Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Preliminary serological evidence for Schmallenberg virus infection in China.

Schmallenberg virus (SBV) is an emerging and rampant arbovirus in Europe, and even Africa and West Asia. Investigating whether SBV existed in new regions or countries, it was very helpful for the early warning and control of SBV. In this study, we collected 317 serum samples (n = 242 for dairy cattle, n = 13 for yellow cattle, n = 21 for buffalo, and n = 41 for goats) from Guangdong province of southern China, which is located in a subtropical region and is an important distribution area for arboviral diseases. A commercial competitive enzyme-linked immunosorbent assay (cELISA) kit and a previously established real-time PCR were used to detect SBV antibody and RNA in those serum samples. Via testing, serological evidence of SBV was confirmed, with total positive rates (57.4, 15.4, 19, and 9.8%) in dairy cattle, yellow cattle, buffalo, and goats, respectively, while no positive signal for SBV RNA was found. To summarize, this study for the first time provided preliminary serological evidence of SBV infection in China, East Asia. Further investigations on molecular evidence, origin, and pathogenesis of SBV in ruminants needed to be studied in China.

Influenza D Virus in Animal Species in Guangdong Province, Southern China.

Molecular tests revealed influenza D viruses of D/OK lineage widely circulating in farmed animal species in Guangdong Province, southern China. In particular, we found high levels of influenza D virus infection in goats and pigs. We also detected viral RNA in serum specimens and feces of animals with certain severe diseases.

Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations.

BACKGROUND: Porcine circovirus type 3 (PCV3), as an emerging circovirus species, was reported to be widely circulating in the United States, China, South Korea and Poland. Previous studies revealed that PCV3 was mainly concentrated in sick animals with respiratory disease, skin disease, reproductive disorders and so on. However, the circulating status of PCV3 in pigs with other clinical presentations (especilly asymptomatic or diarrhea) was not well established. FINDINGS: In this study, to conduct a comparative epidemiological survey of PCV3, 80 weaned pig serum samples with severe respiratory disease (SRD), 175 weaned pig serum samples with mild respiratory disease (MRD), 216 asymptomatic weaned pig serum samples, 35 diarrheal weaned pig samples and 35 non-diarrheal weaned pig samples were collected from eight provinces of China. Via qPCR testing, PCV3 was circulating in all sampling provinces, with total positive rates varying from 1.04% to 100%. Interestingly, the PCV3-positive rate was significantly higher in weaned pigs with SRD (63.75%, 51/80) than in those weaned pigs with MRD (13.14%, 23/175) and asymptomatic pigs (1.85%, 4/216) (P < 0.01). Similarly, the PCV3-positive rate was significantly higher in diarrheal weaned pigs (17.14%, 6/35) than in non-diarrheal weaned pigs (2.86%, 1/35) (P < 0.05). Moreover, the lower Ct values of qPCR were frequently found in those weaned pigs or fattening pigs with respiratory disease and diarrhea rather than that in asymptomatic pigs. Sequence analysis showed that low genetic diversity existed among those PCV3 sequences collected from pigs with different clinical presentations. CONCLUSIONS: The present study further extends evidence that newly described PCV3 widely circulates in six additional provinces of Southern and Northern China and has high similarity to previously reported isolates. As an emerging virus of swine, although the present case-control study reveals that PCV3 has a potential association with swine respiratory disease and diarrhea, further investigations into the pathogenesis are needed to ascertain the role of PCV3 in swine health.

Occurrence and sequence analysis of porcine deltacoronaviruses in southern China.

