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Chromobacterium haemolyticum is a fatal Gram-negative bacterium which could infect human beings. Our investigation found severe piglet diarrhea in one farm; the morbidity rate of piglets was 30.65%. Then, we isolated a nonpigmented, β-hemolytic Gram-negative bacillus from the clinical samples of this farm, which was designated GDHYZ30 strain. The 16S rRNA gene sequence indicated it was most closely related to Chromobacterium haemolyticum. Similar clinical symptoms were successfully reproduced in experimental piglets with this isolate, and the isolate was also subjected to whole genome-sequencing. It is worth noting that this Chromobacterium haemolyticum has been isolated from several other pig farms with diarrhea of unknown causes. To our knowledge, this is the first report that discusses Chromobacterium haemolyticum as a new pathogen causing diarrhea and death in piglets and transmitting through water sources, and it provides a reference for the prevention and control of human/animal infections as well as food safety.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which inflicts major economic losses on the global pig farming industry. Based on its similarity to highly pathogenic strains, the GDzj strain isolated in this study was predicted to be highly pathogenic. We therefore analyzed the pathogenicity of this strain experimentally in piglets. All piglets challenged with this virus experienced fever or high fever, loss of appetite, decreased food intake, daily weight loss, shortness of breath, and listlessness, and the necropsy results showed that they had experienced severe interstitial pneumonia. We then used the BAC system to construct a full-length cDNA infectious clone of GDzj, and the rescued virus displayed in vitro proliferation characteristics similar to those of the parental PRRSV strain. In summary, we successfully isolated a highly pathogenic PRRSV strain and constructed a full-length infectious cDNA clone from it, thereby providing an effective reverse genetics platform for further study of viral pathogenesis.

Background The circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus emergence in diverse host has been associated with severe disease. Porcine circovirus-like virus (Po-Circo-like [PCL] virus) is a CRESS DNA virus, the prevalence and pathogenicity of which are rarely studied. Methods We obtained two blood samples, four faecal samples, and two intestinal samples from a pig farm suffered from diarrheal disease in the delivery room in September 2020 and attempted to isolate and identify a causative pathogen. Subsequently, only PCL virus was positive, and qRT-PCR was designed to detect the loading titre of PCL virus. We then initiated a heightened surveillance program on the pathogenicity and epidemiology of PCL virus. Results Six PCL virus strains, with severe diarrhoea and haemorrhagic enteritis, have been found in six different pig farms in Guangdong province, China. A multiple sequence alignment of these PCL viruses and bovine circovirus-like virus/CH showed a similarity of 92.5-94.8% for the Rep protein, indicating these PCL viruses are highly homologous to Bo-Circo-like virus associated with calf diarrhoea. There were striking similarities between the PCL virus and bovine circovirus-like virus outbreaks in aetiological settings and Genomic sequence. We found that 11.2% (20/178) of diarrhoea samples and 13.3% (6/45) of pig farms were positive for PCL virus, suggesting that PCL virus may have spread widely in Pig farms. Moreover, this article underscores the risk of PCL virus spilling over and adapting to new species. Conclusions Porcine circovirus-like virus was found to be associated with porcine diarrheal disease in China.

Porcine epidemic diarrhea virus (PEDV) causes severe economic loss in the pig industry each year. To better understand the relationship between cytokines and PEDV replication, in this study, pro-inflammatory cytokine and chemokine expression profiles in Vero cells infected with PEDV were analyzed. Real-time quantitative PCR assay indicated that IL-1α, IL-1β, TNF-α, CCL2, CCL5 and CXCL8 expression levels were significantly upregulated. Moreover, overexpression and siRNA silencing assays showed that overexpression of IL-1α, IL-1β, TNF-α, CCL2, CCL5 and CXCL8 could significantly inhibit PEDV replication, while silencing of IL-1α, IL-1β, TNF-α, CCL2, CCL5 and CXCL8 could significantly promote PEDV replication. Finally, a dual-luciferase reporter assay showed that nsp4 contributed to the expression of IL-1α, IL-1β, TNF-α, CCL2, CCL5 and CXCL8 via the NF-κB pathway. Together, these data determined that PEDV nsp4 could upregulate pro-inflammatory cytokine and chemokine expression, inhibiting viral replication in vitro. These results provided novel insights for understanding the roles of cytokines in PEDV replication.

