Detection of atypical porcine pestivirus in Brazil in the central nervous system of suckling piglets with congenital tremor

Atypical porcine pestivirus (APPV ) has been detected in piglets with congenital tremor (CT ) from three different continents including North America, Europe and Asia. Thirteen piglets from four farms in two different states in Brazil with CT were sampled. Viral RNA was detected by quantitative real‐time PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT , and APPV was not detected in any tissue sample from clinically non‐affected piglets with the exception of the cerebellum of one piglet from Farm A. Piglets with CT had an odds ratio of 99.0 (95% CI 3.4, 2823.8; p  =  .0072) compared to piglets without CT to test positive for APPV by qRT ‐PCR . A subset of positive samples was selected for sequencing of the NS 3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS 3 formed an independent cluster and had the highest sequence identity with a sequence from the United States. This is the first identification of APPV infection in piglets with CT in South America.     

Phylogenetic and genomic characterization of a novel atypical porcine pestivirus in China

Atypical porcine pestivirus (APPV ) has been considered a novel pestivirus and causative agent of congenital tremor type A‐II . An APPV CH ‐GX 2016 strain was characterized from newly born piglets with clinical symptoms of congenital tremor in Guangxi, China. The genome of APPV CH ‐GX 2016 strain was 11,475 bp in length and encoded a polyprotein composed of the 3,635 amino acids. This genome sequence exhibited 88.0% to 90.8% nucleotide sequence homology with other APPV reference sequences in GenBank. Phylogenetic analysis further showed that APPV CH ‐GX is a novel pestivirus compared with previously described classical pestivirus strains. Therefore, APPV is present in pigs in China.     

Presence of atypical porcine pestivirus (APPV) in Brazilian pigs

Recently, a putative new pestivirus species, provisionally named as Atypical Porcine Pestivirus (APPV ), was associated with the congenital tremor in piglets in North America and consequently in Europe and Asia. The present research aimed to describe the detection and characterization of APPV employing NS 5B gene partial sequencing, gross pathology and histologic examination of piglets displaying congenital tremor from two different farms of Southern Brazil. No gross lesions were observed, and the histological findings revealed moderate vacuolization of the white matter of the cerebellum. RT ‐PCR followed by DNA sequencing and a phylogenetic analysis confirmed the presence of APPV in samples from the two farms, which the samples were distinct in nature. Phylogenetic reconstruction reinforced the high genetic variability within the APPV s previously reported. This is the first report of APPV in South America suggesting that this new group of viruses may be widespread in swine herds in other countries as it is in Brazil.     

First report of the novel atypical porcine pestivirus in Spain and a retrospective study

This study aimed to provide information regarding viral pathogenesis and molecular epidemiology linked with recently reported atypical porcine pestivirus (APPV) strains and to determine the circulation of APPV in Spain from 1997 to 2016. Two‐day‐old piglets with moderate–severe congenital tremor (CT) from a Spanish farm were received for diagnostic purposes. Sera, nasal and rectal swabs and tissue samples were collected. qRT‐PCR was performed in these samples, and a retrospective study to detect APPV RNA was carried out using a serum collection from 1997 to 2016. APPV genome was identified with high and moderate RNA loads in different tissues of the CT affected pigs. High APPV RNA load was detected in lymphoid organs, suggesting that these constitute a target for APPV replication. In 89 of the 642 retrospectively analysed samples (13.9%), APPV genome was detected. CT cases were related to the presence of APPV in viraemic piglets below 1 week of age, in which the viral RNA load was the highest. A considerable number of animals between 4 and 14 weeks of age and some 1‐week‐old piglets were viraemic in the absence of CT, which can act as carriers of the virus. The relative risk of APPV and CT was 8.5 (CI 95% 5.8–12.5). Thus, our data show that APPV infection is epidemiologically related to CT. Phylogenetic analysis from 1615 NS2‐3 nucleotides showed only one defined APPV clade, grouping the most phylogenetically related strains from Europe and China. Of this clade, there are other strains from Europe, USA and China. This data confirm the high APPV genetic diversity, not being able to cluster this virus according to the geographic area. Our result showed that APPV has been circulating in Spain at least since 1997, being the earliest date of detection of this virus worldwide and suggesting that APPV may be widespread.     

