Molecular characterization of two novel atypical porcine pestivirus (APPV) strains from piglets with congenital tremor in China

As one of emerging porcine viruses, atypical porcine pestivirus (APPV) was found in three continents since it emerged in 2015. It is now thought as the causative agent for congenital tremor type A‐II in piglets. At the end of 2017, two APPV strains were identified from piglets with congenital tremor in Guangxi and Yunnan, China. The genome of APPV GX04/2017 strain was so far determined to be 11,534 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a polyprotein comprising 3,635 amino acids. Comparative analysis of ORF, N^pro, E2, and NS3 gene sequences revealed that the APPV GX04/2017 strain shares nucleotide sequence identities of 82.8%–92.8% with other APPV strains, while YN01/2017 strain is 79.4%–97.4% homology to the others. Phylogenetic analysis showed that the APPV GX04/2017 and YN01/2017 are two novel APPV strains with the highest homology to each other, and relative high similarity to the APPV 000515 and JX‐JM01 strains in genome sequence. The current findings provide updated information about APPV epidemiology and divergence in China, which would certainly help to establish reliable diagnosis and surveillance programs for APPV.     

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.     

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.     

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.     

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.     

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 .     

Semi‐quantitative duplex RT‐PCR reveals the low occurrence of Porcine Pegivirus and Atypical Porcine Pestivirus in diagnostic samples from the United States

Porcine Pegivirus (PPgV) and Atypical Porcine Pestivirus (APPV) are two recently identified porcine viruses. In this study, the identification of two viruses by metagenomic sequencing, and a duplex semi‐quantitative RT‐PCR was developed to detect these pathogens simultaneously. The PPgV strain Minnesota‐1/2016 had a 95.5%–96.3% nucleotide identity and clustered with the recently identified US PPgV strains, which is a distant clade from the German PPgV strains. The APPV strain Minnesota‐1/2016 shared an 87.3%–92.0% nucleotide identity with the other global APPV strains identity but only shared an 82.8%–83.0% nucleotide identity with clade II consisting of strain identified in China. Detection of both PPgV and APPV was 9.0% of the diagnostic cases. Co‐infection of PPgV and APPV was identified in 7.5% of the diagnostic cases. The occurrence and genetic characterization of PPgV and APPV further enhance our knowledge regarding these new pathogens in the United States.     

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV‐1), BVDV‐2, border disease virus (BDV), and an atypical pestivirus named HoBi‐like pestivirus. In this study, eighty‐nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5′ untranslated regions (5′UTR), N‐terminal autoprotease (N^pro) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV‐1, 33.7% as BVDV‐2 and 12.4% as HoBi‐like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV‐1a (35.9%), BVDV‐2b (31.4%), BVDV‐1b (10.1%), BVDV‐1d (6.7%), BVDV‐2c (2.2%) and BVDV‐1e (1.1%). BVDV‐2c and BVDV‐1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.     

High Prevalence of Highly Variable Atypical Porcine Pestiviruses Found in Germany

Recently, a novel atypical porcine pestivirus (APPV) with significant distribution was described in the USA. Subsequent screening of the German pig sector showed a high prevalence of APPV with high variability among strains. First indication of a cell culture isolate is provided which will allow further investigations like pathogenesis studies.     

Senecavirus A: An Emerging Vesicular Infection in Brazilian Pig Herds

Vesicular diseases are clinically and economically important infections that affect farm animals. North American studies have suggested that Senecavirus A infection might be associated with a vesicular disease in pigs known as porcine idiopathic vesicular disease (PIVD). In the beginning of 2015, outbreaks of porcine vesicular disease have occurred in six Brazilian states from three geographical regions. Official diagnostic tests were performed with negative results for classical vesicular diseases of compulsory reporting. This study investigated Senecavirus A infection in PIVD outbreaks in which other aetiological agents were ruled out. A primer set was designed to amplify a 542‐bp product size of VP3/VP1 region of Senecavirus A genome in RT‐PCR assay. Primer specificity was analysed in silico and in porcine biological specimens. For this, clinical specimens were collected from eight pig herds affected with PIVD, including vesicular fluid (n = 4) and swabs (n = 7) and scrapings of ruptured vesicles and ulcerative lesions (n = 5) from weaned and adult pigs. Clinically healthy animals (n = 52) of PIVD‐affected and non‐affected pig herds also were evaluated for Senecavirus A infection. The 16 samples from PIVD‐affected animals were positive for Senecavirus A in the RT‐PCR assay, while none of the clinically healthy pigs were detected with the virus. Sequencing analysis revealed high nucleotide (87.6–98.5%) and amino acid (95–99.4%) similarities to SVV‐01 prototype and other Senecavirus A strains from North American pigs. Primer set presented herein was suitable for molecular characterization of Senecavirus A. The results suggest that Senecavirus A was the aetiological agent of the vesicular disease outbreaks in the evaluated pig herds. This is the first study to report the Senecavirus A infection in clinically affected pigs outside of North America. Senecavirus A was considered a novel emerging pathogen associated with an important vesicular disease in Brazil.     

Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans)

Within Eastern Europe, African swine fever virus (ASFV ) has unexpectedly spread to farms with high biosecurity. In an attempt to explain this process, pigs were allowed to ingest flies that had fed on ASFV ‐spiked blood, which had a realistic titre for an infected pig. Some of the pigs became infected with the virus. Thus, ingestion of blood‐sucking flies, having fed on ASFV ‐infected wild boar before entering stables, represents a potential route for disease transmission.     

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.     

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007–2015)

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007–2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single‐copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet‐15, Tet‐17a, Tet‐17b and Tet‐48) were recovered, while a fifth variant (Tet‐20) was the most widely distributed in the country displacing Tet‐36 reported previously in 2003–2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country.     

Distinction Between Persistent and Transient Infection in a Bovine Viral Diarrhoea (BVD) Control Programme: Appropriate Interpretation of Real‐Time RT‐PCR and Antigen‐ELISA Test Results

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)‐specific RNA using a commercial real‐time RT‐PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT‐PCR test and with an antigen‐capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT‐PCR and 72 (1.4%) by antigen‐ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen‐ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen‐ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (C_t) values obtained by RT‐PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT‐PCR test performed on a single individual blood sample.     

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.     

Tuberculosis in Southern Brazilian wild boars (Sus scrofa): First epidemiological findings

Bovine tuberculosis (bTB ) is a zoonosis caused mainly by Mycobacterium bovis that affects domestic and wild animals. In Brazil, there are no epidemiological studies on tuberculosis in wild animal populations and their possible role in the disease maintenance in cattle herds; thus, the aim of this study was to evaluate the occurrence of tuberculosis in wild boars in Rio Grande do Sul, southern Brazil. Tissue samples of animals hunted under government consent were submitted to histopathology and M. bovis polymerase chain reaction (PCR ) as screening tests; the positive samples were subsequently submitted to bacterial isolation, the gold standard diagnosis. Eighty animals were evaluated, of which 27.9% and 31.3% showed histopathological changes and M. bovis genome presence, respectively. Moreover, 23.8% of the animals had at least one organ with isolates classified as Mycobacterium tuberculosis complex (MTC ). Three hunting points were risk factors for positive results on screening tests. This study shows the occurrence of tuberculosis in a wild boars’ population, and raise the possibility of these animals to play a role as disease reservoirs in southern Brazil. These results may help to improve the Brazilian tuberculosis control programme, as well as elucidate the circulation of mycobacteria in this country.     

Rickettsia parkeri in free‐ranging wild canids from Brazilian Pampa

Spotted fevers are tick‐borne diseases associated with various Rickettsia species. Rickettsia parkeri sensu stricto (s.s.) is the agent of an emerging eschar‐associated rickettsiosis in humans from the USA and South American Pampa. Considering that R. parkeri s.s. is restricted to Americas and the potential role of dogs in the epidemiology of the disease, it is thus reasonable to hypothesize that wild canids could be involved in the enzootic cycle of this rickettsiosis. The aim of this work was to investigate the potential role of the wild canids from Pampa, Cerdocyon thous (crab‐eating fox) and Lycalopex gymnocercus (Pampas fox), in the ecology of R. parkeri s.s. For that, 32 live‐trapped free‐ranging wild canids were sampled. Ticks were observed in 30 of the 32 foxes. Of the 292 ticks collected, 22 (7.5%) were positive by PCR for the presence of R. parkeri s.s. DNA . Also, 20 (62%) wild canids showed antibodies against R. parkeri . The results suggest that wild canids are involved in the enzootic cycle of R. parkeri s.s. in the Pampa biome and could be responsible for pathogen (and its vectors) dispersal.     

Experimental infection with high‐ and low‐virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free‐ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high‐virulence (Cadí‐6) and low‐virulence (Freser‐5) BDV strains was performed. Pregnant and non‐pregnant animals with and without antibodies against BDV were included in each group. Cadí‐6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser‐5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí‐6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí‐6 group. Chamois from both groups presented lesions in brain but the ones infected with the low‐virulence Freser‐5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low‐virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross‐protection in chamois against high‐virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.