BACKGROUND: Following the initial isolation of porcine deltacoronavirus (PDCoV) from pigs with diarrheal disease in the United States in 2014, the virus has been detected on swine farms in some provinces of China. To date, little is known about the molecular epidemiology of PDCoV in southern China where major swine production is operated. RESULTS: To investigate the prevalence of PDCoV in this region and compare its activity to other enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis coronavirus (TGEV), and porcine rotavirus group C (Rota C), 390 fecal samples were collected from swine of various ages from 15 swine farms with reported diarrhea. Fecal samples were tested by reverse transcription-PCR (RT-PCR) that targeted PDCoV, PEDV, TGEV, and Rota C, respectively. PDCoV was detected exclusively from nursing piglets with an overall prevalence of approximate 1.28 % (5/390), not in suckling and fattening piglets. Interestingly, all of PDCoV-positive samples were from 2015 rather than 2012-2014. Despite a low detection rate, PDCoV emerged in each province/region of southern China. In addition, compared to TGEV (1.54 %, 5/390) or Rota C (1.28 %, 6/390), there were highly detection rates of PEDV (22.6 %, 88/390) in those samples. Notably, all five PDCoV-positive piglets were co-infected by PEDV. Furthermore, phylogenetic analysis of spike (S) and nucleocapsid (N) gene sequences of PDCoVs revealed that currently circulating PDCoVs in southern China were more closely related to other Chinese strains of PDCoVs than to those reported in United States, South Korea and Thailand. CONCLUSIONS: This study demonstrated that PDCoV was present in southern China despite the low prevalence, and supported an evolutionary theory of geographical clustering of PDCoVs.

Emergence of reticuloendotheliosis virus in pigeons in Guangdong Province, Southern China.

Reticuloendotheliosis virus (REV), an important immunosuppressive pathogen, has many hosts, including chickens, ducks, geese, turkeys, and wild birds. Clinically, REV may lead to increased susceptibility to other pathogens, resulting in serious tissue damage (especially tumors) and the death of its host. In this study, we encountered a disease outbreak resulting in a large number of deaths of pigeons in Guangdong Province, Southern China. Histopathological analysis revealed apparent tumor-like lesions in multiple organs of pigeons. PCR assays for detection of tumor-associated pathogens (REV, avian leukosis virus, and Marek's disease virus) in poultry revealed the presence of REV sequences only. Moreover, fowlpox virus (FPV) with an insertion of REV long terminal repeat (LTR) sequences was also considered, but it was excluded using a specific PCR assay. To gain more genetic information, two full-length REV genome sequences were determined and found to have the highest nucleotide sequence similarity (99.9 %) and the closest genetic relationship to a vaccine strain (MD-2) and had a more distant genetic relationship (94.3 %) to a duck-origin strain (ATCC-VR775). To confirm the presence of REVs in pigeons, specific-pathogen-free (SPF) chickens and healthy pigeons were inoculated with microfiltered tumor tissue homogenates and were found to be susceptible to infection with REV. To our knowledge, this is the first report of REV in pigeons, and the data suggest that pigeons may be the natural host of REV.

Molecular detection and genome characterization of porcine circovirus type 2 in rats captured on commercial swine farms.

Porcine circovirus type 2 (PCV2) is considered the major etiological pathogen of porcine circovirus-associated diseases (PCVADs) in pigs. Recently, PCV2 was also found in non-porcine animals such as cattle, rats, and mice. However, there was no record of PCV2 in rats in China. The goal of this study was to investigate whether PCV2 was present in rats (Rattus norvegicus, RN) on three swine farms, using molecular tools. PCR results showed that 30 of 95 (31.6 %) rat samples were positive for PCV2. Moreover, further genotype analysis suggested that 10 of 30 (33.3 %) were positive for PCV2a, 19 of 30 (63.3 %) were positive for PCV2b, and only one sample (1/30, 3.33 %) was co-infected by PCV2a and PCV2b. To determine the possible origin of PCV2, 60 serum samples were also collected from weaned pigs on those swine farms, and 23 out of 60 samples were positive for PCV2. In addition, two distinct RN-origin and two distinct porcine-origin PCV2 full-length nucleotide sequences were obtained from the farms. Sequence and phylogenetic analysis indicated that they had the highest nucleotide similarity and closest genetic relationships to each other. In this study, we report the infection and genome characterization of PCV2 in rats and compare RN-origin and porcine-origin PCV2 sequences obtained from the same pig farm, revealing possible cross-species transmission of PCV2.

Genome characterization and phylogenetic analysis of a lineage IV peste des petits ruminants virus in southern China.