Identification of atypical porcine pestivirus infection in swine herds in China

Atypical porcine pestivirus (APPV) have been detected in swine herds from the USA, Germany, the Netherlands, Spain and most recently in Austria, suggesting a wide geographic distribution of this novel virus. Here, for the first time, we reported APPV infection in swine herds in China. Newborn piglets from two separate swine herds in Guangdong province were found showing typical congenital tremors in July and August 2016. RT‐PCR, sequencing and phylogenetic analysis showed APPV infection occurred. Phylogenetic analysis showed that Chinese APPV strains, GD1 and GD2, formed independent branch from the USA, Germany and the Netherlands. Nucleotide identities between members of the APPV ranged between 83.1% and 83.5%, and this showed APPV is highly diverse. It is apparent that this provides the first molecular evidence of APPV infection in swine herds in China.     

Viruses associated with congenital tremor and high lethality in piglets

The recently described atypical porcine pestivirus (APPV ) has been associated with congenital tremor (CT ) type A‐II in piglets in different countries. Another important neurological pathogen of pigs is porcine teschovirus (PTV ), which has been associated with non‐suppurative encephalomyelitis in pigs with severe or mild neurological disorders. There have been no reports of APPV and/or PTV coinfection associated with CT or encephalomyelitis in Brazilian pig herds. The aim of this study was to describe the pathological and molecular findings associated with simultaneous infection of APPV and PTV in piglets with clinical manifestations of CT that were derived from a herd with high rates of CT ‐associated lethality. In 2017, three piglets from the same litter with CT died spontaneously. The principal pathological alterations in all piglets were secondary demyelination and hypomyelination at the cerebellum, brainstem and spinal cord confirmed by histopathology and luxol fast blue–cresyl violet stain. Additional significant pathological findings included multifocal neuronal necrosis, neuronophagia and gliosis found in the cerebral cortex and spinal cord of all piglets, while atrophic enteritis and mesocolonic oedema were observed in some of them. APPV and PTV RNA were detected in the central nervous system of affected piglets, and PTV was also detected in the intestine and faeces. The pathological alterations and molecular findings together suggest a dual infection due to APPV and PTV at this farm. Moreover, the combined effects of these pathogens can be attributed to the elevated piglet mortality, as coinfections involving PTV have a synergistic effect on the affected animals.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Frequent infection of wild boar with atypical porcine pestivirus (APPV)

The recently identified atypical porcine pestivirus (APPV ) was demonstrated to be the causative agent of the neurological disorder “congenital tremor” in newborn piglets. Despite its relevance and wide distribution in domestic pigs, so far nothing is known about the situation in wild boar, representing an important wild animal reservoir for the related classical swine fever virus. In this study, 456 wild boar serum samples obtained from northern Germany were investigated for the presence of APPV genomes and virus‐specific antibodies. Results of real‐time RT ‐PCR analyses revealed a genome detection rate of 19%. Subsequent genetic characterization of APPV (n  = 12) from different hunting areas demonstrated close genetic relationship and, with exception of APPV from one location, displayed less than 3.3% differences in the analysed partial NS 3 encoding region. Furthermore, indirect E^rns ELISA revealed an antibody detection rate of approx. 52%, being in line with the high number of viremic wild boar. Analysis of fifteen wild boar samples from the Republic of Serbia by E^rns antibody ELISA provided evidence that APPV is also abundant in wild boar populations outside Germany. High number of genome and seropositive animals suggest that wild boar may serve as an important virus reservoir for APPV .     

Genome characterization of a porcine circovirus type 3 in South China

Porcine circovirus type 3 (PCV 3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. Recently, a PCV 3 strain was identified from pyretic and pneumonic piglets in Guangdong province, China. This virus strain was sequenced and designated PCV 3‐China/GD 2016. The complete genome of PCV 3‐China/GD 2016 is 2,000 bp in length and shared 99.1% and 99.1% nucleotide identities with PCV 3/29160 and PCV 3/2164, 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 PCV 3‐China/GD 2016 clustered with the emerging PCV 3 and separated with other virus in genus Circovirus . The results of this study suggest that PCV 3 has existed within the pigs of China. It is urgent to investigate the pathogenicity and epidemiology of this novel circovirus China.     

Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China

A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV‐SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV‐SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59–61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80–224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%–98.9%. ORF3 protein of both HLJBY strain and PEDV‐SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY‐P10 or PEDV CV777‐P8 inoculated piglets.     

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.     

