies described in this review demonstrate the overarching importance of ant-FIV T-cell immunity more than anti-FIV antibody immunity in affording protection. Thus, future efforts in developing the next generation FIV vaccine and the first effective HIV-1 vaccine should consider incorporating highly conserved protective T-cell epitopes together with the conserved protective B-cell epitopes, but without inducing adverse factors that eliminate efficacy.

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.

Major Histocompatibility Complex Class I (FLA-E*01801) Molecular Structure in Domestic Cats Demonstrates Species-Specific Characteristics in Presenting Viral Antigen Peptides.

Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag positions 40 to 48; RMANVSTGR [RMA9]). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular-dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and in vitro refolding of the mutant FLA-E*01801 complex demonstrated that Glu63 and Trp167 in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine.IMPORTANCE Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC-I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp167 blocks the left side of pocket A, causing P1D to conflict with Glu63 We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We identified 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC-I molecules present peptides.