Detection of a novel sapelovirus in central nervous tissue of pigs with polioencephalomyelitis in the USA

An approximately 3,000 finishing swine operation in the United States experienced an outbreak of an atypical neurologic disease in 11‐weeks‐old pigs with an overall morbidity of 20% and case fatality rate of 30%. The clinical onset and progression of signs in affected pigs varied but included inappetence, compromised ambulation, ataxia, incoordination, mental dullness, paresis, paralysis and decreased response to environmental stimuli. Tissues from affected pigs were submitted for diagnostic investigation. Histopathologic examination of the cerebrum, cerebellum and spinal cord revealed severe lymphoplasmacytic and necrotizing polioencephalomyelitis with multifocal areas of gliosis and neuron satellitosis, suggestive of a neurotropic viral infection. Bacterial pathogens were not isolated by culture of neurologic tissue from affected pigs. Samples tested by polymerase chain reaction (PCR) were negative for pseudorabies virus and atypical porcine pestivirus. Immunohistochemistry for porcine reproductive and respiratory syndrome virus, porcine circovirus and Listeria was negative. Porcine sapelovirus (PSV) was identified in spinal cord by a nested PCR used to detect porcine enterovirus, porcine teschovirus and PSV. Next‐generation sequencing of brainstem and spinal cord samples identified PSV and the absence of other or novel pathogens. In addition, Sapelovirus A mRNA was detected in neurons and nerve roots of the spinal cord by in situ hybridization. The PSV is genetically novel with an overall 94% amino acid identity and 86% nucleotide identity to a recently reported sapelovirus from Korea. This is the first case report in the United States associating sapelovirus with severe polioencephalomyelitis in pigs.     

Vitamin E and Selenium Levels are Within Normal Range in Pigs Diagnosed with Mulberry Heart Disease and Evidence for Viral Involvement in the Syndrome is Lacking

Mulberry heart disease (MHD) in pigs is characterized by lesions of acute haemorrhagic myocarditis and myocardial necrosis. The objectives of this study were to determine the levels of vitamin E and selenium and 13 other trace minerals in heart and liver tissues and to determine the prevalence of certain viral infections in heart tissues from MHD‐affected and MHD‐unaffected pigs and the vitamin E and selenium concentration in feed samples from selected farms with MHD. Based on the pathological examination, 114 pigs were separated into MHD lesion‐negative (L‐NEG) (n = 57) and MHD lesion‐positive (L‐POS) (n = 57) groups. Seventy‐three samples (40 L‐NEG and 33 L‐POS) were subjected to chemical analysis, and 66 (32 L‐NEG and 34 L‐POS) were subjected to PCR detection for viral pathogens. Lower (P < 0.05) levels of myocardial copper, lower (P < 0.05) levels of hepatic magnesium and higher (P < 0.05) levels of myocardial and hepatic sodium were detected in the L‐POS cases. Although lower (P < 0.05) levels of hepatic selenium were detected in L‐POS group, all were within the normal range. Analysis of feed samples (n = 22) revealed that selenium levels in all the samples were above the legal limit (0.3 ppm) for pigs. Vitamin E levels in all feed samples were above 20 IU/kg. Among the 66 pigs subjected to PCR detection, there were 19, 4, 13, 8, 2 and 1 animals positive for porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pan‐herpes virus, porcine enterovirus, pan‐pestivirus and porcine parvovirus, respectively. Clear evidence of viral association with L‐POS was lacking.     

First detection of porcine circovirus type 3 on commercial pig farms in Poland

Porcine circovirus type 3 (PCV3) is a novel circovirus species recently discovered in USA and China in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, respiratory disease and multisystemic inflammation. This study reports on the first identification of PCV3 in Europe, in serum from pigs from Polish farms. A total of 1,050 serum samples were collected between 2014 and 2017 from sows and 3–20 weeks old pigs from 14 commercial farms representing different regions of Poland, different size and health status. The samples were pooled by 4–6 and tested with real‐time PCR for PCV3. PCV3 DNA was detected in 12 of 14 farms (85.7%). On the PCV3‐positive farms, the virus was detected in 5.9% to 65% serum pools. PCV3 was most common among weaned pigs and finishers (26.1% and 28.0% of serum pools, respectively). Sequence analysis of 359 nucleotide fragment of ORF2 showed highest identity of 99.7% to PCV3‐US/SD2016 from USA. Our results indicate that PCV3 is a common virus among Polish pigs but no links to unexplained disease conditions were established.     

