Pesq. Vet. Bras. 36(5):357-362, maio 2016 DOI: 10.1590/S0100-736X2016000500001 First phylogenetic analysis of Avipoxvirus (APV) in Brazil1 2 2 2 2 3 2 2 4 Hiran C. Kunert-Filho *, Samuel P. Cibulski , Fabrine2 Finkler , Tiela T. Grassotti , Fátima R.F. Jaenisch , Kelly C.T. de Brito , Daiane Carvalho , Maristela Lovato ABSTRACT.- and Benito G. de Brito First phylogenetic analysis of Avipoxvi- rus (APV) in Kunert-FilhoBrazil. Pesquisa H.C., Veterinária Cibulski S.P., Brasileira Finkler 36(5):357-362F., Grassotti T.T., Jaenisch F.R.F., Brito K.C.T., Carvalho D., Lovato M. & Brito B.G. 2016. Laboratório de Saúde E-mail:das Aves e Inovação Tecnológica, Instituto de Pesquisas Veterinárias Desidério Finamor, FEPAGRO Saúde Animal, Estrada do Conde 6000, Eldorado do Sul, RS 92990-000, Brazil. [email protected] This study represents the first phylogenetic analysis of avian poxvirus recovered from- turkeys in Brazil. The clinical disorders related to fowlpox herein described occurred in a turkey housing system. The birds displaying characteristic pox lesions which were ob served on the neck, eyelids and beak of the turkeys. Four affected turkeys were randomly chosen, euthanized and necropsied. Tissues samples were submitted for histopathologicalP4b analysis and total DNA was further extracted, amplified by conventional PCR, sequenced- and phylogenetically analyzed. Avian poxviruses specific PCR was performed basedP4b on core protein gene sequence. The histological analysis revealed dermal inflammatory pro- cess, granulation tissue, hyperplasia of epithelial cells and inclusion bodies. The ® gene was detected in all samples. Sequencing revealed a 100% nucleotide and amino acid se quence identity among the samples, andAvipoxvirus the sequences were deposited in GenBank . The four Avian poxviruses fragments sequenced in this study clustered along the A1 clade of avipoxviruses, and were classified as (APV). Additional studies, such as virus isolation, PCR and sequencing includinga large number of specimens from the Brazilian- turkey production must be conducted due to the hazardous risk that poxvirus infections may cause to the Brazilian poultry production scenario, given that Brazil’s turkey produc tion attracts attention due to its economic importance worldwide. Our findings point to- the need to identify the prevalence of APV in Brazilian turkey production, to perform risk assessment studies and continued surveillance of APV infections in both wild and commer Poxviridae Avipoxvirus cial avian species. RESUMO.- [PrimeiraINDEX análiseTERMS: Turkey, filogenética de ,Avipoxvirus , APV, PCR,Brasil. phylogenetic analysis. - (APV) no Brasil.] Este trabalho representa a primeira aná- Os distúrbios clínicos relacionados com bouba aviá ria aqui descritos ocorreram em um sistema de alojamento lise filogenética de Poxvirus aviário detectado em perus no de perus. As aves apresentaram lesões características de- 1 varíola observadas no pescoço, pálpebras e bico das aves. 2 Received on October 22, 2015. - Quatro perus com sinais característicos foram escolhi- Accepted for publication on February 4, 2016. - Laboratório de Saúde das Aves & Inovação Tecnológica (LSAIT), Insti dos aleatoriamente, sacrificados e submetidos à autópsia.- tuto de Pesquisas Veterinárias Desidério Finamor (IPVDF), FEPAGRO Saú Amostras de tecido foram submetidas à análise histopato de3 Animal, Estrada do Conde 6000, Eldorado do Sul, RS 92990-000, Brazil. lógica e o DNA total foi extraído, amplificado por PCR con *Corresponding author: [email protected] vencional e os amplicons foram sequenciados e analisados ​​ 4 Laboratório de Patologia, Embrapa Suínos e Aves, Rodovia BR-153 Km filogeneticamente. A PCR específica para Poxvírus aviário 10, Cx. Postal 21, Concórdia, SC 89700-000, Brazil. P4b Laboratório Central de Diagnóstico de Patologias Aviárias (LCDPA), foi realizada com base na seqüência do gene da proteína Departamento de Medicina Veterinária Preventiva (DMVP), Centro de do núcleo . A análise histológica revelou um processoP4b Ciências Rurais (CCR), Universidade Federal de Santa Maria (UFSM), Av. inflamatório dérmico, tecido de granulação, hiperplasia de Roraima 1000, Santa Maria, RS 97105-900, Brazil. células epiteliais e corpúsculos de inclusão. O gene foi 357 Hiran C. Kunert-Filho et al. 358 - mopoxvirus Gammaentomopoxvirus one ( Avipoxvirus detectado em todas as amostras. O sequenciamento reve- , and a not assingned lou uma identidade® entre nucleotídeos e aminoácido de- www.ictvonline.org/virusTaxonomy.asp). 