Detection and genome sequencing of porcine circovirus 3 in neonatal pigs with congenital tremors in South China

Porcine circovirus 3 (PCV3) is a novel circovirus first discovered in the United States in piglets and sows with porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multisystemic inflammation. Here, seven PCV3 strains were identified for the first time from neonatal pigs with clinical signs of congenital tremors (CT) in South China. The tissue tropism of PCV3 in CT‐affected piglets was analysed by the real‐time quantitative PCR, and the result showed that high loads of viral genomes were detected in the brains and hearts. The complete genomes of seven new PCV3 revealed 96.8%–99.6% nucleotide identities with eleven other PCV3 strains previously reported from the United States and China. Phylogenetic analysis based on the complete genome sequences showed that all PCV3 strains clustered together and were clearly separated from other circovirus species. This study reports on the first identification of PCV3 in CT‐affected newborn piglets and provides the epidemiological information of neonatal piglets with CT in Guangdong and Guangxi Provinces of China.     

Isolation and phylogenetic analysis of porcine deltacoronavirus from pigs with diarrhoea in Hebei province,China

Porcine deltacoronavirus (PDC oV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease with clinical signs including diarrhoea, vomiting, dehydration and mortality in neonatal piglets. Although evidence of the prevalence of PDC oV in China is accumulating, little published information about Chinese PDC oV isolates is available. In this study, we investigated the presence of PDC oV in 49 faecal/intestinal samples from piglets with diarrhoea on different farms in Hebei province. Five samples (10.2%) were positive for PDC oV, but no coinfection of PDC oV with other enteropathogens was observed. A PDC oV strain named HB ‐BD was successfully isolated from the intestinal contents of a diarrhoeic piglet and serially propagated in swine testicular (ST ) cells for >40 passages. The complete genome of the HB ‐BD strain was sequenced and analysed. Genomic analysis showed that the HB ‐BD strain had a closer relationship with Chinese strains than those from other countries and was grouped within the Chinese PDC oV cluster. The results of this study will be valuable for further research of PDC oV genetic evolution and development of effective diagnostic reagents, assays and potential vaccines against newly emerged PDC oV strains.     

First report of a novel goose astrovirus outbreak in Cherry Valley ducklings in China

A highly acute disease broke out in ducklings in Shandong Province in March 2019. The disease was characterized as visceral gout, with a mortality rate of 30%. The causative agent, which has given rise to similar symptoms in goslings, has been confirmed to be a novel goose astrovirus. The novel goose astrovirus, which was designated as the SDXT strain, was identified from a diseased duck farm using duck embryo primary cells in an experimental infection test. Genomic sequence analysis, as well as phylogenetic analysis of the viral proteins, revealed that the SDXT strain was closely related to a novel goose astrovirus of the Avastrovirus three species. These results indicate that the novel goose astrovirus may cross‐host infect ducklings. Further studies are needed to define its host range and transmission route.     

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.     

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.     

Detection and phylogenetic analysis of porcine bocaviruses carried by murine rodents and house shrews in China

Bocaparvovirus infections of humans and both wild and domestic animals have been widely reported around the world. In this study, we detected and genetically characterized porcine bocavirus (PBoV) carried by murine rodents (Rattus norvegicus, Rattus tanezumi, and Rattus losea) and house shrews (Suncus murinus) in China. Between May 2015 and May 2017, 496 murine rodents and 23 house shrews were captured in four Chinese provinces. Nested polymerase chain reaction was used to investigate the prevalence of PBoV in throat swab, faecal and serum samples. A total of 7.5% (39/519) throat swab samples, 60.5% (309/511) faecal samples, and 22.9% (52/227) serum samples were PBoV‐positive. The prevalence among R. norvegicus and R. tanezumi was higher than that among R. losea and house shrews. PBoV‐positive samples were found in all four provinces. Phylogenetic analysis based on partial viral capsid protein 1/2 (VP1/VP2) showed that sequences obtained in this study formed a novel group (PBoV G4). In addition, five near full‐length PBoV genomes (4,715–4,798 nt) were acquired. These genomes encoded two non‐structural proteins, NS1 (1,908 nt in four genomes and 1,923 nt in the remaining genome) and NP1 (600 nt), and the structural proteins, VP1/VP2 (1,851 nt). Phylogenetic analysis showed that PBoV G4 is distinct from rodent, human, and other bocaviruses. In conclusion, PBoV G4 prevalence was high among two common murine rodents in China, and the pathogenecity of PBoV G4 need to be further clarified.