Background: The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J). Findings: The expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF. Conclusions: The RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

Porcine respiratory disease complex (PRDC), a common piglet disease, causes substantive economic losses in pig farming. To investigate the viral diversity associated with PRDC, the viral communities in serum and nasal swabs from 26 PRDC-affected piglets were investigated using metagenomics. By deep sequencing and de novo assembly, 17 viruses were identified in two pooled libraries (16 viruses from serum, nine from nasal swabs). Porcine circovirus (PCV)-2, porcine reproductive and respiratory syndrome virus (PRRSV) and pseudorabies virus, all commonly associated with PRDC, were identified in the two pooled samples by metagenomics, but most viruses comprised small linear and circular DNAs (e.g. parvoviruses, bocaviruses and circoviruses). PCR was used to compare the detection rates of each virus in the serum samples from 36 PRDC-affected piglets versus 38 location-matched clinically healthy controls. The average virus category per sample was 6.81 for the PRDC-affected piglets and 4.09 for the controls. Single or co-infections with PCV-2 or PRRSV had very high detection rates in the PRDC-affected piglets. Interestingly, porcine parvovirus (PPV)-2, PPV-3, PPV-6 and torque teno sus virus 1a were significantly associated with PRDC. These results illustrate the complexity of viral communities in the PRDC-affected piglets and highlight the candidate viruses associated with it.

The yak (Bosgrunniens) is a unique domestic bovine species that plays an indispensable role for herdsmen in the Qinghai–Tibet Plateau. Here, 336 diarrhoeic samples were collected from yaks on 29 farms in the Qinghai–Tibet Plateau from 2015 to 2017. Approximately 69.05 % (232/336) of the diarrhoeic samples were assessed as bovine coronavirus (BCoV)-positive by RT-PCR assay, and most of the detected strains showed a unique evolution based on 40 spike (S), nucleocapsid (N) and haemagglutinin-esterase (HE) gene fragments. Notably, the 12 complete S genes detected shared 1 identical amino acid mutation (E121V) in the S1 subunit compared with the other 150 complete S genes in the GenBank database. Furthermore, a BCoV strain (designated YAK/HY24/CH/2017) was isolated from one diarrhoeic sample (virus titre : 108.17TCID50 ml−1), and a phylogenetic analysis based on complete genome sequences revealed that strain YAK/HY24/CH/2017 has the closest genetic relationship with the BCoV prototype strain Mebus. Interestingly, 2 significant characteristics were observed in the genome of strain YAK/HY24/CH/2017 : (1) the strain had 26 unique amino acid variations in the S gene compared with the other 150 BCoV S genes in the GenBank database and (2) a recombination event was identified between the esterase and lectin domains of the HE gene. In conclusion, this study revealed the high prevalence of BCoV in yaks in the Qinghai–Tibet Plateau. To the best of our knowledge, this is the first description of the molecular prevalence of BCoV in yaks and of a BCoV genome with an HE gene recombination.