Isolation and characterization of a genotype 4 Hepatitis E virus strain from an infant in China

In the present study, a genotype 4 HEV strain was identified in the fecal specimen from a seven months old infant with no symptom of hepatitis in Shanghai Children's hospital. The full capsid protein gene (ORF2) sequence of this strain was determined by RT-PCR method. Sequence analysis based on the full ORF2 sequence indicated that this HEV strain shared the highest sequence identity (97.6%) with another human HEV strain isolated from a Japanese patient who was infected by genotype 4 HEV during traveling in Shanghai. Phylogenetic analysis showed that this genotype 4 HEV was phylogenetically far from the genotype 4 HEV strain that was commonly prevalent in Shanghai swine group, suggesting that this strain may not come from swine group and not involved in zoonotic transmission in this area

Hepatitis E Virus Infection in Central China Reveals No Evidence of Cross-Species Transmission between Human and Swine in This Area

Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus. Since 2000, Genotype 4 HEV has become the dominant cause of hepatitis E disease in China. Recent reports showed that genotype 4 HEV is freely transmitted between humans and swine in eastern and southern China. However, the infection status of HEV in human and swine populations in central China is still unclear. This study was conducted in a rural area of central China, where there are many commercial swine farms. A total of 1476 serum and 554 fecal specimens were collected from the general human and swine populations in this area, respectively. The seroepidemiological study was conducted by enzyme-linked immunosorbent assay. Conserved genomic sequences of open reading frame 2 were detected using reverse transcription-PCR. The results indicated that the overall viral burden of the general human subjects was 0.95% (14/1476), while 7.0% (39/554) of the swine excreted HEV in stool. The positive rate of anti-HEV IgG and IgM in the serum samples was 7.9% (117/1476) and 1.6% (24/1476), respectively. Phylogenetic analysis based on the 150 nt partial sequence of the capsid protein gene showed that the 53 swine and human HEV isolates in the current study all belonged to genotype 4, clustering into three major groups. However, the HEV isolates prevalent in the human and swine populations were classified into known distinct subgenotypes, which suggested that no cross-species transmission between swine and humans had taken place in this area. This result was confirmed by cloning and phylogenetic analysis of the complete capsid protein gene sequence of three representative HEV strains in the three major groups. The cross reactivity between anti-HEV IgG from human sera and the two representative strains from swine in central China was confirmed by Dot-blot assay. In conclusion, although all the HEV strains prevalent in central China belonged to genotype 4, there is no evidence of cross-species transmission between human and swine in this area

Screening and identification of the dominant antigens of the African swine fever virus

African swine fever is a highly lethal contagious disease of pigs for which there is no vaccine. Its causative agent African swine fever virus (ASFV) is a highly complex enveloped DNA virus encoding more than 150 open reading frames. The antigenicity of ASFV is still unclear at present. In this study, 35 proteins of ASFV were expressed by Escherichia coli, and ELISA was developed for the detection of antibodies against these proteins. p30, p54, and p22 were presented as the major antigens of ASFV, positively reacting with all five clinical ASFV-positive pig sera, and 10 pig sera experimentally infected by ASFV. Five proteins (pB475L, pC129R, pE199L, pE184L, and pK145R) reacted well with ASFV-positive sera. The p30 induced a rapid and strong antibody immune response during ASFV infection. These results will promote the development of subunit vaccines and serum diagnostic methods against ASFV

Genomic Characterization and High Prevalence of Bocaviruses in Swine

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ∼2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45–75% and 55–70% respectively, with 30–47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses

The Fecal Virome of Pigs on a High-Density Farm ▿ †

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

Detection of a new tet(X6)-encoding plasmid in Acinetobacter towneri

ABSTRACT: Objectives: The aim of this study was to characterise a novel tet(X6)-carrying plasmid detected in a livestock-associated Acinetobacter towneri isolate. Methods: PCR screening was performed to detect tet(X) variants in livestock-associated Acinetobacter spp. isolates. The tet(X6)-positive isolate was analysed by whole-genome sequencing. Functional cloning was performed to detect the activity of Tet(X6). Antibiotic susceptibility was determined by broth dilution and microbiological degradation assays. Site-directed mutagenesis was performed to identify the role of 23-Ala residue of Tet(X6) in tigecycline resistance. Results: The tet(X6) gene was detected on a 159-kb plasmid (pAT205) carried by a tigecycline-susceptible A. towneri isolate recovered from a swine faecal sample. The genetic context of tet(X6) [ΔISVsa3–tet(X6)–abh–guaA–ISVsa3] is highly similar to that of the reported plasmid-borne tet(X) variants, suggesting that it may represent a common structure mediating the dissemination of plasmid-borne tet(X) genes. Additional resistance genes detected on pAT205 were carried by a Tn6205-like region and a disrupted class 2 integron. Gene expression and microbiological degradation assays consistently suggested that the activity of tet(X6) is weaker than that of tet(X3) and tet(X4). The 23-Ala residue of the first FAD-binding site conferred higher activity to Tet(X6) than the Gly reside conserved in the other plasmid-borne tet(X) variants, indicating that the site might be under selection. Conclusion: This study alerts to the silent dissemination possibility of tigecycline resistance mediated by a novel plasmid. Continuous monitoring of plasmid-borne tet(X) is imperative for tackling its dissemination

Comparative genomic analyses of a virulent pseudorabies virus and a series of its in vitro passaged strains

Abstract Background Pseudorabies virus (PRV) of the family Herpesviridae is the causative agent of Aujeszky’s disease. Attenuation of PRV by serial passaging in vitro is a well-established method; however, the dynamic variations occurring on viral genome during this process have not been characterized. Methods Genome sequencing and comparative genomic analyses of a virulent pseudorabies virus and a series of its plaque-purified strains via serial passaging in vitro were performed, and the properties in vitro and in vivo of which were further characterized. Results Compared to the parental virus, replication in vitro was enhanced in the highly passaged F50, F91, and F120. In contrast, lethality in mice decreased gradually with passage number. Genome sequencing of F50, F91, and F120 showed deletion of a large fragment containing gE, which is likely related to their attenuation. In addition, single nucleotide variations were identified in many genes of F50, F91, and F120. In-frame and frameshift indels were also detected in specific genes of passaged strains. Particularly frameshift mutations were observed in highly passaged strains, resulting in a truncated but overexpressed pUL46. Conclusion During attenuation of PRV by serial passaging in Vero cells, dynamic variation patterns including a large deletion, single nucleotide variations, small in-frame indels, and also frameshifts mutations successively emerged, contributing to evolution of the viral population and enabling the gradual attenuation of the virus. These data provide clues to better understand PRV attenuation during passaging

Metagenomic survey of viral diversity obtained from feces of piglets with diarrhea

Pigs are natural host to various zoonotic pathogens including viruses. In this study, we analyzed the viral communities in the feces of 89 piglets with diarrhea under one month old which were collected from six farms in Jiangsu Province of the Eastern China, using the unbiased virus metagenomic method. A total of 89 libraries were constructed, and 46937894 unique sequence reads were generated by Illumina sequencing. Overall, the family Picornaviridae accounted for the majority of the total reads of putative mammalian viruses. Ten novel virus genomes from different family members were discovered, including Parvoviridae (n = 2), Picobirnaviridae (n = 4) and CRESS DNA viruses (n = 4). A large number of phages were identified, which mainly belonged to the order Caudovirales and the family Microviridae. Moreover, some identified viruses were closely related to viruses found in non-porcine hosts, highlighting the potential for cross-species virus dissemination. This study increased our understanding of the fecal virus communities of diarrhea piglets and provided valuable information for virus monitoring and preventing