Since 2013, the second outbreak of peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) has spread over more than 20 provinces, municipalities, and autonomous regions in China, resulting in major economic losses for livestock industry. In 2014, we encountered a clinical PPR case on a goat farm in Guangdong province, southern China. The complete genome of this PPRV strain, named CH/GDDG/2014, was sequenced to determine its similarities and differences with other strains. The CH/GDDG/2014 genome comprised 15,954 nucleotides (six nucleotides more than classical PPRVs identified before 2013, but complying with the rule of six) with six open reading frames encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. The whole-genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus (99.8 %) to China/XJYL/2013 and the least consensus (87.2 %) to KN5/2011. The phylogenetic analysis showed that CH/GDDG/2014 was clustered in one branch (lineage IV) with other emerging strains during the second outbreak. This study is the first report describing the whole-genome sequence of PPRV in Guangdong province, southern China and also suggests the PPR outbreak may be closely related to illegal cross-regional importation of goats.

Porcine circovirus type 2 in China: an update on and insights to its prevalence and control.

Currently, porcine circovirus type 2 (PCV2) is considered the major pathogen of porcine circovirus associated-diseases (PCVAD) that causes large economic losses for the swine industry in the world annually, including China. Since the first report of PCV2 in 1998, it has been drawing tremendous attention for the government, farming enterprises, farmers, and veterinary practitioners. Chinese researchers have conducted a number of molecular epidemiological work on PCV2 by molecular approaches in the past several years, which has resulted in the identification of novel PCV2 genotypes and PCV2-like agents as well as the description of new prevalence patterns. Since late 2009, commercial PCV2 vaccines, including the subunit vaccines and inactivated vaccines, have already been used in Chinese swine farms. The aim of this review is to update the insights into the prevalence and control of PCV2 in China, which would contribute to understanding the epidemiology, control measures and design of novel vaccines for PCV2.

First molecular detection of porcine circovirus type 2 in bovids in China.

For the worldwide pig industries, porcine circovirus type 2 (PCV2) is an economically important pathogen. At present, the prevalence of PCV2 is common in Chinese swine herds. However, there is little information on PCV2 prevalence in non-porcine animals in China, such as bovids. Therefore, the goal of this study is to obtain the firsthand prevalence data of PCV2 in bovids in China. Two hundred and eighty serum and muscle samples from dairy cows (n = 180), buffalo (n = 50), and yellow cattle (n = 50) were analyzed by PCR. The detection results show that PCV2 infections (16 %, 8/50) only exist in buffaloes. In addition, there are different PCV2 viral DNAs identified by differential PCR in the same buffalo sample. Nucleotide sequencing and phylogenetic analysis results based on partial ORF1 and ORF2 sequences suggest that PCV2 strains have genetic diversity in buffaloes and they are divided into three different genotypes (PCV2b, PCV2d, and PCV2e, respectively). Moreover, to our knowledge, the PCV2d and PCV2e genotypes have not been previously reported in bovids. Through this study, the firsthand data of PCV2 prevalence in bovids in China was documented.

An fusaric acid-based CRISPR library screen identifies MDH2 as a broad-spectrum regulator of Fusarium toxin-induced cell death.

Fusarium mycotoxins are of great concern because they are the most common food-borne mycotoxins and environmental contaminants worldwide. Fusaric acid (FA), Deoxynivalenol (DON), Zearalenone (ZEA), T-2 toxin (T-2), and Fumonisin B1 (FB1) are important Fusarium toxins contaminating feeds and food and can cause serious health problems. FA can synergize with some other Fusarium toxins to enhance overall toxicity. However, the underlying molecular mechanism remains poorly understood. In this study, our CRISPR screening revealed Malate dehydrogenase 2 (MDH2) and Pyruvate dehydrogenase E1 subunit beta (PDHB) are the key genes for FA-induced cell death. Pathways associated with mitochondrial function, notably the TCA cycle, play a significant role in FA cytotoxicity. We found that MDH2 and PDHB depletion reduced FA-induced cell death, ROS accumulation, and the expression of caspase-3 and HIF-1α. The cell viability assays and flow cytometry demonstrated that MDH2 knockout but not PDHB decreased DON, ZEA, T-2, and FB1-induced cytotoxicity, apoptosis, and ROS accumulation. MDH2 inhibitor LW6 also decreased DON, ZEA, T-2, and FB1-induced toxicity. This suggested that MDH2, but not PDHB, is a common regulator of broad-spectrum Fusarium toxin (FA, DON, ZEA, T-2, and FB1)-induced cell death. Our work provides new avenues for the treatment of Fusarium toxin toxicity.

