Epidemiological and genetic characteristics of porcine circovirus 3 in 15 provinces and municipalities of China between 2016 and 2020.

Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection.

Investigation and analysis of etiology associated with porcine respiratory disease complex in China from 2017 to 2021.

Porcine respiratory diseases complex (PRDC) is a highly serious threat to the pig industry. In the present study, we investigated and analyzed the etiology associated with PRDC and explored the role of viruses in respiratory bacterial infections. From 2017 to 2021, clinical samples were collected from 1,307 pigs with typical respiratory symptoms in 269 farms in China and screened for pathogens related to PRDC by PCR and bacterial isolation. The results indicated that PRRSV (41.16%, 95%CI: 38.49~43.83%), PCV2 (21.58%,95%CI: 19.35~23.81%), S. suis (63.50%, 95%CI: 60.89~66.11%), and G. parasuis (28.54%, 95%CI: 26.09~30.99%) were the most commonly detected pathogens in pigs with PRDC in China. The dominant epidemic serotypes (serogroups) of S. suis, G. parasuis, and P. multocida were serotype 2, serotype 1, and capsular serogroups D, respectively. Pigs of different ages exhibited different susceptibilities to these pathogens, e.g., PRRSV, PCV2, and G. parasuis had the highest detection rates in nursery pigs, whereas fattening pigs had the highest detection rates of P. multocida and A. pleuropneumoniae. Among the 1,307 pigs, the detection rates of S. suis, G. parasuis, P. multocida, and B. bronchiseptica were higher in virus-positive pigs, especially G. parasuis and P. multocida were significantly (p < 0.01) higher than in virus-negative pigs. In addition, a strong positive correlation was found between coinfection by PRRSV and G. parasuis (OR = 2.33, 95%CI: 1.12~2.14), PRRSV and P. multocida (OR = 1.55, 95%CI: 1.12~2.14), PCV2 and P. multocida (OR = 2.27, 95%CI: 1.33~3.87), PRRSV-PCV2 and S. suis (OR = 1.83, 95%CI: 1.29~2.60), PRRSV-PCV2 and G. parasuis (OR = 3.39, 95%CI: 2.42~4.74), and PRRSV-PCV2 and P. multocida (OR = 2.09, 95%CI: 1.46~3.00). In summary, PRRSV, PCV2, S. suis, and G. parasuis were the major pathogens in pigs with PRDC, and coinfections of two or more PRDC-related pathogens with strong positive correlations were common in China, such as PRRSV and G. parasuis, PRRSV and P. multocida, PCV2 and P. multocida, and also PRRSV-PCV2 and G. parasuis and PRRSV-PCV2 and P. multocida.

The epidemiological investigation of co-infection of major respiratory bacteria with pseudorabies virus in intensive pig farms in China.

Porcine respiratory disease complex (PRDC), a respiratory disease caused by a variety of factors, is one of the most common problems in the intensive pig farms. To investigate the mixed infection incidence of wild-type pseudorabies virus (WT PRV) and respiratory bacteria, a total of 1,293 clinical samples were collected from pigs with typical respiratory signs from 14 different provinces of China from September 2016 to February 2018. The WT PRV was detected by ELISA targeting gE antibody while the bacteria were detected by bacterial isolation and serotyping by PCR. The results revealed that the detection rate of A. pleuropneumoniae and B. bronchiseptica infection associated with WT PRV infection were 6.30% and 15.99%, respectively, which were significantly higher than those without WT PRV infection (3.41% and 4.41%) at the farm level (p < .05). There were no significant differences in the detection rate of H. parasuis, S. suis or P. multocida between WT PRV positive and negative farms (p > .05). However, the detection rate of attenuated H. parasuis and S. suis strains were 68.19% and 64.75%, respectively, in WT PRV infected farms, which were significantly higher than those (41.56% and 52.25%) in WT PRV free farms (p < .05). The prevalent serotypes of H. parasuis-5/12 and S. suis-2 were also investigated by multiplex PCR. These results indicated that the presence of WT PRV increased the chance of bacterial infection and the number of pathogenic strains in the respiratory system of pigs. Therefore, the eradication of pseudorabies is an effective approach to prevent and control the bacterial respiratory diseases in the intensive pig farms in China.

Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0-20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017.

Bacterial diseases of swine are a kind of multifactorial and uncontrollable diseases that commonly exist in pig farms all over the world and will lead to huge economic losses every year. In this study, a detailed and overall survey was carried out to better understand the prevalence and antimicrobial susceptibilities of bacterial diseases from 2013 to 2017 in China. A total of 19673 bacterial strains were isolated from 44175 samples collected from 9661 pig farms that distributed in 16 Chinese major pig breeding provinces. The results showed that the average isolation rates of Streptococcus suis (SS), Haemophilus parasuis (HPS), Escherichia coli (E. coli), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae (APP), Brodetella bronchiseptica (Bb), Salmonella enteria (SE), Erysipelothrix rhusiopathiae (E. rhusiopathiae) were 16.9%, 9.7%, 6.3%, 3.4%, 0.3%, 1.5%, 2.3% and 0.9%, respectively. The isolate rates of E. coli, APP and SE showed an increasing trend from 2013 to 2017. The seasonal prevalence characteristics of SS, HPS and Pm were obviously higher from April to August for first two bacteria and higher at February, March, April, and October for Pm. The dominant serotypes for SS, HPS were serotype 2 and serotype 5 (changed from serotype 4), respectively. The SS, HPS, and Pm showed very high antibiotic resistance rates to almost 8 common antibiotics (ß-lactam, aminoglycoside, macrolides, lincomycin, tetracycline, quinolone, polymyxin, and sulfonamide) and an obvious increasing trend of antibiotic resistance rates from 2013 to 2017. In conclusion, the study provides detailed information on the prevalence and antimicrobial susceptibilities of different bacterial pathogens of swine from 2013 to 2017 in China. These data can provide a foundation for monitoring epidemiological patterns of bacterial diseases in the Chinese swine herds, as well as provide insight into potential antibiotic resistance profiles in these pathogens.

Establishment and application of a multiplex RT-PCR to differentiate wild-type and vaccine strains of porcine epidemic diarrhea virus.

Due to outbreaks of porcine epidemic diarrhea (PED) and the wide use of attenuated live vaccine, both wild-type and vaccine strains (CV777) are believed to circulate in Chinese pig farms. Thus, identification of different PEDV strains is of epidemiological importance. In this study, a multiplex RT-PCR method was established based on the sequence features of spike (S) gene and ORF3 gene of PEDVs. The method could identify PEDV variant strains, classical wild-type strains and classical vaccine strains. The limit of detection of the RT-PCR was 1.51 × 104 copies/uL for plasmids and 1 × 101.7 TCID50/100 u L for PEDV, respectively. There were no cross-detections among three different PEDVs and no false detections among six swine pathogens. This assay was used to test 940 samples from China of which 303 samples were PEDV positive, and 289, 5, 10 were positive for variant, classical wild, classical vaccine, respectively. One sample was positive for both variant and classical vaccine PEDV. The variant PEDVs could be detected in samples from 13 provinces, while classical PEDVs were detected from nine provinces, supporting the prevalence of variant PEDV in China. In summary, this multiplex RT-PCR was a useful tool for the clinical detection and epidemiological survey of PEDV.

The AI-2/luxS Quorum Sensing System Affects the Growth Characteristics, Biofilm Formation, and Virulence of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.

Co-infection with porcine bocavirus and porcine circovirus 2 affects inflammatory cytokine production and tight junctions of IPEC-J2 cells.

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.

Complete Genome Sequence of a Novel Porcine Circovirus Type 3 Strain, PCV3/CN/Hubei-618/2016, Isolated from China.

In October 2016, porcine circovirus type 3 (PCV3) was identified as a pathogen agent for pigs in the United States. Here, we report the genome sequence of a Chinese PCV3 strain, PCV3/CN/Hubei-618/2016. This will help us better understand the epidemiology and genetic characteristics of PCV3.

Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 102.5 TCID50/mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

Porcine Epidemic Diarrhea Virus Induces Autophagy to Benefit Its Replication.

The new porcine epidemic diarrhea (PED) has caused devastating economic losses to the swine industry worldwide. Despite extensive research on the relationship between autophagy and virus infection, the concrete role of autophagy in porcine epidemic diarrhea virus (PEDV) infection has not been reported. In this study, autophagy was demonstrated to be triggered by the effective replication of PEDV through transmission electron microscopy, confocal microscopy, and Western blot analysis. Moreover, autophagy was confirmed to benefit PEDV replication by using autophagy regulators and RNA interference. Furthermore, autophagy might be associated with the expression of inflammatory cytokines and have a positive feedback loop with the NF-κB signaling pathway during PEDV infection. This work is the first attempt to explore the complex interplay between autophagy and PEDV infection. Our findings might accelerate our understanding of the pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies.

Genetic characteristics of porcine epidemic diarrhea virus in Chinese mainland, revealing genetic markers of classical and variant virulent parental/attenuated strains.

Since October 2010, porcine epidemic diarrhea (PED) caused by variant porcine epidemic diarrhea virus (PEDV) has led great economic losses to the global pig industry, especially in China. To study the genetic characteristics of PEDV strains in Chinese mainland, a total of 603 clinical samples from nine provinces/districts of Chinese mainland from January 2014 to December 2015 were collected for RT-PCR detection and 1-1323bp of S gene of 91 isolates and ORF3 gene of 46 isolates were sequenced. The results showed that the variant PEDV were the dominant pathogens of viral diarrhea diseases in these areas. Six novel variant PEDV strains (FJAX1, FJAX2, HeNPDS1, HeNPDS2, HeNPY3, and HeNPY4) with two amino acids (aa) deletion at the 56-57 aa of S protein were identified. A total of 405 Chinese PEDV strains were subjected to phylogenetic and phylogeographic analysis. The results revealed that the subgroup Va in variant PEDV group were the dominant subgroup and the spread trend of variant PEDV strains seemed to be from the southeast coastal districts to other coastal districts and interior districts. The N-terminal of S gene (1-750bp), to some extent, could represent S1 or full length S gene for phylogenetic, similarity, antigen index, hydrophilicity plot, and differentiation analyses. The 404-472bp of S gene contained the three genetic markers, i.e., "TAA" insertion at 404-405bp, "ACAGGT" deletion at 430-435bp, and "ATA" deletion at 455-457bp can be used to differentiate the classical and variant virulent parental/attenuated PEDV strains and help us to learn the infectious and genetic characteristics of PEDV strains more convenient and cheaper. This study has important implication for understanding the infectious, genetic, and evolutionary aspects of PEDV strains in Chinese mainland.

Growth characteristics and complete genomic sequence analysis of a novel pseudorabies virus in China.

Swine pseudorabies (PR) re-emerged in Bartha-vaccinated pig herds and caused death of millions of piglets in China since the later part of 2011. We isolated a novel pseudorabies virus (PRV), named HNX strain, from the brain of abortion fetuses to diagnose the disease. To reveal the genomic organization and characterize the HNX strain, the complete genomes of HNX and Fa strain, an isolate in the 1960s, were sequenced and analyzed. The genomic size of HNX and Fa strains were 142,294 and 141,930 nt, respectively, with corresponding G + C contents of 73.56 and 73.70 %. The two strains consistently possessed 70 open reading frames. In addition, comparative genomic analysis between HNX and Bartha strains was performed to understand the possible reason of immune failure. The major virulence-associated genes of HNX strain had slight changes, whereas glycoprotein B and glycoprotein C genes of HNX strain had 73 mutations; the homology at the whole genomic level between HNX and Bartha strains was 90.6 %. Genome-wide comparison between HNX and Fa strains indicated that the strains shared about 96.4 % of homology and clustered in a separate Chinese isolate group; the two strains are also distant from the isolates from other countries. Similarity plot and bootscanning analysis of complete genome sequences of nine PRV strains, including HNX and Fa, four newly Chinese strains, and three traditional reference strains, revealed that non-recombination events occurred in the HNX strain. The PRV HNX strain with genomic variations might contribute to the PR outbreak in China since the later part of 2011.

