RESUMO
Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Animais , Febre Suína Africana/virologia , Suínos , Vírus da Febre Suína Africana/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Perfilação da Expressão Gênica , Genes Essenciais/genéticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF) are economically important diseases of pigs throughout the world. During an outbreak, all age groups of animals except piglets < 1 month of age were affected with symptoms of high fever, cutaneous hemorrhages, vomition with blood, diarrhea, poor appetite, ataxia, and death. The outbreak was confirmed by the detection of the N gene of the porcine reproductive and respiratory syndrome virus (PRRSV) and the VP72 gene of the African swine fever virus (ASFV) by PCR in representative blood samples from affected pigs followed by Sanger sequencing. Mixed infection was also confirmed by simultaneous detection of both the viruses using multiplex PCR. Phylogenetic analysis of both the viruses revealed that the outbreak was related to ASFV and PRRSV strains from China which were also closely related to the PRRSV and ASFV strains from the recent outbreak from India. The study confirmed the involvement of genotype II of ASFV and genotype 2 of PRRSV in the present outbreak. Interestingly, PRRSV associated with the present outbreak was characterized as a highly pathogenic PRRSV. Therefore, the present study indicates the possibility of future waves or further outbreaks of these diseases (PRRS and ASF) in this region. This is the first report of ASFV and PRRSV co-infection in pigs from India.