Background Since 2010, porcine epidemic diarrhea virus (PEDV) has caused severe diarrhea disease in piglets, which has led to large economic losses in China. To understand the genetic characteristics of PEDV strains that have been circulating in some provinces of China from 2015 to 2018, 362 feces and small intestine samples in pigs were collected and tested. Methods The S gene of PEDV in collected samples were amplified by RT-PCR. A phylogenetic tree was constructed using MEGA6.0 software with the neighbor-joining method to analyze the evolutionary relationship. Nucleotide and deduced amino acid (AA) sequences of S were aligned using the MegAlign program of DNASTAR7.1 software to determine sequence homology. PEDV GDgh16 strain isolation, IFA identification and titer detection were performed in Vero cells. Six 4-day-old healthy colostrum deprived suckling piglets were used for challenging experiment of PEDV GDgh16 strain. Virus copies from the small intestine were detected by RT-qPCR. The other section was stained with the anti-N protein McAb at 1:1000 dilution for immunohistochemical (IHC) examinations. Results The results showed that 160 samples tested positive and the PEDV-positive prevalence was 44.20%. Phylogenetic tree analysis of entire S genes showed that these strains were clustered into four subgroups, G1-b, G1-c, G2-a and G2-b, and that the G2-b strains had become dominant in recent years. Compared with previous strains, these strains had multiple variations in the SP and S1-NTD domains and in the neutralizing epitope of the S protein. Furthermore, we successfully isolated and identified a new virulent G2-b strain, GDgh16, which was well adapted to Vero cells and had a high mortality rate in piglets through challenge experiments. Conclusions Our study provides full insights into the genetic characteristics of prevalent PEDV strains in parts of China, suggesting that the development of novel effective vaccines is necessary and urgent.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404-1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe pathogen that causes enormous economic losses to the swine industry worldwide. Since its first report in the 1980s, PRRSV has undergone extensive variation. In the previous study, we demonstrated that the GDsg strain was a new recombined virus between the low pathogenic field strain QYYZ and the vaccine strain JXA1-P80, belonging to the newly emerging type 2 PRRSV subgenotype III. In this study, the pathogenicity of a new recombined strain GDsg for pigs was analyzed. The results of in vivo experiments indicated that GDsg could cause persistently high fever, severe interstitial pneumonia, and high viremia and antibody levels in inoculated piglets. In particular, the brains of inoculated pigs exhibited serious hemorrhage and microscopic lesions. These results suggested that compared with the low pathogenic field strain QYYZ and the vaccine strain JXA1-P80, the new recombined GDsg strain had higher virulence in pigs. This study will help to characterize the relationship between recombination and evolution of PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) is highly genetically diverse; however, little is known about the molecular epidemiology of PRRSV in the boar farms of South China. In this study, 367 samples were collected from boar farms in South China in 2015. The Nsp2 hypervariable region and ORF5 gene were PCR amplified from 66 PRRSV-positive samples, followed by sequencing and analysis. The percentage of PRRSV antigen-positive samples was 17.98%; 8.72% were positive for highly pathogenic PRRSV (HP-PRRSV), and 9.26% were positive for low pathogenic PRRSV (LP-PRRSV). Sequence alignment and phylogenetic tree analyses revealed three novel patterns of deletion in the hypervariable region of Nsp2, which had not been identified previously. Furthermore, numerous amino acid substitutions were identified in the putative signal peptide and extravirion regions of GP5. These results demonstrate for the first time that the existence of multiple different strains on the same boar farm, and extensive genetic mutation and high infection rate of PRRSV in boars from South China. Our research contributes to the understanding of the epidemiology and genetic characteristics of PRRSV on boar farms.

Porcine epidemic diarrhea (PED) is an acute enteric disease caused by porcine epidemic diarrhea virus (PEDV). In China, variant PEDV causes severe watery diarrhea, vomiting, and dehydration in piglets, leading to very high morbidity and mortality. However, the pathogenesis of PEDV is still not fully understood. In our study, we analyzed the long noncoding RNA (lncRNA) and mRNA expression profiles of PEDV GDgh16 in infected Vero cells at 60 h postinfection. A total of 61,790 annotated mRNAs, 14,247 annotated lncRNAs and 1290 novel lncRNAs were identified. A total of 227 annotated lncRNAs and 13 novel lncRNAs were significantly and differentially expressed after viral infection. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used to identify genes adjacent to the lncRNAs, and it was found that these lncRNAs were enriched in pathways related to immune and antiviral responses. Next, we selected candidate lncRNAs and their predicted target genes for study. RT-qPCR demonstrated that these lncRNAs and genes were differentially expressed after PEDV infection. Our study investigated the function of lncRNAs involved in PEDV infection, providing new insight into the pathogenic mechanisms of PEDV.