Complete Genome Sequence of Novel Pseudorabies Virus Strain HNB Isolated in China.

The complete genome sequence of a novel pseudorabies virus, strain HNB, isolated from a dead weaned pig in China, was determined using next-generation sequencing. The viral genome sequence of HNB shared 90.6% nucleotide similarity with that of the traditional vaccine strain, the Bartha strain.

Comparative Genomic Analysis of Classical and Variant Virulent Parental/Attenuated Strains of Porcine Epidemic Diarrhea Virus.

Since 2010, the variant porcine epidemic diarrhea virus (PEDV) has been the etiological agent responsible for the outbreak of porcine epidemic diarrhea (PED) worldwide. In this study, a variant PEDV strain YN1 was isolated, serially propagated on the Vero cells and was characterized for 200 passages. To better elucidate the molecular basis of Vero cell adaptation of variant PEDV strains, we sequenced, compared, and analyzed the full-genome sequences of parental YN1 and passages 15, 30, 60, 90, 144, and 200. The results showed that the variations increased with the viral passage. The nucleotides sequences of non-structural protein (NSP)2, NSP4-7, NSP10, NSP12 and NSP13 genes did not change during the Vero cell adaptation process. After comparison of the variation characteristic of classical, variant virulent/attenuated strains, it was found that attenuation of PEDV virus was associated with 9-26 amino acid (aa) changes in open reading frames (ORF) 1a/b and S protein, early termination in ORF3, 1-3 aa changes in E, M and N protein and some nucleotide sequences' synonymous mutations. The aa deletion at about 144 aa of S protein could be the attenuation marker for the PEDV. The pig study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation.

Full-Length Genome Characterization of Chinese Porcine Deltacoronavirus Strain CH/SXD1/2015.

A porcine deltacoronavirus (PDCoV) was identified in the Chinese mainland and found to be closely related to Hong Kong strain HKU15-155 but differed from PDCoV strains in the United States and South Korea. The complete genome of PDCoV strain CH/SXD1/201 was sequenced and analyzed to further characterize PDCoV in Chinese swine.

Gene expression profiling of Cecropin B-resistant Haemophilus parasuis.

Synthetically designed antimicrobial peptides (AMPs) present the potential of replacing antibiotics in the treatment of bacterial infections. However, microbial resistance to AMPs has been reported and little is known regarding the underlying mechanism of such resistance. The naturally occurring AMP cecropin B (CB) disrupts the anionic cell membranes of Gram-negative bacteria. In this study, CB resistance (CBR) was induced in Haemophilusparasuis SH0165 by exposing it to a series of CB concentrations. The CB-resistant H.parasuis strains CBR30 and CBR30-50 were obtained. The growth curves of SH0165 and CBR30 showed that CBR30 displayed lower growth rates than SH0165. The result of transmission electron microscopy showed cell membranes of the CB-resistant CBR30 and CBR30-50 were smoother than SH0165. Microarrays detected 257 upregulated and 254 downregulated genes covering 20 clusters of orthologous groups (COGs) of the CB-resistant CBR30 compared with SH0165 (>1.5-fold change, p < 0.05). Sixty genes were affected in CBR30-50 covering 18 COGs, with 28 upregulated and 32 downregulated genes. Under the COG function classification, the majority of affected genes in the CB-resistant CBR30 and CBR30-50 belong to the category of inorganic ion transport, amino acid transport, and metabolism. The microarray results were validated by real-time quantitative reverse transcription PCR. This study may provide useful guidance for understanding the molecular mechanism underlying H.parasuis resistance to CB.

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets.

BACKGROUND: Post-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS. METHODS: In the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 107 TCID50 of a virulent PCV2 isolate. RESULTS: The vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group. CONCLUSIONS: The obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.