Detection of novel viruses in porcine fecal samples from China
© Yu et al; licensee BioMed Central Ltd. 2013
Received: 27 April 2012
Accepted: 26 November 2012
Published: 30 January 2013
Pigs are well known source of human infectious disease. To better understand the spectrum of viruses present in pigs, we utilized the 454 Life Sciences GS-FLX high-throughput sequencing platform to sequence stool samples from healthy pigs.
Total nucleic acid was extracted from stool samples of healthy piglets and randomly amplified. The amplified materials were pooled and processed using a high-throughput pyrosequencing technique. The raw sequences were deconvoluted on the basis of the barcode and then processed through a standardized bioinformatics pipeline. The unique reads (348, 70 and 13) had limited similarity to known astroviruses, bocaviruses and parechoviruses. Specific primers were synthesized to assess the prevalence of the viruses in healthy piglets. Our results indicate extremely high rates of positivity.
Several novel astroviruses, bocaviruses and Ljungan-like viruses were identified in stool samples from healthy pigs. The rates of isolation for the new viruses were high. The high detection rate, diverse sequences and categories indicate that pigs are well-established reservoirs for and likely sources of different enteric viruses.
Many emerging viruses are of zoonotic origin and can cause epidemics in humans after overcoming the inter-species barrier through mutation or other genetic events, including recombination. The identification of previously unknown viruses in animal hosts is important for understanding their potential for cross-species transmission and emergence. Pigs are known to harbor a diverse array of viruses, including coronaviruses, astroviruses and kobuviruses [1–4], among which coronaviruses and astroviruses are well known viruses that can infect human. To better understand the spectrum of viruses present in pigs, we utilized the 454 Life Sciences GS-FLX high-throughput sequencing platform (454 Life Sciences/Roche, Branford, CT, USA), which has emerged as a non-biased, comprehensive and powerful tool for virus discovery in complex environmental samples [5, 6], to sequence stool samples from healthy pigs.
With the cooperation of the Lulong County CDC, stool samples were collected from healthy piglets < 3 months of age from several farms and sporadically distributed families that raised pigs in Lulong County during 2006–2009. The samples were transported on dry ice and stored at −80°C at the Chinese CDC.
The samples were screened for rotavirus, calicivirus and astrovirus by ELISA or routine PCR. Nine of the samples negative for these viruses were pooled, diluted (1:5 ratio, wt/vol) and then sequentially filtered through 0.45- and 0.22-μm membranes. The pooled sample included two samples that were previously demonstrated to contain a novel porcine bocavirus (6V/7V CHN) that was partially sequenced in our lab . Total nucleic acid was extracted from the pooled sample and cDNA was generated using random hexamers. Random PCR amplification  was performed and the amplified cDNA was used as the template for standard library construction and sequencing using Roche Genome Sequencer FLX Titanium pyrosequencing technology.
Human bocavirus (HBoV) was first identified in respiratory samples from patients with respiratory infections in 2005 . Subsequently, various bocaviruses have been found in respiratory and fecal specimens of human or animal origin, including HBoV 2–4, Gorilla bocavirus (GBoV1) and porcine bocavirus (PBoV1–4, 6V, and 7V) [19, 20]. The sizes of the 23 bocavirus contigs in this study ranged from 147 to 1256 bp, and they shared 74.6–99.2% nucleotide sequence identities with the best hit in the NCBI nt database. Contigs 2, 5, 18 and 19 mapped to the same portion of the NS1 region of the reference genome but shared less than 80% identity among themselves at the nucleotide level. A phylogenetic analysis of partial NS1 sequences showed that contig 2 was closely related to porcine bocavirus 1–2 while contig 18 was closely related to PBoV3 . Contigs 5 and 19 shared 74.6 and 80.0% nucleotide identities with PBoV4, respectively (Figure 2b). When we compared the contigs to the sequences from porcine bocavirus 6V and 7V, one and two contigs, respectively, exhibited high nucleotide homologies (> 99%) indicating that we were able to recover these viruses once again with our methods. According to the most recent International Committee on Taxonomy of Viruses species demarcation criteria for the genus Bocavirus (at least 5% nucleotide sequence divergence in their nonstructural gene), our data indicate the existence of multiple new Bocavirus species in pigs.
