Abstract
Porcine astroviruses (PAstVs) are prevalent in pigs worldwide, and five genotypes have been reported to circulate in China. However, little is known about the coinfection status of PAstVs. For differential and simultaneous diagnoses of these five genotypes of PAstVs, a multiplex RT-PCR method was established on the basis of the ORF2 gene of type 1 PAstV, and the ORF1ab genes of type two to five PAstVs. This quintuple PCR system was developed through optimization of multiplex PCR and detection sensitivity and specificity. The results showed that this multiplex RT-PCR method could specifically detect all the five PAstV genotypes without cross-reaction to any other major viruses circulating in Chinese pig farms. The detection limit of this method was as low as 10 pg of standard plasmids of each PAstV genotype. In addition, a total of 275 fecal samples collected from different districts of Guangxi, China, between April 2019 and November 2020, were tested by this newly established multiplex RT-PCR. Moreover, the sensitivity and specificity of monoplex and multiplex RT-PCR methods were compared by detecting the same set of clinical positive samples. The results revealed that PAstV1 (31/275), PAstV2 (49/275), PAstV3 (36/275), PAstV4 (41/275), and PAstV5 (22/275) were all detected, and dual (PAstV1+PAstV2, PAstV1+PAstV3, PAstV2+PAstV3, PAstV2+PAstV4, PAstV3+PAstV4, and PAstV4+PAstV5) or triple genotypes (PAstV1+PAstV2+PAstV3 and PAstV2+PAstV3+PAstV4) of coinfections were also unveiled in this study. The detection result of multiplex PCR was consistent with that of monoplex PCR. Compared with monoplex PCR, this multiplex PCR method showed obvious advantages such as time and cost efficiency and high sensitivity and specificity. This multiplex RT-PCR method offered a valuable tool for the rapid and accurate detection of PAstV genotypes circulating in pig herds and will facilitate the surveillance of PAstV coinfection status.
Highlights
Astroviruses are non-enveloped, positive-sense, single-stranded RNA (+ssRNA) viruses whose genomes are 6–7 kb in length and contain three open reading frames (ORFs), namely, ORF1a, ORF1b, and ORF2 [1]
The monoplex RT-PCR result showed that the fragments at expected sizes of each genotype (124 bp for PAstV1, 573 bp for PAstV2, 175 bp for PAstV3, 485 bp for PAstV4, and 305 bp for PAstV5) were successfully amplified from the stored positive samples (Figure 1A)
The standards and primer sets of type one to five Porcine astroviruses (PAstVs) were added to the reaction tube one by one, and the results demonstrated that all these target genes were wellamplified without any interference, indicating good amplification and high efficacy of this multiplex RT-PCR method (Figure 1B)
Summary
Astroviruses are non-enveloped, positive-sense, single-stranded RNA (+ssRNA) viruses whose genomes are 6–7 kb in length and contain three open reading frames (ORFs), namely, ORF1a, ORF1b, and ORF2 [1]. The overall prevalence rates and the dominant genotypes of PAstV in different countries or districts varied on geographic locations. 70.4% of pigs were detected to be PAstV4 positive in five European countries [13]. All the five known PAstV genotypes have been detected in China [6, 14], and the overall prevalence rate ranged from 17.5% in Sichuan Province to 56.4% in Guangxi Province [6, 15, 16]. PAstV2 and PAstV4 were detected from the blood and fecal samples, causing viremia and circulate in pig herds [19]. Multiple genotypes of PAstV coinfections will further accelerate the genetic variation of this virus and bring challenges to the monitoring of PAstVs. In addition, the interspecies barrier of PAstV may not be strict. Results of genetic evolution analysis suggest that PAstV may have crossed the interspecies barrier between humans and other animals [23, 25, 26]
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