Detection and phylogenetic analysis of porcine circovirus type 3 in central China

Porcine circovirus type 3 (PCV3) is the pathogen responsible for a new infectious disease that was first reported in 2016 in the United States. To further investigate the epidemic profile and genetic diversity of the virus, one hundred and seventy clinical samples (110 tissue samples and 60 serum samples) were collected from 41 different pig farms in 14 cities in central China, and a SYBR Green I‐based quantitative real‐time PCR method was developed to detect PCV3. The partial cap genes of four field strains from four different farms were sequenced and analysed. The results showed the detection limit was 2.19 × 10¹ genome copies/μl. Fifty‐three of 170 samples were detected as positive for PCV3, giving a PCV3‐positive rate of 31.18%, with 48.78% (20/41) of pig farms harbouring PCV3, which varied from 20% to 42.86% between 2013 and 2017. PCV3 could be detected in samples from pigs with different clinical presentations, and the PCV3‐positive rates varied for these different clinical presentations. The partial capsid genes of four PCV3 strains (designated YZ, LY‐03, NY and SP) shared 96.3%–99.4% nucleotide identity with those available in GenBank. Phylogenetic analysis based on the capsid gene of 32 PCV3 strains showed that the four PCV3 strains in this study were clustered with the China/GD2016 and South Korea Ku‐1606 strains. The results of this study will aid our understanding of the molecular epidemiology of PCV3.     

The prevalence of novel porcine circovirus type 3 isolates in pig farms in China

The emerging porcine circovirus type 3 (PCV3) has been reported in Chinese swine herds since 2017. We performed a nationwide investigation on the prevalence of PCV3 in pig breeding farms and slaughterhouses in China. A total of 4,040 tonsil samples were collected from 89 farms in 25 provinces, and 1,419 lymph node samples were collected from 50 slaughterhouses in 27 provinces. The PCR results showed that in pig breeding farms, the positive rate was 41.6% (37/89) at the farm level and 5.0% (201/4040) at the individual level. In the slaughterhouses, the positive rate was 62.0% (31/50) at the farm level and 8.0% (114/1419) at the individual level. The PCR‐positive samples were further sequenced, and 19 new PCV3 isolates were identified. The complete genomes of the 19 virus isolates showed 97.4%–99.7% nucleotide identity with other PCV3 isolates. The phylogenetic analysis revealed that the 19 isolates were divided into PCV3a and PCV3b genotype clusters based on the PCV3 complete genome sequences. This study indicated that PCV3 has spread extensively in both pig breeding farms and slaughterhouses. The positive rate of PCV3 was higher in eastern China compared to other regions in China. Furthermore, this study will help us understand the prevalence and genetic variation of PCV3 in Chinese swine herds.     

The detection of porcine circovirus 3 in Guangxi,China

Porcine circovirus type 3 (PCV 3) is a novel circovirus that was firstly detected in the USA . PCV 3 is associated with porcine dermatitis and nephropathy syndrome (PDNS ), reproductive failure and cardiac and multisystemic inflammation. Latterly, PCV 3 was detected in Guangxi, China. Forty‐one of 108 (37.96%) samples and nine of 47 (19.14%) samples were PCV 3 positive in pig farms and pig slaughter houses, respectively. Three PCV 3 strains were sequenced and designated PCV 3‐China/GX 2016‐1, PCV 3‐China/GX 2016‐2 and PCV 3‐China/GX 2016‐3. The complete genome of PCV 3‐China/GX 2016‐2 and PCV 3‐China/GX 2016‐3 is both 2,000 bp in length, while PCV 3‐China/GX 2016‐1 is of 1,999 bp and has a G deletion at position of 1,155 in its genome. The complete genome and capsid nucleotide of the three PCV 3 strains identified in this study shared 97.5%–99.4% and 96.7%–99.1% identities with that of the other PCV 3 strains available in NCBI , respectively. Phylogenetic analysis based on complete genome and capsid gene of 35 PCV 3 strains showed that the three PCV 3 sequences from Guangxi Province were divided into two clusters. The results of this study contribute to the understanding of PCV 3 molecular epidemiology.     

