Metagenomics to Identify Viral Communities Associated with Porcine Respiratory Disease Complex in Tibetan Pigs in the Tibetan Plateau, China.

Tibetan pig is a unique pig breed native to the Qinghai-Tibet Plateau. To investigate viral communities associated with porcine respiratory disease complex (PRDC), 167 respiratory samples were collected from Tibetan pigs in the Ganzi Tibetan autonomous prefecture of Sichuan province. Following library construction and Illunima Novaseq sequencing, 18 distinct viruses belonging to 15 viral taxonomic families were identified in Tibetan pigs with PRDC. Among the 18 detected viruses, 3 viruses were associated with PRDC, including porcine circovirus type 2 (PCV-2), Torque teno sus virus (TTSuV), and porcine cytomegalovirus (PCMV). The genomic sequences of two PCV-2 strains, three TTSuV strains, and one novel Porprismacovirus strain were assembled by SOAPdenovo software (v2). Sequence alignment and phylogenetic analysis showed that both PCV-2 strains belonged to PCV-2d, three TTSuVs were classified to TTSuV2a and TTSuV2b genotypes, and the Porprismacovirus strain PPMV-SCgz-2022 showed a close genetic relationship with a virus of human origin. Recombination analysis indicated that PPMV-SCgz-2022 may have originated from recombination events between Human 16,806 × 66-213 strain and Porcine 17,668 × 82-593 strain. Furthermore, the high proportion of single infection or co-infection of PCV2/TTSuV2 provides insight into PRDC infection in Tibetan pigs. This is the first report of the viral communities in PRDC-affected Tibetan pigs in this region, and the results provides reference for the prevention and control of respiratory diseases in these animals.

High genotype diversity and zoonotic potential of Enterocytozoon bieneusi in yaks (Bos grunniens) from Ganzi Tibetan Autonomous Prefecture, Sichuan Province.

Enterocytozoon bieneusi is a common pathogen in humans and various animals, threatening the breeding industry and public health. However, there is limited information on the molecular characteristics of E. bieneusi in yaks, an economically important animal mainly domesticated in the Qinghai Tibet Plateau in China. In the present study, nested PCR targeting the ITS gene region was applied to investigate the positive rates and genetic diversity of E. bieneusi in 223 faecal samples of yaks from three locations in Ganzi Tibetan Autonomous Prefecture, Sichuan Province. The total positive rate of E. bieneusi was 23.8% (53/223). Significant differences in positive rates were identified among yaks from three locations (χ2 = 8.535, p = 0.014) and four age groups (χ2 = 17.259, p = 0.001), with the highest positive rates in yaks from Yajiang and aged < 6 months, respectively. Sequence analysis identified seven known (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J and BEB4) and five novel (Ganzi1-5) ITS genotypes. Phylogenetic analysis showed eight genotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 and Ganzi4) in group 1 and three genotypes (J, BEB4 and Ganzi3) in group 2, indicating high genotype diversity and zoonotic potential of E. bieneusi in yaks from Ganzi. Considering the increasing zoonotic genotypes in yaks in the present study compared with previous findings, interventions should be developed to reduce the potential transmission of E. bieneusi between humans and animals.

Title: Grande diversité génotypique et potentiel zoonotique d'Enterocytozoon bieneusi chez les yaks (Bos grunniens) de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Abstract: Enterocytozoon bieneusi est un agent pathogène courant chez l'homme et chez divers animaux, menaçant l'industrie de l'élevage et la santé publique. Cependant, il existe peu d'informations sur les caractéristiques moléculaires d'E. bieneusi chez les yaks, un animal important pour l'économie, principalement domestiqué sur le plateau du Qinghai au Tibet en Chine. Dans la présente étude, une PCR imbriquée ciblant la région du gène ITS a été appliquée pour étudier la positivité et la diversité génétique d'E. bieneusi dans 223 échantillons fécaux de yaks provenant de trois sites de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Le taux total de positivité pour E. bieneusi était de 23,8 % (53/223). Des différences significatives dans les taux positifs ont été identifiées parmi les yaks de trois emplacements (χ2 = 8,535, P = 0,014) et de quatre groupes d'âge (χ2 = 17,259, P = 0,001), avec les taux positifs les plus élevés respectivement chez les yaks de Yajiang et ceux âgés de moins de 6 mois. L'analyse de séquence a identifié sept génotypes ITS connus (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J et BEB4) et cinq nouveaux (Ganzi1­5). L'analyse phylogénétique a montré huit génotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 et Ganzi4) dans le groupe 1 et trois génotypes (J, BEB4 et Ganzi3) dans le groupe 2, indiquant une diversité génotypique élevée et un potentiel zoonotique d'E. bieneusi chez les yaks de Ganzi. Compte tenu de l'augmentation des génotypes zoonotiques chez les yaks dans la présente étude par rapport aux résultats précédents, des interventions devraient être développées pour réduire la transmission potentielle d'E. bieneusi entre les humains et les animaux.