Porcine circovirus‐like virus P1 in cattle,goats and rabbits in China

Porcine circovirus‐like virus P1, a novel single‐stranded circular DNA virus, is a potential causative agent of post‐weaning multisystemic wasting syndrome in swine. In this study, we detected P1 strains in serum samples from cattle, goats and rabbits suffering different clinical symptoms, in Jiangsu Province, China. We sequenced the whole genomes of six P1 strains, which showed 99.4%–100% nucleotide sequence similarity and were closely related to other porcine‐origin P1 isolates. The sequences of six non‐porcine‐origin P1 viruses were most closely phylogenetically related to other porcine‐origin P1 sequences. Therefore, the P1 virus has a wide host range in China.     

Presence of Torque teno sus virus 1 and 2 in porcine circovirus 3‐positive pigs

In this study, the co‐infection of Torque teno sus virus (TTS uV) and porcine circovirus type 3 (PCV 3) was reported. One hundred and ten of 132 (83.3%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 1 (TTS uV1). Ninety‐four of 132 (71.2%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 2 (TTS uV2). Sixty‐six of 132 (50.0%) of PCV 3‐positive samples were co‐infected with both TTS uV1 and TTS uV2. There were no clinical signs of infection in pigs that were both PCV 3‐positive and PCV 2‐negative, in either multiparous sows or live‐born infants. The high co‐infection rate provides valuable information for the further study of the pathological correlation between PCV 3 and TTS uVs.     

Rapid specific and visible detection of porcine circovirus type 3 using loop‐mediated isothermal amplification (LAMP)

In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV 3) was established using loop‐mediated isothermal amplification (LAMP ). Four primers were specifically designed to amplify PCV 3. The LAMP assay was effectively optimized to amplify PCV 3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10¹ copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV 2), both gE and gD genes of pseudorabies virus (PRV ) and porcine parvovirus (PPV ), the LAMP assay showed a high specific detection of PCV 3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency.     

Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province,China

In pigs, three circovirus species within the genus Circovirus have been identified so far, including the non‐pathogenic Porcine circovirus 1 (PCV1), the pathogenic Porcine circovirus 2 (PCV2) and the recently identified Porcine circovirus 3 (PCV3). In April 2019, a new circovirus with a distinct relationship to other circoviruses was identified in several pigs with severe clinical disease in Hunan province, China. The size of the viral genome, tentatively designated as porcine circovirus type 4 (PCV4), is 1,770 nucleotides (nt). PCV4 shows the highest genomic identity to mink circovirus (66.9%) and has identities of 43.2%–51.5% to the other PCV genomes. Two major genes, a replicase (Rep) gene spanning 891 nt and a capsid (Cap) gene spanning 687 nt, were predicted. Furthermore, a TaqMan^® real‐time polymerase chain reaction (PCR) targeting the replicase gene was developed to investigate the prevalence of PCV4 in 187 clinical samples from Hunan province, China. The results revealed an overall PCV4 prevalence of 12.8%, with the highest positive rates in nasal swabs (28.5%, 6/21) followed by serum samples (13.4%, 11/82). The clinical significance and pathogenesis of this virus needs further investigation.     

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.     