Influenza D virus infection in China, 2022-2023.

Influenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022-2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022-2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.

High prevalence of torque teno sus virus in China and genetic diversity of the 5' non-coding region.

Members of the family Anelloviridae are emerging circular DNA viruses infecting many species of vertebrates including pigs. To date, members of two distinct genera, Iotatorquevirus, including torque teno sus virus 1a and torque teno sus virus 1b (TTSuV1a and TTSuV1b), and Kappatorquevirus, including torque teno sus virus k2a and torque teno sus virus k2b (TTSuVk2a and TTSuVk2b), have been identified in domestic pigs and wild boars. The goal of this study was to evaluate the prevalence and genetic diversity of these viruses based on 5' non-coding genes in Chinese swine herds experiencing clinical symptoms. One hundred eighty-five clinical samples from 11 different regions, collected during 2008-2009, were analyzed using a PCR method, and the results revealed a high TTSuV-positive rate of 78.9 % (146/185) in pigs. Moreover, we detected co-infection with multiple TTSuV strains in the same pig. Nucleotide sequencing results revealed greater genetic diversity within the genus Kappatorquevirus than within the genus Iotatorquevirus. In addition, TTSuVk2b, a novel virus discovered in New Zealand in 2012, was also identified in this study. In summary, the present work helps us obtain more knowledge about the epidemiology and genetic diversity of TTSuVs.

Identification of D/Yama2019 Lineage-Like Influenza D Virus in Chinese Cattle.

Outbreaks of influenza D virus (IDV) continue to be reported in many countries. On the basis of the hemagglutinin-esterase fusion (HEF) gene, five IDV genetic lineages have been identified: D/OK, D/660, D/Yama2016, D/Yama2019 and D/CA2019 lineages. Previously reported IDV strains in China all form a sub-clade (D/China sub-lineage) within D/OK lineage. From October 2021 to February 2022, nasal swab samples (n = 250) were collected from apparently healthy cattle in slaughterhouses around the city of Guangzhou, China, and screened for IDV by RT-PCR. Ten samples were positive for IDV. An IDV strain with nearly complete genome sequences was identified and designated as D/bovine/CHN/JY3001/2021. Importantly, sequence alignments and phylogenetic analyses revealed that this IDV strain is genetically close to the strains (>98% homology) in the D/Yama2019 lineage that has been found only in Japan, but distant from the previously reported Chinese IDV strains (~95% similarity). These results demonstrate the emergence of D/Yama2019 lineage IDV in Chinese cattle herds, highlighting a need for future surveillance of D/Yama2019-like viruses toward better understanding both epidemiology and diversity of IDV in China.

Co-existence of multiple strains of porcine circovirus type 2 in the same pig from China.

Pigs are often co-infected by different viral strains from the same virus. Up to now, there are few reports about co-existence of different porcine circovirus type 2 (PCV2) strains in China. The aim of this study was to evaluate it in Chinese swine herds. 118 PCV2 positive DNAs isolated from diseased pigs identified by classic PCR were re-detected using a modified differential PCR assay. The results indicated that co-existence rates of PCV2 were 32.2% (38/118) in diseased pigs and 0% (0/41) in asymptomatic pigs. Four PCV2 complete genomes were cloned from two co-infected samples and their nucleotide (nt) identities were 95%-97.3%. The phylogenetic analysis showed that four PCV2 strains were divided into different genotypes, PCV2a, PCV2b, PCV2d and PCV2e, respectively. In addition, co-existence were not detected in 41 serum samples from healthy pigs but PCV2 single infection (31.7%, 13/41) existed. These data revealed that the co-existence of different strains of PCV2 might contribute to the development of more severe clinical symptoms for pigs. This is the first report confirming the co-existence of different PCV2 strains in Chinese swine herds. Meanwhile, this study could help us to understand new infection and prevalence forms of PCV2 clinically.

Genome Characterization and Phylogenetic Analysis of the First Bovine Rhinitis B Virus Isolate in China.