A novel NADC34-like porcine reproductive and respiratory syndrome virus 2 with complex genome recombination is highly pathogenic to piglets.

The NADC34-like porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) first emerged in China in 2017 and has the potential to become the dominant PRRSV strain in China. Here, a novel PRRSV-2, SCcd2020, was isolated from diseased piglets in Sichuan province, southwest China in 2020. The complete viral genome was determined and analyzed. An ORF5-based phylogenetic analysis showed that SCcd2020 clustered with NADC34-like strains, whereas the genome sequence clustered the isolate with NADC30-like viruses and it contains a discontinuous 131-aa deletion in NSP2 when compared to NADC30 strain. Notably, recombination analyses indicated that SCcd2020 is a multiple recombinant virus from NADC30-like, NADC34-like and JXA1-like strains, which is the first description of Chinese domestic HP-PRRSV involving the recombination event of an NADC34-like strain. Importantly, an animal challenge study in 4-week-old piglets showed that SCcd2020 causes high fever and severe hemorrhagic pneumonia with pulmonary consolidation and edema, and it has a high mortality rate (60%), which indicated that SCcd2020 is a highly pathogenic PRRSV strain. The study reports the emergence of a novel highly pathogenic NADC34-like recombinant strain, and it highlights the importance of monitoring newly emerging PRRSV strains in China.

Characterization of gut microbiota dysbiosis of diarrheic adult yaks through 16S rRNA gene sequences.

The ruminant gut microbial community has a strong impact on host health and can be altered during diarrhea disease. As an indigenous breed of the Tibetan Plateau, domestic yak displays a high diarrhea rate, but little research has been done to characterize the bacterial microbial structure in diarrheic yaks. In the present study, a total of 30 adult yaks, assigned to diarrhea (case, N = 15) and healthy (control, N = 15) groups, were subjected to gut microbiota profiling using the V3-V4 regions of the 16S rRNA gene. The results showed that the gut microbiome of the case group had a significant decrease in alpha diversity. Additionally, differences in beta diversity were consistently observed for the case and control groups, indicating that the microbial community structure was changed due to diarrhea. Bacterial taxonomic analysis indicated that the Bacteroidetes, Firmicutes, and Proteobacteria were the three most dominant phyla in both groups but different in relative abundance. Especially, the proportion of Proteobacteria in the case group was increased as compared with the control group, whereas Spirochaetota and Firmicutes were significantly decreased. At the genus level, the relative abundance of Escherichia-Shigella and Prevotellaceae_UCG-003 were dramatically increased, whereas that of Treponema, p-2534-18B5_gut_group, and Prevotellaceae_UCG-001 were observably decreased with the effect of diarrhea. Furthermore, based on our linear discriminant analysis (LDA) effect size (LEfSe) results, Alistipes, Solibacillus, Bacteroides, Prevotellaceae_UCG_003, and Bacillus were significantly enriched in the case group, while the other five genera, such as Alloprevotella, RF39, Muribaculaceae, Treponema, and Enterococcus, were the most preponderant in the control group. In conclusion, alterations in gut microbiota community composition were associated with yak diarrhea, differentially represented bacterial species enriched in case animals providing a theoretical basis for establishing a prevention and treatment system for yak diarrhea.

