| Astrovirus is considered to be the secondary cause of the viral diarrhea among infants and elders. Astrovirus distributes wildly in the world while it can infect various of mammals and avians and has a high variation in genome. Researches have revealed that genomic recombination may exist between different serologic types of astrovirus. So it may has the ability to spread between different species of animals or from animals to humans, which makes astrovirus become a potential zoonotic agent. In addition, the porcine astrovirus(PAstV) can cause a watery diarrhea in piglets when coinfected with porcine epidemic diarrhea virus and rotavirus, and thus causing sustantial economic losses in pig industry.To establish a novel reverse transcription-polymerase chain reaction(RT-PCR) for detection of porcine astroviruses, a pair of primers specific to RNA dependent RNA polymerase(RDRP) gene of PAstV, the most conserved region, was designed and then a rapid and efficient RT-PCR method for detecting PAstV was established and optimized based on the designed primers. The RT-PCR products, designated as fragment H, was cloned into pMD18-T vector as standards for the determination of the sensitivity of the RT-PCR method established. The results showed that the detection limit of this method was 103 copies/μl. Afterwards, small intestine and fecal samples(N=219) were examined by using the established assay. The results showed that the positive rate of PAstV was 51.7% for intestine samples and 29.2% for fecal samples.To address the molecular characteristics of PAstVs at the basis of the complete genome, 5 pairs of primer for amplification of full-length of genome and 2 pairs of primer for 5’ and 3’ RACE were designed. Therewith, two strains of PAstV, one from Jiangxi Ji’an(JXJA) and another from Jiangxi Zhangshu(JXZS) were sequenced, The full-length genome of JXJA and JXZS were 6,675 nt and 6,700 nt in size, respectively after annotated with DNAStar. The results of multialignment analyses of these two PAstV strains indicated that the nucleotide identity and the deduced amino acid identity of the complete genome were 82.2% and 66.3%, respectively. The identity of ORF1 a was the highest(94.6%(nt) and 97.6%(aa)), while the identity of ORF2 was the lowest(62.6%(nt) and 49.9%(aa)). Due to the variability of the ORF2, the presumed ORF2 capsid protein of PAstV strains of JXJA and JXZS in hydrophobicity and antigenicity were compared. In addition, a phylogenetic tree based on the complete genome of the two PAstV strains sequenced in this study and 30 reference astrovirus strains retreaved from Gen Bank was constructed. The phylogenetic tree showed that the sequenced strains were most closed with type 4 PAstVs. Phylogenetic tree based on the ORF2 gene showed that JXZS strain was fallen into the different branch unlike JXJA strain and other type 4 PAstVs, which were clustered into another group, suggesting the JXZS strain might be a novel genotype of PAstV.Together, the results from this study revealed the genetic characteristics of the genome of PAstVs currently circulting in Jiangxi province. Up to date, the reports on PAstV in China are very limited, e.g., there are only two complete genome sequences of PAstVs, from Shanghai and Guangxi, respectively available in GenBank. As the great variability of the sequence at the complete genome level when compared with other reference PAstV strains, the PAstVs determined in this study might be a novel genotype. The findings of this study provide a useful tool for detection of PAstVs and molecular basis which will enrich our knowledge on PAstV virology, which will benefit the prevention and control of the disease caused by PAstVs. |
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