Tongling Shan

The Fecal Virome of Pigs on a High-Density Farm

ABSTRACT Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials.

Picornavirus salivirus/klassevirus in children with diarrhea, China.

To learn more about salivirus/klassevirus, we tested feces of children with diarrhea in China during 2008–2009. We isolated the virus from 9/216 diarrhea samples and 0/96 control samples. The nearly full polyprotein of 1 isolate, SH1, showed 95% identity with a salivirus from Nigeria, indicating widespread distribution and association with diarrhea.

Host miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons.

Abstract MicroRNAs (miRNAs) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. To identify host miRNAs important to controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection, we screened 15 miRNAs that were previously implicated in innate immunity or antiviral functions. Over-expression of the miR-26 family strongly inhibited PRRSV replication in vitro, as shown by virus titer assays, Western blotting, and qRT-PCR assays. MiR-26a inhibited the replication of both type 1 and type 2 PRRSV strains. Mutating the seed region of miR-26 restored viral titers. Luciferase reporters showed that miR-26a does not target the PRRSV genome directly but instead affects the expression of type I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection. These results demonstrate the important role of miR-26a in modulating PRRSV infection and also support the possibility of using host miR-26a to achieve RNAi-mediated antiviral therapeutic strategies.

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platforms utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

Emergence of a Pseudorabies virus variant with increased virulence to piglets

Pseudorabies virus (PRV) causes Pseudorabies (PR), an economically important disease in domestic swine. PR outbreaks on pig farms caused by PRV variant strains in Bartha-K61-vaccinated pigs have resulted in considerable economic losses in China since 2011. In this study, the pathogenicity of the PRV variant JS-2012 strain to pigs was investigated by experimentally inoculating piglets of different ages in comparison with a classic virulent PRV SC strain. The JS-2012 strain caused an earlier onset of clinical signs and higher mortality in 15, 30, and 60-day-old pigs, as compared with a classic virulent PRV SC strain. The Bartha-K61 vaccination provided complete protection against challenge with classical virulent PRV, but only partial protection against challenge with the JS-2012 strain in piglets. In conclusion, the increased virulence of the PRV variant may have partly contributed to the PR outbreak in China.

Molecular cloning and characterizations of porcine SAMHD1 and its roles in replication of highly pathogenic porcine reproductive and respiratory syndrome virus.

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity.

Complete Genome Sequence of a Novel Porcine Enterovirus Strain in China

ABSTRACT The porcine enteroviruses (PEVs) belong to the family Picornaviridae. We report a complete genome sequence of a novel PEV strain that is widely prevalent in pigs at least in central and eastern China. The complete genome consists of 7,390 nucleotides, excluding the 3′ poly(A) tail, and has an open reading frame that maps between nucleotide positions 812 and 7318 and encodes a 2,168-amino-acid polyprotein. Phylogenetic analysis based on the 3CD and VP1 regions reveals that this PEV strain belongs to a species of PEV9 but may represent a novel sero-/genotype in CPE group III. We also report the major findings from bootscan analysis based on the whole genomes of PEVs in the present study and those available in GenBank.

Complete Genome of a Novel Porcine Astrovirus

ABSTRACT Astroviruses have been widely described in mammalian and avian species. Here, we report a complete genome sequence of a novel porcine astrovirus (PoAstV) isolated from a porcine fecal sample in China. The genome consists of 6,611 nucleotides, excluding the 3′ poly(A) tail, and has two open reading frames (ORFs). ORF1 maps between nucleotide positions 19 and 4211 and encodes a 1,396-amino-acid (aa) polyprotein precursor consisting of nonstructural protein and putative RNA-dependent RNA polymerase, and ORF2 maps between nucleotide positions 4202 and 6531 and encodes a 775-aa polyprotein which is a capsid precursor protein. The genome sequence of the virus was distinct enough from those of the known PoAstVs to be considered a novel sequence. Phylogenetic analysis based on the predicted amino acid sequence of the complete capsid region showed that this strain may be a novel porcine astrovirus.

Virome comparisons in wild-diseased and healthy captive giant pandas

BackgroundThe giant panda (Ailuropoda melanoleuca) is a vulnerable mammal herbivore living wild in central China. Viral infections have become a potential threat to the health of these endangered animals, but limited information related to these infections is available.MethodsUsing a viral metagenomic approach, we surveyed viruses in the feces, nasopharyngeal secretions, blood, and different tissues from a wild giant panda that died from an unknown disease, a healthy wild giant panda, and 46 healthy captive animals.ResultsThe previously uncharacterized complete or near complete genomes of four viruses from three genera in Papillomaviridae family, six viruses in a proposed new Picornaviridae genus (Aimelvirus), two unclassified viruses related to posaviruses in Picornavirales order, 19 anelloviruses in four different clades of Anelloviridae family, four putative circoviruses, and 15 viruses belonging to the recently described Genomoviridae family were sequenced. Reflecting the diet of giant pandas, numerous insect virus sequences related to the families Iflaviridae, Dicistroviridae, Iridoviridae, Baculoviridae, Polydnaviridae, and subfamily Densovirinae and plant viruses sequences related to the families Tombusviridae, Partitiviridae, Secoviridae, Geminiviridae, Luteoviridae, Virgaviridae, and Rhabdoviridae; genus Umbravirus, Alphaflexiviridae, and Phycodnaviridae were also detected in fecal samples. A small number of insect virus sequences were also detected in the nasopharyngeal secretions of healthy giant pandas and lung tissues from the dead wild giant panda. Although the viral families present in the sick giant panda were also detected in the healthy ones, a higher proportion of papillomaviruses, picornaviruses, and anelloviruses reads were detected in the diseased panda.ConclusionThis viral survey increases our understanding of eukaryotic viruses in giant pandas and provides a baseline for comparison to viruses detected in future infectious disease outbreaks. The similar viral families detected in sick and healthy giant pandas indicate that these viruses result in commensal infections in most immuno-competent animals.

A novel species of torque teno mini virus (TTMV) in gingival tissue from chronic periodontitis patients

A new species of torque teno mini virus, named TTMV-222, was detected in gingival tissue from periodontitis patients using a viral metagenomics method. The 2803-nucleotide genome of TTMV-222 is closely related to TTMV1-CBD279, with 62.6% overall nucleotide similarity. Genetic analyses of the new virus genome revealed a classic genomic organization but a weak identity with known sequences. The prevalence of TTMV-222 in the periodontitis group (n = 150) was significantly higher than that in the healthy group (n = 150) (p = 0.032), suggesting that the new virus may be associated with inflammation in chronic periodontitis patients. However, this finding requires further investigation.