Background Porcine reproductive and respiratory syndrome (PRRS) has leaded to an enormous loss per year to the swine industry, its etiology porcine reproductive and respiratory syndrome virus (PRRSV) is a highly mutated virus in pigs. To fully understand the genetic characteristics of PRRSV genome in South China, this study collected the lung samples infected with PRRSV in Guangdong and Hainan province from 2014 to 2015 and tried to isolate the PRRSV. Finally, the complete genomes of isolated strains were sequenced and analyzed. Methods Virus isolation was performed in MARC-145 cells. The 13 fragments of PRRSV genome were amplified by RT-PCR and the complete PRRSV genome sequence was obtained by SeqMan program of DNASTAR7.0 software. Nucleotide and deduced amino acid (AA) sequences of NSP2 and ORF5 were aligned using the MegAlign program of DNASTAR7.0 software to determine sequence homology. A phylogenetic tree was constructed using MEGA5.2 software with the neighbor-joining method to analyze the evolutionary relationship. Results 11 PRRSV strains were isolated in South China from 2014 to 2015. All the isolated strains clustered into subgenotype V along with the HP-PRRSV representative strains JXA1, HuN4 and JXwn06. The subgenotype V was furtherly divided into two groups. AA sequence alignment analysis indicated that all the isolated strains had 1 AA deletion and 29 AA continuous deletion at position 481 and 533-561. Notably, GDHY strain had another 120 AA continuous deletion at position 629-748. All the isolated strains had an A137S mutation in the residue A137 of GP5 which was considered to differentiate vaccine strains. All the isolated strains had a L39I mutation in the primary neutralizing epitope (PNE) of GP5. Except GDHZ had a N34T mutation, all the other isolated strains had conserved N30, N44 and N51 glycosylation sites in the four potential N-glycosylation sites (N30, N34, N44 and N51) of GP5. Conclusions Our study showed that the prevalent strains in this region were highly pathogenic PRRS virus-like. Moreover, one new strain having another 120 amino acids continuous deletion except the discontinuous 30 (29+1) amino acids deletion in NSP2 region had emerged. Besides, the isolated strains had extensive amino acids substitutions in the putative signal, extravirion and intravirion regions of GP5. These results showed that PRRSV has undergone extensive variation in South China, providing some theoretical basis for researching effective vaccince to better controling the PRRSV in this area.

Summary Porcine circovirus type 3 (PCV3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. Recently, a PCV3 strain was identified from pyretic and pneumonic piglets in Guangdong province, China. This virus strain was sequenced and designated PCV3-China/GD2016. The complete genome of PCV3-China/GD2016 is 2,000 bp in length and shared 99.1% and 99.1% nucleotide identities with PCV3/29160 and PCV3/2164, respectively. [Corrections added after initial online publication on 13 March 2017: The numbers ‘98.5%’ and ‘97.4%’ has been changed to ‘99.1%’ and ‘99.1%’ in the previous sentence.] Phylogenetic analysis based on the complete genome showed that PCV3-China/GD2016 clustered with the emerging PCV3 and separated with other virus in genus Circovirus. The results of this study suggest that PCV3 has existed within the pigs of China. It is urgent to investigate the pathogenicity and epidemiology of this novel circovirus China.

Staphylococcus hyicus has caused great losses in the swine industry by inducing piglet exudative epidermitis (EE), sow mastitis, metritis, and other diseases and is a threat to human health. The pathogenesis of EE, sow mastitis, and metritis involves the interaction between the host and virulent protein factors of S. hyicus, however, the proteins that interact with the host, especially the host immune system, are unclear. In the present study, immunoproteomics was used to screen the immunogenic proteins of S. hyicus strain ZC-4. The cellular and secreted proteins of S. hyicus strain ZC-4 were obtained, separated by 2D gel electrophoresis, and further analyzed by western blot with S. hyicus strain ZC-4-infected swine serum. Finally, 28 specific immunogenic proteins including 15 cellular proteins and 13 secreted proteins, 26 of which were novel immunogenic proteins from S. hyicus, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. To further verify their immunogenicity, two representative proteins (acetate kinase [cellular] and enolase [secreted]) were chosen for expression, and the resultant recombinant proteins could react with S. hyicus ZC-4-infected swine serum. In mice, both acetate kinase and enolase activated the immune response by increasing G-CSF and MCP-5 expression, and acetate kinase further activated the immune response by increasing IL-12 expression. Enolase can confer better protection against S.hycius than acetate kinase in mice. For the first time to our knowledge, our results provide detailed descriptions of the cellular and secreted proteins of S. hyicus strain ZC-4. These immunogenic proteins may contribute to investigation and elucidation of the pathogenesis of S. hyicus and provide new candidates for subunit vaccines in the future.

Recently, a novel atypical porcine pestivirus (APPV) in pig was reported. In this study, two APPV strains, APPV-China/GZ01/2016 (GZ01) and APPV-China/GD-SD/2016 (GD-SD), were identified in two newborn piglet herds with congenital tremor from China. The open reading frame of the two strains shared an 83.5% nucleotide identity. Phylogenetically, the APPV strains were placed into two groups: GZ01 belonged to group I and GD-SD belonged to group II. A high viral load was detected in the cerebellum (quantification cycles ＜ 26). Further studies should be carried out to thoroughly elucidate the development of congenital tremors caused by APPV.