Molecular detection and phylogenetic analysis of porcine circovirus type 3 in 21 Provinces of China during 2015–2017

The emerging Porcine circovirus type 3 (PCV3) is associated with porcine dermatitis and nephropathy syndrome, reproductive failure and cardiac and multisystemic inflammation. To trace the prevalence and evolution of PCV3 in pigs with respiratory diseases or digestive diseases in China, 616 samples were collected from 21 provinces or municipalities of China from 2015 to 2017. All samples were analysed with PCR and a cap‐gene‐based phylogeny. The results indicated that the positive rate of PCV3 was 12.2% (75/616) at the sample level; 24.1% (42/174) at the farm level; 10.4% (50/480) in the digestive‐disease‐affected samples; 26.6% (25/94) in the respiratory‐disease‐affected samples; all 42 healthy samples were negative for PCV3. A statistical analysis showed that PCV3 infection was closely associated with both digestive diseases (p < 0.05) and respiratory diseases (p < 0.01). A sequence analysis revealed that the cap genes of the 51 PCV3 strains identified in our study shared nucleotide homologies of 97.2%–100% and amino acid homologies of 96.3%–100%. A total of 17 amino acid mutations were observed among the Cap proteins of the 51 PCV3 strains, of which R¹⁰/K, A²⁴/V, R²⁷/K, T⁷⁷/S, F¹⁰⁴/Y, I¹⁵⁰/L are mutations among worldwide strains. A phylogenetic analysis demonstrated that the 51 PCV3 strains formed three clades, including PCV3a (15/51, 29.4%), PCV3b (21/51, 41.2%) and PCV3c (15/51, 29.4%). These data provide evidence that PCV3 exhibits high prevalence and genetic diversity and is associated with digestive diseases and respiratory diseases in pig.     

Epidemiological investigation of outbreaks of fowl adenovirus infections in commercial chickens in China

One hundred and five fowl adenovirus (FAdV) strains were isolated in China from 2015 to 2016 from poultry with inclusion body hepatitis (IBH ) and hydropericardium syndrome (HPS ). Polymerase chain reactions determined that 68 were FA dV species C, five were FA dV species D, two were FA dV species E, and 30 contained two or more different FA dV strains. A phylogenetic analysis showed that the isolated FA dV strains clustered into three major groups: FA dV‐C, FA dV‐D and FA dV‐E. Based on a hexon gene sequencing analysis, these viruses were genetically related to FA dV‐4, FA dV‐7, FA dV‐8b and FA dV‐11, of which FA dV‐4 was dominant (93% of the strains). An epidemiological analysis showed that FA dVs had been circulating in broilers, domestic chickens, and layers, and co‐infections with other immunosuppressive pathogens, such as chicken infectious anaemia virus, Marek's disease virus and reticuloendotheliosis virus, were identified. To control FA dVs, strict biosecurity protection measures are necessary, and a continued surveillance of FA dVs is needed to increase our understanding of the epidemiology of the viruses that are associated with IBH and HPS .     

Epidemiological investigation of H9 avian influenza virus,Newcastle disease virus,Tembusu virus,goose parvovirus and goose circovirus infection of geese in China

To deepen the knowledge about epidemic prevalence in the goose breeding field, a triplex PCR assay was established, and 478 samples were collected from scaled goose farms in 11 provinces in China. The results of this epidemiological investigation showed that incidence rates of H9 avian influenza and goose circovirus were the highest among five infectious diseases that were evaluated. In addition, the triplex PCR assay established remarkable sensitivity, rapidity and versatility compared to other diagnostic methods. Dual infection comprised a large proportion of the co‐infections in the field, of which the combinations of H9/Tembusu, H9/goose circovirus and goose circovirus/Tembusu co‐infected cases were more common. Epidemics were more severe in winter and spring. Additionally, significant differences in the prevalence of these infectious diseases were observed in association with different age groups. In addition, phylogenetic analysis, determined by the neighbour‐joining method, was carried out to investigate the evolution of these viruses during the study period. For the most part, virus strains isolated during the study were consistent with most goose‐origin strains isolated from the Chinese mainland over the past few years. However, mutations were observed between isolated H9 avian influenza virus strains and sequences available from GenBank, which should draw much attention.     

Molecular epidemiological survey and phylogenetic analysis of bovine influenza D virus in Japan

The influenza D virus, a new member of the Orthomyxoviridae family, is predominantly found in cattle. Although viral pathology and clinical disease in cattle appear mild, this virus plays an important role as a trigger of bovine respiratory disease (BRD ). BRD is a costly illness worldwide. Thus, epidemiological surveys of the influenza D virus are necessary. Here, we conducted a molecular epidemiological survey for the influenza D virus in healthy and respiratory‐diseased cattle in Japan. We found that 2.1% (8/377) of the cattle were infected with influenza D. The cattle with and without respiratory symptoms had approximately equal amounts of the virus. A full‐genome sequence analysis revealed that the influenza D virus that was isolated in Japan formed an individual cluster that was distinct from the strains found in other countries. These results suggest that this virus might have evolved uniquely in Japan over a long period of time and that the viral pathology of Japanese strains might be different from the strains found in other countries. Continuous surveillance is required to determine the importance of this virus and to characterize its evolution.     