Bovine rhinitis B virus (BRBV) is an emerging viral species in the genus Aphthovirus, family Picornaviridae. Studies suggested that BRBV was considered a potential etiological agent of bovine respiratory disease complex (BRDC). BRBV has been reported in the United States, Sweden, Canada, Japan, and Mexico. However, little information of BRBV was available in China. In this study, we performed viral metagenomic analysis in a calf with respiratory disease. The results showed high abundance (3.85) of BRBV nucleotide and 248 mapped reads in calf samples. Online BLASTn analysis showed that three contigs of those had the highest nucleotide similarity (95%) with one Swedish BRBV isolate (BRBV_SWE1, GenBank accession no. KY432299). To identify the genome characterization of the Chinese BRBV isolate (designated CHN1), six couples of overlapping RT-PCR primers were designed according to genome sequences of BRBV_SWE1. Through gene cloning and splicing, we obtained the genome information of CHN1, possessing 7,465 nucleotides (46.6% G+C). Although CHN1 had the highest nucleotide similarity (95.1%) with BRBV_SWE1, one 11-nucleotide (ACATTTGTTGT) deletion occurred in the 5' untranslated region compared to SWE1. Phylogenetic analysis showed that CHN1 clustered together with BRBV_SWE1, and far from other BRBV isolates. This study recorded the first discovery of BRBV infection in China. Further investigation should be made in order to evaluate the infection status and epidemiological significance of BRBV in China.

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/µl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

Reservoirs of Porcine Circoviruses: A Mini Review.

Porcine circovirus (PCV) is one of the smallest known DNA viruses in mammals. At present, PCVs are divided into three species, PCV1, PCV2, and PCV3. PCV1 and PCV2 were found in the 1970s and the 1990s, respectively, whereas PCV3 was discovered recently in 2016. PCV1 does not cause diseases in pigs. However, PCV3, similar to PCV2, is reported to be associated with several swine diseases, including porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCVs are very common in domestic pigs as well as wild boars. However, PCVs have been occasionally isolated from non-porcine animals, including ruminants (such as cattle, goats, wild chamois, and roe deers), rodents (such as NMRI mice, BALB/c mice, Black C57 mice, ICR mice, Mus musculus, and Rattus rattus), canines (such as dogs, minks, foxes, and raccoon dogs), insects (such as flies, mosquitoes, and ticks), and shellfish. Moreover, PCVs are frequently reported in biological products, including human vaccines, animal vaccines, porcine-derived commercial pepsin products, and many cell lines. PCVs are also abundant in the environment, including water samples and air samples. Interestingly, PCV1 and/or PCV2 antibody or antigen has also been detected in sera, stool samples and respiratory swab samples of human, revealing zoonotic potential of PCVs. Thus, PCVs inhabit many types of reservoirs. In this review, we summarize the reservoirs of PCVs, and this information would be helpful in understanding the natural circulating status and possible cross-species transmission of PCVs.

A Novel Enzootic Nasal Tumor Virus Circulating in Goats from Southern China.

Enzootic nasal tumor virus (ENTV) has two types, ENTV-1 in sheep and ENTV-2 in goats, respectively. In China, the incidence of ENTV-2 related diseases has increased year by year. In this study, we reported an outbreak of ENTV-2 in a commercial goat farm in Qingyuan city, Guangdong province, southern China. A full-length genome of ENTV-2 (designated GDQY2017), with 7479 base pairs, was sequenced. Although GDQY2017 shared the highest nucleotide identity with a Chinese ENTV-2 isolate (ENTV-2CHN4, GenBank accession number KU258873), it possesses distinct genome characteristics undescribed, including a non-continuous 21-nucleotide insertion in the gag gene and a non-continuous 12-nucleotide deletion in the env gene. Notably, most of these indel nucleotide sequences were originated from a Chinese jaagsiekte sheep retrovirus (JSRV) isolate (GenBank accession number DQ838494). In the gag and env genes, GDQY2017 was phylogenetically related to those Chinese ENTV-2 isolates and a Chinese JSRV isolate (DQ838494). For GDQY2017-like viruses, more surveillance work should be made to explain their pathogenicity in goat herds. To our knowledge, this study represents the first to demonstrate the circulating pattern of ENTV-2 in Guangdong province, China, which will help to better understand the epidemiology and genetic diversity of ENTV-2.