100% entre as amostras e as sequências foram deposita is the only genus capable of infecting birds (Gubser et al. das no GenBank . Os quatro fragmentos de poxvírusAvipoxvirus aviá 2004). Avipoxvirus: Canarypox virus Fowlpox virus rio sequenciado neste estudo foram agrupados no clado- JuncopoxTo date, virus ten Mynahpoxspecies have virus beenPigeonpox described virus as belongingPsittaci- A1 de avipoxvirus e foram classificados como nepoxto the virusgenusQuailpox virus Sparrowpox virus, Starlingpox, (APV). Estudos adicionais, como isolamento viral, PCR e se virus Turkeypox, virus , , quenciamento, incluindo um grande número de perus da , , , - produção brasileira devem ser conduzidos devido ao grave and Peacockpox. There virus are Penguinpoxthree other virusspeciesCro as- risco que a infecção por poxvírus pode causar ao cenário de wpoxadditional virus attempt ( by the International Commitee on Taxo produção avícola brasileira, tendo em vista que a produção nomy of Viruses: Fowlpox, virus , - brasileira de perus atrai atenção devido a sua importância www.ictvonline.org/virusTaxonomy.asp). The mundial. Nossos resultados apontam para a necessidade de- prototype of the APV family is (FWPV), whi identificar a prevalência da APV na produção de peru no ch causes lesions on the skin and in the upper respiratory- Brasil, para realizar estudos de avaliação de risco e conti tract (Tadese et al. 2008). nuada monitoração de infecções por APV nas espécies de Poxvirus infections cause cutaneous and internal le Poxviridae, Avipoxvirus, aves comerciais e silvestres. sions and may affect chickens and turkeys (Tripathy & Reed TERMOS DE INDEXAÇÃO: Peru, APV, PCR, 2013). Microscopically, the skin lesions are characterized- análise filogenética. INTRODUCTION by severe hypertrophy and hyperplasia of epidermal cell,- many of which undergoing ballooning degeneration and of- - ten containing intracytoplasmic eosinophilic inclusion bo dies called Bollinger bodies. These inclusion bodies are pa Avian poxviruses cause a common viral disease in bird spe thognomonic for Avian poxvirus infection (Fletcher 2008). cies. It is worldwide distributed, affecting at least 3% of Histopathological examination is considered the major 232 species of birds (Bolte et al. 1999, Tripathy et al. 2000, method for APV diagnosis, but other techniques, such as Kim et al. 2003, Godoy et al. 2013). APV may manifest in- cell tissue cultures, virus isolation in CAM of embryonated two different ways: lesions on the skin, commonly named chicken eggs, serologic methods and electron microscopy- as cutaneous form, and lesions in the mouth, pharynx, la (Tripathy & Reed 2013) are available to isolate or detect rynx, esophagus and trachea, called diphtheritic form. Avipoxvirus the virus. To date, the most sensitive techniques includeP4b se These two forms may occur simultaneously (Biswas et al. veral molecular approaches (Manarolla et al. 2010). Hence,- 2011). When (APV) affects poultry production, the amplification of a 578-base pair (bp) region of , a it can lead to decreased egg production, reduced growth,- highly conserved gene of APV, is commonlyP4b used to diag and increased mortality. In canaries, APV causes severe nose APV infections, which is highly conserved amongst all- pulmonary damage, leading to extremely high mortality ra- poxviruses (Binns et al. 1989). The gene has already tes (Manarolla et al. 2010). been reported in other phylogenetic studies of APV to dis Viruses belonging to the APV family are large, oval, bri- tinguish between clades A, B, C, A1-4 and B1-2 (Lüschow et ck-shaped, and enveloped. Double-stranded DNA ranging al. 2004, Weli et al. 2004, Jarmin et al. 2006). In addition, from 130 to 375 kb in linear configuration is the main cha conventional and real time PCR techniques provide results racteristic of the members of this family (Bolte et al. 1999, more rapidly than virus isolation. Manarolla et al. 2010). APV DNA usually presents a low The aim was to characterize Avian poxvirus isolates (about 30e.g. to 40%) GC-content (Bolte et al. 1999), encoding from turkeys in Southern Brazil. For this purpose we used about 150 genes (Lefkowitz et al. 2006). Avian cell tissue histopathologicalMATERIALS analysis, PCR, AND and METHODS phylogenetic analysis. cultures ( , chicken embryo fibroblasts, chicken embryo dermis and kidney cells, and duck embryo fibroblast) and Sample collection. - Meleagris gallopavo - chorioallantoic membrane (CAM) of embryonated eggs are In this survey, about 80 out of 120- the tissues of choice for APV isolation, due to its easily re 140-days-old turkeys ( ) housed in the Insti plication. Nonetheless, turkeys isolates may fail to grow in- tuto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Eldo- these cell lines, even after repeated passages (Tripathy &- rado do Sul, Rio Grande do Sul State, Brazil, demonstrated clinical- Reed 2013). According to Jarmin et al. (2006), APV repli signs consistent with Pox. The characteristic lesions were obser catesChordopoxvirinae