Detection of porcine circovirus‐like virus P1 in Hebei,China

Porcine circovirus‐like virus P1 is a novel unclassified circovirus that was first detected in China and may be associated with post‐weaning multisystemic wasting syndrome (PMWS ) and congenital tremor. In this study, we detected P1 infection in pigs in Hebei Province, China, in 2017. One hundred and forty of 500 (28.0%) serum samples from 25 pig farms with different PMWS status in seven cities were P1 positive on PCR . Twelve P1 strains were sequenced, and the complete genomes of 11 P1 strains were 648 nucleotides (nt) in length, whereas that of strain ZJK 02 was 647 nt, with a G deletion at position of 183 in its genome. The complete genomic and capsid protein sequences of the 12 P1 strains analysed in this study shared 98.8%–100.0% and 86.5%–100.0% identity, respectively. A phylogenetic analysis based on the complete genomic and capsid sequences of 26 P1 strains showed that the 12 P1 sequences from Hebei Province clustered on two small branches. Further studies of the evolution and pathogenesis of P1 are required.     

Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China

Swine acute diarrhoea syndrome coronavirus (SADS‐CoV), a novel coronavirus, was first discovered in southern China in January 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. Here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of SADS‐CoV in pigs in China. Our results suggest that SADS‐CoV has emerged in China at least since August 2016. Meanwhile, we detected a prevalence of SADS‐CoV (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (PEDV) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of SADS‐CoV and PEDV occurred most frequently with the rate of 17.65%. We screened and obtained two new complete genomes, five N and five S genes of SADS‐CoV. Phylogenetic analysis based on these sequences revealed that all SADS‐CoV sequences in this study clustered with previously reported SADS‐CoV strains to form a well defined branch that grouped with the bat coronavirus HKU2 strains. This study is the first retrospective investigation for SADS‐CoV and provides the epidemiological information of this new virus in China, which highlights the urgency to develop effective measures to control SADS‐CoV.     

Genetic and phylogenetic analysis of reemerged novel Seneca Valley virus strains in Guangdong province, 2017

From June to July 2017, six Seneca Valley virus (SVV ) strains were isolated from swine herds exhibiting SVV ‐associated porcine idiopathic vesicular disease (PIVD ) in Guangdong province, China. Complete genomic sequences of these six newly identified strains were genetically and phylogenetically analysed. The results revealed that these six SVV strains were genetically closely related to USA /GBI 29/2015 and notably distinct from all previous Chinese strains, indicating the reemergence of new SVV strains in Guangdong province.     

The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in Southern China

Porcine deltacoronavirus (PDC oV) is a newly discovered coronavirus that causes diarrhoea, vomiting and dehydration in sucking and nursing piglets. It was first reported in Hong Kong in 2012 and has since been discovered in the United States, Canada, South Korea, mainland China, Thailand and Laos. PDC oV has been experimentally proved to lead to diarrhoea in swine and it was detected positive in pigs in Guangdong, southern China. In our study, 252 faecal and intestinal samples from sucking piglets and sows with diarrhoea were surveyed for common enteric viruses. We found a prevalence of PDC oV (21.8%), porcine epidemic diarrhoea virus (65.5%), transmissible gastroenteritis virus (0%), rotavirus group A (25.0%) and porcine kobuvirus (68.7%). We isolated 13 PDC oV strains and discovered that PDC oV infections were often co‐infections with kobuvirus rather than the commonly linked porcine epidemic diarrhoea virus. Phylogenetic analysis of S gene and N gene revealed that 11 of 13 PDC oV strains belonged to Chinese lineage. As for the left two strains, one single strain (CHN ‐GD 16‐05) belonged to American and Korean lineages while another strain (CHN ‐GD 16‐03) was similar to a Thai strain, but only in the S gene. This suggested a possible recombination event between the Thai and the newly described Chinese strain.     

