High-resolution melt curve analysis to confirm the presence of co-circulating isolates of avian nephritis virus in commercial chicken flocks

Abstract

Avian Nephritis Virus (ANV) has been implicated in poor growth and renal disease of young chickens. This paper describes the development of a reverse-transcriptase polymerase chain reaction for the detection of ANV in commercial meat chickens and the use of high-resolution melt curves to detect the presence of genetically different ANVs. Pooled cloacal swabs from both healthy and ill commercial chicken broiler flocks were tested for the presence of ANV using a combination of polymerase chain reaction, molecular cloning, high-resolution melt curve analysis and sequencing. Except for one, all specimens were found to contain two genetically different ANVs. Phylogenetic analysis of the capsid amino acid sequences revealed the presence of four of six groups of ANV identified previously in other countries as well as in two novel groups of ANV. Phylogenetic analysis of nucleotide sequences of partial polymerase, capsid and 3′ untranslated regions reveal that the genes of individual ANV virus isolates have different ancestors. This was shown to be due to a template-switching event in the capsid gene that resulted in the 3′ end of the capsid gene and the 3′ untranslated region of one ANV isolate being transferred to another ANV. These results reveal that infection of chicken flocks with multiple ANV isolates is common and this needs to be taken into consideration in diagnosis of ANV using molecular techniques and in future epidemiological investigations.

Primer design for complete sequencing of the Avastrovirus ORF2 gene

“…The complete sequencing of ORF2 by primer walking is a well-known strategy used in ANV and CAstV strains (TODD et al, 2011;SMYTH et al, 2012). The 3´ Rapid Amplification of cDNA Ends (RACE) technique has also been used for the complete sequencing of ORF2 in ANV strains (CHAMINGS et al, 2015). In fact, the unique complete TAstV-1 ORF2 sequence available in GenBank was obtained by RACE methodology (JONASSEN et al, 2001).…”

<b>Detecção e genotipagem de astrovírus em amostras fecais de aves de produção procedentes de diferentes criações comerciais brasileiras</b>

“…Adicionalmente, como ferramenta para a detecção de cepas co-circulantes, além de uso da clonagem, têm se relatado em outro estudo outras técnicas como o Análise de Curvas de Dissociação de Alta Resolução (HRMA, High Resolution Melting Analysis), sendo descrito para Astrovírus por Chamings et al (2015). A detecção de mais de uma cepa circulante num mesmo lote de aves ou amostra é importante porque representa uma fonte de possíveis eventos recombinatórios e co-infecções, que podem gerar novos tipos de vírus e se disseminar a outras espécies.…”

“…Estas variantes virais são produzidas como consequência de uma alta taxa de mutações durante a replicação viral, e em alguns casos por recombinação viral e/ou rearranjos de fragmentos genômicos (DOMINGO; SHELDON; PERALES, 2012). Este tipo de eventos, já foi observado em astrovírus humanos (MARTELLA et al, 2014), sendo considerado por alguns científicos como uma importante assinatura de infecção por astrovírus em geral (CHAMINGS et al, 2015). Este tipo de eventos acontece majoritariamente em vírus RNA devido à alta propensão a erros da polimerase viral RNA dependente RNA polimerase (RdRp), que por sua vez são devidos a um baixa fidelidade intrínseca e falta de mecanismos de correção da enzima (RAMIREZ et al, 2005).…”

>b<Detection and molecular characterization of enteric viruses in commercial birds >/b<

“…An aliquot of each supernatant was used for DNA and RNA extraction, using the organic phenol-chloroform method, according to procedures described by Green et al, (2017) and Chomczynski and Sacchi (1987). The concentration and quality of nucleic acids were evaluated in a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA).…”

“…ChPV was detected in the liver, intestines, and pancreas, which are areas commonly used for ChPV detection due its pathogenicity as an enteric virus, considering the variety of organs where this virus can be found, including the brain, duodenal loop, and even on cloacal swabs (MARUSAK et al, 2010;NUÑEZ et al, 2016b). Astroviruses, such as CAstV and ANV, are commonly detected in the digestive organs and fecal samples of sick and healthy birds (ZHAO et al, 2011;HUNGARICA et al, 2015;CHAMINGS et al, 2015), supporting our findings of astrovirus in the liver, intestines, and pancreas (Table 13). IBV can be detected in the intestinal content, trachea, lung, liver, bursa of Fabricius, pancreas, thymus, kidney, proventriculus, and spleen of affected birds FAN et al, 2012), according to the dynamic distribution of the virus, resulting in a wide range of useful organs for molecular detection of this virus, which in our case helped to determine the presence of the pathogen in liver, intestine, pancreas, cloacal swabs, and caecal tonsils (Table 13).…”