Parechovirus is a genus in the family Picornaviridae that comprises two species: human parechovirus (HPEV) and Ljungan virus (LV). HPEV causes mild gastrointestinal or respiratory illness, and has been implicated in cases of myocarditis and encephalitis . LV can cause significant morbidity and mortality in wild rodents as well as in laboratory animals, and it is a suspected human pathogen . Bank voles infected with LV in captivity develop several different pathologic signs and symptoms, including myocarditis, diabetes and encephalitis . Thirteen unique viral reads with similarities to both HPEV and LV were identified in this study. They spanned the LV reference genome (GenBank NC_003976.2), to which the amino acid identities ranged from 22.3 to 55.8%. Based on the sequences, we infer the existence of at least two parecho-like viruses. Reads 4, 9 and 11 belong to one virus, and overlap with read 2 over 190–231 bp but with only 81.1–82.7% nucleotide identity. Although each of the read pairs 1 and 12, 3 and 10 and 6 and 13 overlap when aligned with a common reference genome, the two reads in each pair share less than 50% nucleotide identity. Phylogenetic analyses using partial 3C region sequences demonstrated that reads 6 and 13 form a distinct branch in the Parechovirus genus (Figure 2c). It is worthy to mention that these divergent sequences represent some of the first parecho-like viruses reported in pigs till now.
List of primers used in both the 1 st and 2 nd rounds of PCR in this study
Astroviruses ORF1a region
5′- STTRCCWTGGSTYTGGGAGAT −3′
5′- ATMAGCCTCCAGCAGAAGCA −3′
5′- ACTGCYTCTACYTWGCAGCAG −3′
5′- ACATCWGCCARGAACATACC −3′
Astroviruses ORF1a region
5′- AGGCTAAACCTCAAGTTAG −3′
5′- CCTACAGGAAAAGTTACTC −3′
5′- CACGGATCTCAATGACATGACC −3′
5′- GCTATTGCACCTATTCAGAC −3′
Bocaviruses NS1 region
5′- TCTGCCTSAGGTSRGTGAGAA −3′
5′- KATGCATCTCGAGMAYCTCKT −3′
5′- GAGAAYTCKCTGGCTAGACAGA −3′
5′- AYCTCKTCKAGCGTYCGAGA −3′
Bocaviruses NS1 region
5′- TCTCTAGCTAGCGAGTTCTC −3′
5′- TAGATGAGGTGCTCGAGATGCA −3′
5′- ACCTCATCTAGCGTTCGAGA −3′
5′- GCCTGCTAAGATCACCAAA −3′
LV-like virus 2C region
5′- CTCTTCTGCTATGGAACTGCT −3′
5′- TTGCCGAAGTATCAGGCTTC −3′
5′- CTGCTTGGCAATGATGTGTCGA −3′
5′- ACAAACGTGTGCAGTTTCTGG −3′
In this study, we demonstrated the existence of different novel astroviruses, bocaviruses and parechovirus in porcine feces using 454 massively-parallel pyrosequencing. These viruses were highly prevalent in healthy pigs < 30 days old. The great diversity and high detection rate of these viruses in the porcine samples indicate that pigs are well-established reservoirs for many different viruses. Human astroviruses, bocaviruses and parechovirus are well-known pathogens that cause a variety of human diseases. It is unknown if the novel viruses detected in this study are pathogenic or have zoonotic potential. Given the diversity of viruses detected in this preliminary study, more attention should be paid to the viromes of pigs and other agricultural animals due to the potential for transmission of these viruses to humans in close proximity to these animals.
We thank David Wang and Guoyan Zhao from the Department of Pathology and Immunology, Washington University School of Medicine, for performing the bioinformatics analysis. This work was partly supported by the China Mega-Project for Infectious Disease (2011ZX10004).
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