Ciência Rural,Felis catusSanta gammaherpesvirus Maria, v.48: 03, 1 (FcaGHV1) e20170480, and 2018coinfections with feline viral pathogens http://dx.doi.org/10.1590/0103-8478cr20170480 in domestic cats in Brazil. 1 ISSNe 1678-4596 MICROBIOLOGY Felis catus gammaherpesvirus 1 (FcaGHV1) and coinfections with feline viral pathogens in domestic cats in Brazil Jacqueline Kazue Kurissio1, 2* Marianna Vaz Rodrigues1, 2 Sueli Akemi Taniwaki3 Marcelo de Souza Zanutto4 Claudia Filoni1, 2 Maicon Vinícius Galdino5 João Pessoa Araújo Júnior 1, 2* 1Departamento de Microbiologia e Imunologia, Instituto de Biociências, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), 18618-691, Botucatu, SP, Brasil. 2Instituto de Biotecnologia, Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Botucatu, SP, Brasil. E-mail: [email protected] 3Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Universidade de São Paulo (USP), SP, Brasil. 4Departamento de Clínicas Veterinárias, Centro de Ciências Agrárias, Universidade Estadual de Londrina (UEL), Londrina, PR, Brasil. 5Departamento de Bioestatística, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), 18618-693, Botucatu, SP, Brasil. ABSTRACT: Felis catus gammaherpesvirus 1 (FcaGHV1) may causes an asymptomatic infection that result in an efficient transmission and subsequently dissemination of the virus in feline population. This study used molecular detection by qPCR (quantitative PCR) based on DNA polymerase gene fragment amplification to evaluate the occurrence of FcaGHV1 and its correlation with other feline viral pathogens, such as Carnivore protoparvovirus 1 (CPPV-1), Felid alphaherpesvirus 1 (FeHV-1), and feline retroviruses such as feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Of the 182 blood samples evaluated 23.6% (43/182) were positives for FcaGHV1. Approximately 37.9% (33/87) of the samples that tested positive for retrovirus were also were positive for FcaGHV1 infection (P<0.0001). Among FIV- infected samples, 49% (24/49) were positive for FcaGHV1 (P<0.0001). FcaGHV1 infection was not associated with FeLV (P>0.66) or CPPV- 1 (P>0.46) coinfection. All samples were negative for FeHV-1. Male felines were significantly associated to FcaGHV1 (P<0.0001) and their risk of infection with FcaGHV1 was about of 7.74 times greater compared to females. Kittens (≤ 1year) were the least affected by FcaGHV1 infection, being verified a rate of 7.7% (4/52). Therefore, male cats over one year old and infected with FIV were considerably more likely to be infected with FcaGHV1. To our knowledge, this is the first study to report the occurrence and molecular detection of FcaGHV1 infection in domestic cats in Brazil and in South America. Key words: gammaherpesvirus, herpesvirus, feline pathogen, infectious diseases, diagnosis. Ocorrência de Felis catus gammaherpesvirus 1 (FcaGHV1) e coinfecções com patógenos virais felinos em gatos domésticos no Brasil RESUMO: Felis catus gammaherpesvirus 1 (FcaGHV1) pode causar uma infecção assintomática, que resulta em uma transmissão eficiente e consequente disseminação do virus na população felina. Este estudo utilizou a detecção molecular por qPCR (PCR quantitativa) baseado na amplificação de um fragmento do gene da DNA polimerase para avaliar a ocorrência de FcaGHV1, sendo correlacionado a outros patógenos virais felinos como Carnivore protoparvovirus 1 (CPPV-1), Felid alphaherpesvirus 1 (FeHV-1) e aos retrovírus felinos como vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV). Das 182 amostras de sangue avaliadas, 23,6% (43/182) foram positivas para FcaGHV1. Aproximadamente 37,9% (33/87) das amostras positivas para retrovirus também foram positivas para FcaGHV1 (P<0,0001). Entre as amostras FIV-infectadas, 49% (24/49) foram positivas para FcaGHV1 (P<0,0001). A infecção por FcaGHV1 não foi associada à coinfecção por FeLV (P>0,66) e CPPV-1 (P>0,46). Todas as amostras foram negativas para FeHV-1. Felinos machos foram significativativamente associados à infecção por FcaHV1 (P <0,0001) e o risco de infecção com FcaGHV1 foi aproximadamente 7,74 vezes maior comparados às femeas. Os filhotes (≤1 ano) foram os menos acometidos pela infecção por FcaGHV1 sendo verificado uma proporção de 7.7% (4/52). Assim, gatos machos com mais de um ano de idade e infectados por FIV foram, consideravelmente, mais susceptíveis a serem infectados com FcaGHV1. Para nosso conhecimento, este é o primeiro estudo que relata a ocorrência de infecção e detecção molecular de FcaGHV1 em gatos domésticos no Brasil e na América do Sul. Palavras-chave: gamaherpesvirus, herpesvirus, patógenos felinos, doenças infecciosas, diagnóstico. INTRODUCTION subfamilies: alpha-, beta- and gamma herpesvirus (DAVISON, 2010). The majority of members of Herpesviruses are large and enveloped the Gammaherpesvirinae subfamily are potentially viruses that have a linear double-stranded DNA oncogenic (DAVISON et al., 2009). Thus, gamma genome. They are divided into three distinct herpesviruses are associated with the development Received 06.12.17 Approved 01.12.18 Returned by the author 02.28.18 CR-2017-0480.R3 Ciência Rural, v.48, n.3, 2018. 