Viral communities associated with porcine diarrhoeal disease and genetic characterization of a bufavirus in Tibetan pigs in China.

To investigate the viral communities in diarrhoeal faeces of Tibetan pigs, 146 diarrhoeic samples were collected from 16 pigs farms on the Tibetan plateau. Nineteen viruses belonging to eleven viral taxonomic families were identified in a pooled library. Metagenomics analysis revealed that the viruses were mainly small linear and circular DNA viruses. Furthermore, sequences of 10 NS1 genes and two complete genomes of PBuVs were obtained by PCR amplification. Sequence comparisons and phylogenetic analysis showed that the PBuVs from Tibetan pigs displayed more abundant genetic diversity than those from domestic pigs. This is the first description of the faecal viral community in Tibetan pigs associated with diarrhoea.

Hepatitis E virus in Tibetan pigs on the Qinghai-Tibetan Plateau: detection, molecular characterization and infection of BALB/c mice.

This study was performed to investigate the prevalence and genetic variation of hepatitis E virus (HEV) in Tibetan pigs and to determine its ability to infect mice. A total of 38 out of 229 (16.59%; 95% CI = 12.00%-22.10%) fecal samples from Tibetan pigs from the Qinghai-Tibetan Plateau in 2018 were positive for HEV RNA, which was detected by RT-nPCR. Significantly different detection rates were observed between samples from diarrheic and clinically healthy animals (OR = 9.56; 95% CI, 2.84-32.14; p < 0.001), suggesting a potential association between HEV infection and diarrhea in Tibetan pigs. Phylogenetic analysis showed that the HEV isolates were clustered into subtypes 4a (31 samples), 4b (1), 4d (2), and 4j (4). HEV-4a was the predominant subtype, indicating that it might be circulating in Tibetan pigs. Nine complete HEV genome sequences obtained from Tibetan pigs were found by phylogenetic analysis to be closely related to those of genotype 4 HEV isolates from humans. Two recombinant events were identified in both HEV-4a strains; a novel recombination breakpoint was first identified at the 3' end of the ORF2 region in the SWU/L9/2018 strain, and a common recombination region was found at the junction of the ORF1 and ORF2 regions in the SWU/31-12/2018 strain. Furthermore, HEV-4a could be detected in all BALB/c mice that were experimentally infected by gavage and contact exposure. The information presented here about the prevalence and genotype diversity of HEV from Tibetan pigs provides important insights into the epidemic features of HEV on the Qinghai-Tibetan Plateau.

Emergence of porcine epidemic diarrhea viruses with the novel S genes in Tibetan pigs in the Qinghai-Tibetan plateau in China.

The purpose of present study was to investigate the prevalence and genetic variation of porcine epidemic diarrhea virus (PEDV) in Tibetan pigs on the Qinghai-Tibetan Plateau in 2018. The PCR yielded a significantly high detection rate (38.34%, 95%CI=31.5-45.6%) for PEDV from 193 fecal samples from Tibetan pigs. The novel PEDVs were discovered in Tibetan pigs and seven complete S genes were obtained and analyzed. The unique multiple mutations were detected in S genes of PEDV from Tibetan pigs, one of which led to a new amino acid substitution of a neutralizing epitope. Phylogenetic analysis showed that seven S genes had significant genetic distance to other PEDV. Specially, two S genes formed a novel subgroup on the genogroup 2 (G2) branch, of which same recombination event occurred between different strains from genotype G2. The remaining five S genes formed a new subgroup on the G1 branch, among which distinct recombination events occurred between genotypes G1 and G2 strains. The result indicated that the new recombination events were detected in the S genes of PEDV from Tibetan pigs, which could be circulating in the Qinghai-Tibetan Plateau. Notably, the four complete PEDV genomes obtained in this study had an identical recombination region spanning S2, ORF3 and E genes. This is the first report of the crossover regional recombination event in PEDV genome. Our findings not only augmented current understanding of the genetic evolution of PEDV, but also indicated that new variants of PEDV strains have been emerging in Tibetan pigs.