First report and phylogenetic analysis of porcine deltacoronavirus in Mexico

Porcine deltacoronavirus has caused great economic losses in the swine industry worldwide. In this study, we carried out the first detection, sequencing and characterization of this virus in Mexico. We analysed 885 rectal samples by multiplex RT‐PCR to determine coinfections. In addition, the Spike gene was amplified, sequenced and analysed phylogenetically. We found 85 positive samples for porcine deltacoronavirus, representing 9.6% of the total samples, and we determined that the most frequent coinfection was with porcine epidemic diarrhoea virus (54.1%). Four sequences of Mexican isolates were most closely related to those of the United States. The antigenic regions and the glycosylation site of the strains obtained coincide with those previously reported. This relationship is probably related to the commercial exchange of pigs between the US and Mexico and the geographical proximity of these two countries.     

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.     

我国及广西手足口病流行病学研究进展

自从1948年Dalldorf在美国纽约州格林县的柯萨奇镇（Coxsackie New York）的患儿中发现并描述了一种后来被命名为柯萨奇病毒（Coxsackie virus）以来,直到1960 Alsop将由萨科奇病毒引起的疾病命名为手足口病（HFMD）,随后在世界各地均发现存在手足口病的流行。我国于1980年首次报告手足口病,广西于1989年第一次发现并报告了该病,自2008年手足口病列为国家法定丙类传染病以来,广西对手足口病的病原体的基因型进行了详细的研究,结果显示其中肠道病毒71型（EV71）占41.70%,柯萨奇病毒A16型（CoxA16）阳性8.50%。同时证实广西分离的EV71与国内其他省流行的EV71流行株具有高度的同源性,同属于C4基因亚型C4b分支。资料显示手足口病在国内呈现自然流行消长趋势,尽管该病的疫苗研究取得了多方面的进展,但离现场应用仍然存在多方面的挑战。     

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full‐length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016–2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild‐type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.     

Detection of reassortant avian influenza A (H11N9) virus in wild birds in China

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra‐subtype and inter‐subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds.     

Genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from South Korea, 2017

Porcine epidemic diarrhoea virus (PEDV ) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. Since the re‐emergence of massive PEDV outbreaks in South Korea in 2013−2014, domestic pig farms have continued to experience PED epidemics or endemics. This study represents the molecular characterization of PEDV isolates identified in diarrhoeic animals collected across the country in 2017. Initial sequencing analysis of the full‐length S genes revealed that 70% of the 2017 isolates (7/10) belong to the G2b subgroup, while the remaining isolates were classified as G1b. The data indicated that both variant G1b and global epidemic G2b strains were responsible for current PED outbreaks in South Korea. The 2017 G1b and G2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the S gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding Korean prototype G1b and G2b strains, respectively. In an interesting manner, one G2b‐like KNU ‐1705 strain was found to possess a large 39‐nucleotide deletion in the ORF 1a region theoretically encoding nonstructural protein 3. Phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global G1b or G2b strains but formed different branches within the same genogroup. These results indicate that PEDV s undergo continuous evolution in the field. In addition, one 2017 PEDV strain, KOR /KNU ‐1705/2017, was successfully isolated and propagated in Vero cells. The antisera raised against the Korean prototype 2014 G2b strain efficiently neutralized KNU ‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 PEDV strains. Our data advance the understanding of the molecular epidemiology and antigenicity of PEDV circulating in South Korea.     

Molecular characterization of Peste des petits ruminants viruses in the Marmara Region of Turkey

Recent outbreaks of Peste des petits ruminants (PPR) in the Marmara region of Turkey including the European part of Thrace is important due to its proximity to Europe (Greece and Bulgaria) and the potential threat of spread of PPR into mainland Europe. In order to investigate the circulation of PPRV in the region suspect clinical and necropsy samples were collected from domestic sheep (n = 211) in the Marmara region of Turkey between 2011 and 2012. PPR virus (PPRV) genome was detected in 10.4% (22 out of 211) of sheep samples by real‐time RT‐PCR, and PPR virus was isolated from lungs of two sheep that died from infection. Of the 22 positive samples nine were used for partial N‐gene amplification and sequencing. The phylogenetic analyses indicated that the virus belongs to lineage IV, the same lineage that is circulating in eastern and central part of Turkey since its first official report in 1999. In addition, samples from 100 cattle were collected to investigate potential subclinical circulation of PPRV. However all were found to be negative by real‐time RT‐PCR, and also in serological tests indicating the large ruminants were likely not exposed or infected with the virus. The impact of these findings on the potential threat of spread of PPR to Europe including the first PPR outbreak in Europe in Bulgaria on 23rd June 2018 is discussed.