2 Kurissio et al. of lymphoproliferative disorders that persist were from cats that had infectious disease suspect after the viruses became latent in B lymphocytes and were from several localities of the country. (BARTON et al., 2011; SPECK & GANEM, 2010). DNA was extracted from the blood samples using a These viruses can be effectively contained by the commercial kit (GE HealthCare, Buckinghamshire, immune system of infected healthy individuals who UK) following the manufacturer’s recommendations. remain asymptomatic (USHERWOOD et al., 2001). Through molecular techniques using qPCR However, in immunosuppressed individuals, infection (quantitative PCR), were detected proviral DNA in with gammaherpesvirus, either as a sole agent or 87 feline retrovirus-positive DNA samples, while 95 by coinfection with other viruses, may lead to the samples were considered negative for the retrovirus. development of neoplasms and lymphoproliferative Of those 87 samples retrovirus-positive, 49 were diseases (MEANS et al., 2007; KAYE et al., 2016). FIV-positive, 32 were FeLV-positive and 6 samples Felis catus gammaherpesvirus 1 were positive for both FIV and FeLV. All samples (FcaGHV1) was first identified in the United were then tested for FcaGHV1, CPPV-1 and FeHV-1. States by TOYER et al (2014) and has since been Detection of FIV proviral DNA was performed based associated with coinfections, mainly with feline on the method of TANIWAKI & ARAÚJO JR (2011), retroviruses (BEATTY et al., 2014; TROYER et and the FeLV proviral DNA detection method used al., 2014). Coinfections involving retroviruses and primers described by TANDON et al. (2005) using an herpesviruses are common and can occur in humans ABI 7500 Fast thermal cycler (Applied Biosystems, and animals (ISFORT et al., 1992). Synergistic CA, USA). The CPPV-1 VP2 gene and the FeHV- interactions between herpes and retroviruses can 1 glycoprotein B gene were detected based on the favor disease progression producing a long latency methods described by DECARO et al. (2008) and period and efficient immune system evasion VÖGTLIN et al. (2002), respectively, using an ABI (EVERMANN et al., 1991). However, the clinical 7300 thermal cycler (Applied Biosystems). signs and pathogenesis related to FcaGHV1 infection The qPCR assay was used as qualitative are still not clear (KAYE et al., 2016; STUTZMAN- method for detecting the presence of FcaGHV1 RODRIGUEZ et al. 2016). DNA. To detect FcaGHV1 DNA, specific primers FcaGHV1 DNA has been detected in cats were designed to target the DNA polymerase from countries on most continents including Europe, gene based on the GenBank sequence KF840715, Oceania, North America and Asia (TROYER et al., resulting in a product of 174bp in length. The 2014; BEATTY et al., 2014; ERTL et al., 2015; primer sequences used for FcaGHV1 gene TATENO et al., 2017). Until date, no cases of amplification were FcaGHV1-P1 (forward) FcaGHV1 infection have been reported in felids in - 5’CCACATTGAAAGGCCCAAGG-3’ and Brazil or elsewhere in South America. Thus, this FcaGHV1-P2 (reverse) - 5’TGTTGTCTCCAGAT study investigated the occurrence of FcaGHV1 GCTGACG-3’. The qPCR reaction was then infection among domestic cats and correlated it prepared with 10μL of GoTaq® qPCR Master Mix with feline retroviruses (feline immunodeficiency (Promega, WI, USA), 0.2 µM of each FcaGHV1 virus (FIV) and feline leukemia virus (FeLV)) primer, 2.5% of DMSO (≥99.5%), 4μL of extracted and with other viral pathogens such as Carnivore DNA and nuclease free water to a total volume protoparvovirus 1 (CPPV-1), previously known of 20μL. The thermal cycling conditions were as as feline panleukopenia virus (FPV), and Feline follows: 95°C for 2min followed by 40 cycles at alphaherpesvirus 1 (FeHV-1). This research also 95°C for 10s and 60°C for 30s using an ABI 7500 evaluated potential risk factors of domestic cats for Fast thermal cycler (Applied Biosystems). Samples FcaGHV1 infection, regarding sex and age. evaluated were considered positive when they showed a single melting curve peak at a melting MATERIALS AND METHODS temperature (Tm) of approximately 77.20ºC (±0.55). During qPCR testing, amplicons of We analyzed EDTA-treated whole blood samples considered