First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

Comprehensive phylogeographic and phylodynamic analyses of global Senecavirus A.

Senecavirus A (SVA) is a member of the genus Senecavirus in the family Picornaviridae that infects pigs and shows symptoms similar to foot and mouth diseases and other vesicular diseases. It is difficult to prevent, thus, causing tremendous economic loss to the pig industry. However, the global transmission routes of SVA and its natural origins remain unclear. In this study, we processed representative SVA sequences from the GenBank database along with 10 newly isolated SVA strains from the field samples collected from our lab to explore the origins, population characteristics, and transmission patterns of SVA. The SVA strains were firstly systematically divided into eight clades including Clade I-VII and Clade Ancestor based on the maximum likelihood phylogenetic inference. Phylogeographic and phylodynamics analysis within the Bayesian statistical framework revealed that SVA originated in the United States in the 1980s and afterward spread to different countries and regions. Our analysis of viral transmission routes also revealed its historical spread from the United States and the risk of the global virus prevalence. Overall, our study provided a comprehensive assessment of the phylogenetic characteristics, origins, history, and geographical evolution of SVA on a global scale, unlocking insights into developing efficient disease management strategies.

Clinical sequencing uncovers the genomic characteristics and mutation spectrum of the 2018 African swine fever virus in Guangdong, China.

African swine fever (ASF) outbreak have caused tremendous economic loss to the pig industry in China since its emergence in August 2018. Previous studies revealed that many published sequences are not suitable for detailed analyses due to the lack of data regarding quality parameters and methodology, and outdated annotations. Thus, high-quality genomes of highly pathogenic strains that can be used as references for early Chinese ASF outbreaks are still lacking, and little is known about the features of intra-host variants of ASF virus (ASFV). In this study, a full genome sequencing of clinical samples from the first ASF outbreak in Guangdong in 2018 was performed using MGI (MGI Tech Co., Ltd., Shenzhen, China) and Nanopore sequencing platforms, followed by Sanger sequencing to verify the variations. With 22 sequencing corrections, we obtained a high-quality genome of one of the earliest virulent isolates, GZ201801_2. After proofreading, we improved (add or modify) the annotations of this isolate using the whole genome alignment with Georgia 2007/1. Based on the complete genome sequence, we constructed the methylation profiles of early ASFV strains in China and predicted the potential 5mC and 6mA methylation sites, which are likely involved in metabolism, transcription, and replication. Additionally, the intra-host single nucleotide variant distribution and mutant allele frequency in the clinical samples of early strain were determined for the first time and found a strong preference for A and T substitution mutation, non-synonymous mutations, and mutations that resulted in amino acid substitutions into Lysine. In conclusion, this study provides a high-quality genome sequence, updated genome annotation, methylation profile, and mutation spectrum of early ASFV strains in China, thereby providing a reference basis for further studies on the evolution, transmission, and virulence of ASFV.

Insights into the evolutionary history and epidemiological characteristics of the emerging lineage 1 porcine reproductive and respiratory syndrome viruses in China.

The newly emerged lineage 1 porcine reproductive and respiratory syndrome viruses (PRRSVs) (especially the NADC30-like and NADC34-like viruses) have posed a direct threat to the Chinese pig industry since 2013. The phylogenetic, epidemic, and recombinant properties of these viruses have not yet systematically analysed in China. This report presents regular surveillance and field epidemiological studies for PRRSV across China from 2007 to 2019. From over 4,000 detected clinical samples, 70 open reading frame five sequences and four complete genomes of lineage 1 viruses were successfully obtained. Combined with global data, we conducted an extensive and systematic molecular phylogeny analysis using a maximum likelihood tree. The Chinese lineage 1 viruses were clustered, and their temporal and spatial distribution was further explored. Multiple viral introductions of lineage 1 virus from the United States to China were detected, and some became endemic in China. There are three sub-lineage 1 clusters: lineage 1.5 (NADC34-like), lineage 1.6 and New Intro cluster (NADC30-like). These viruses show high genetic diversity and a wide distribution in China, with Henan Province showing the highest diversity. Moreover, Chinese lineage 1 viruses have developed an endemic NADC30-like cluster. The demographic feature of this cluster showed a more or less constant population expansion history with a recent decreasing trend. Moreover, the genome recombination of Chinese lineage 1 with two dominant clusters (Chinese HP-PRRSVs: lineage 8.7 and VR2332-like: lineage 5.1) was frequently detected, both of which have commercial vaccine strains available. Furthermore, recombination hotspots were discovered near NSP9 and ORF2-4 regions of the genome. Overall, these findings provide important insights into the evolution and geographical diversity of Chinese lineage 1 PRRSV. These results will facilitate the development of programmes for the control and prevention of the emerging lineage 1 viruses in China.

Emergence of novel recombination lineage 3 of porcine reproductive and respiratory syndrome viruses in Southern China.

Lineage 3 of porcine reproductive and respiratory syndrome viruses, which belong to North America type 2, has a long epidemic history in China. The novel lineage 3 viruses constantly emerging in recent years are characterized by a high detection rate and significant pathogenicity. In this study, we investigated the prevalence of lineage 3 in southern China and selected two isolated strains for genome and virulence analyses. A cross-sectional epidemiology investigation indicated that the prevalence of lineage 3 antigens was 35.68% (95% CI: 27.6-44.3%) among 227 samples collected from over 100 infected farms from January 2016 to July 2017 in southern China. Two novel isolates of lineage 3 were selected. After 20 passages, Marc-145 cells were not susceptible to those viruses. Full-length genome analysis indicated that the two strains share 95.2% homology with each other and 95.7%-96.2% with highly pathogenic porcine reproductive and respiratory syndrome viruses (HP-PRRSVs; JXA1-like strain, lineage 8.7). Phylogenetic and molecular evolutionary results showed that for the two isolates, HP-PRRSV provides most of the ORF1 gene. Animal experiment revealed discrepancies in virulence between the strains. Although challenge resulted in 100% morbidity, the isolate carrying most of the HP-PRRSV ORF1 caused severe clinical symptoms and 40% mortality, whereas the other isolate containing part of the ORF1 gene caused no mortality. Overall, these findings suggest that lineage 3 viruses might be commonly circulating in most of southern China. Frequent recombination events within HP-PRRSVs of this lineage with changing virulence could represent potential threats to the pig industry.

Phylogeography, phylodynamics and the recent outbreak of lineage 3 porcine reproductive and respiratory syndrome viruses in China.

Novel highly pathogenic porcine reproductive and respiratory syndrome viruses (PRRSVs) have attracted increasing attention owing to their continual high emergence and recent re-emergence. Recently, lineage 3 PRRSVs, belonging to the type 2 viruses, with novel characteristics and increased virulence have been continuously re-emerging in China, thereby posing a great threat to pig farming. However, available information about lineage 3 is limited. Here, we carried out molecular epidemiological investigations for PRRSV surveillance in most regions of China from 2007 to 2017. More than 3,000 samples were obtained, amounting to 73 sequences of lineage 3 viruses. The origin, demographic history and spread pattern of lineage 3 PRRSVs were investigated combining with the database globally. Phylogeography and phylodynamic analyses within a Bayesian statistical framework revealed that lineage 3 viruses originated in Taiwan. Followed by subsequent propagation to different areas and geography, it dichotomized into two endemic clusters. South China has become an epicentre for these viruses, which diffused into China's interiors in recent years. Furthermore, viral dispersal route analysis revealed the risk of viral diffusion. Overall, the origin, epidemic history and geographical evolution of lineage 3 PRRSVs were comprehensively analysed in this study. In particular, the epicentre of southern China and the diffusion routines of the viruses are highlighted in this study, and the possible continuous transmission of the novel lineages poses the biggest threat to pig farmers.