Identification of pig circovirus type 2 in New Zealand pigs

Interest in porcine circovirus has been stimulated by the recent emergence of postweaning multisystemic wasting syndrome (PMWS) in pigs and the potential use of pig organs for xenotransplantation in humans. Porcine circovirus type 1 (PCV1) is considered to be widespread in pigs but nonpathogenic. Circovirus type 2 (PCV2) is a similar virus but has been differentiated only recently as a separate type. High tissue concentrations of PCV2 are associated with lesions in PMWS cases, but the etiological role of this agent in the disease remains unclear. The presence of PCV1 in New Zealand pigs has been previously reported based on serological data. PMWS has been recently recorded in New Zealand pigs. The epidemiology of PCV2 in New Zealand pigs has not been examined. The purpose of the study was to look for evidence of circoviruses in New Zealand pig herds. Pig circovirus DNA was sought in various tissues using the polymerase chain reaction. Circovirus type 2 was found in New Zealand pig herds, without any evidence that PMWS has ever occurred in these herds. Newborn piglets were shown to have infection, suggesting vertical transmission of the virus.     

The retrospective identification and molecular epidemiology of porcine circovirus type 3 (PCV3) in swine in Thailand from 2006 to 2017

Porcine circovirus type 3 (PCV3) has recently been detected in pigs worldwide, with similar clinical manifestations to porcine circovirus‐associated disease (PCVAD) from porcine circovirus type 2 (PCV2) infection. Here, we report the identification and molecular epidemiology of PCV3 in swine in Thailand from clinical samples retrieved from 2006 to 2017. The epidemiological data revealed co‐infection with PCV2, PRRSV, and PCV2/PRRSV was common in our samples. Circulating PCV3 from this study shared a high similarity of nucleotide and deduced amino acid sequences of the partial capsid gene (96.7%–100% and 96.7%–100% respectively), indicated the genetic stability of PCV3 in Thailand. Phylogenetic analysis based on the capsid gene revealed scatter clustering with current PCV3 having no relation to the geographical origin of the virus strains. In this retrospective study, results have demonstrated that PCV3 has spread extensively within Thai swine from as early as 2006 and may also be involved in PRDC and PCVAD.     

Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan

Porcine circovirus (PCV) is a small non‐enveloped virus with a single‐stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2‐systemic disease (PCV2‐SD), previously known as post‐weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2‐SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.     

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.     

Detection of a novel porcine circovirus‐like agent in aborted pig foetuses

A large outbreak of swine abortion, which began in January 2017, is ongoing in Jiangsu and Anhui provinces, China. We identified a new porcine circovirus‐like agent, designated P4, in the aborted foetuses from several of these cases of swine reproductive failure. PCR and qRT ‐PCR analyses confirmed that other common abortogenic agents were not present in these animals. P4 contains a 710‐nucleotide circular viral genome, and all strains examined in this study were closely related to the dominant genotype of porcine circovirus type 2. Further studies, such as in vivo analyses, are needed to confirm P4 as the aetiological agent.     

The porcine circovirus type 1 in porcine kidney 15 cell line is not transferred to mice lymphoid cells after xenoimplantation into the peritoneal cavity

The porcine circovirus type 1 (PCV1) has been identified within lymphoid tissues of experimental infected pigs and suggested to induce an immunosuppressive stage in pigs. The virus does not induce a cytophatic effect in the pig-derived cell line PK-15. Because PCV1 is prevalent in many pig cells and tissues, the risk of inducing a viral xenozoonosis by PCV1 was raised for the xenoimplantation of pig cells into human hosts. The present work evaluated if PCV1 is able to replicate in mice tissues after xenoimplantation of PCV1-infected pig cells. Active growing PK-15 cells harboring PCV1 with or without microencapsulation in sodium alginate were implanted into the peritoneal cavity of mice. After 1 month postimplantation in mice, peritoneal macrophages, spleen, and lymph nodes were harvested and analyzed with the polymerase chain reaction technique (PCR). No evidence of circovirus type 1 DNA was detected within the mice tissues.     

Pathogenicity and Molecular Characterization of Emerging Porcine Reproductive and Respiratory Syndrome Virus in Vietnam in 2007

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the ‘porcine high fever disease’ that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV‐2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7–99.4% identity to Chinese strain HUN4, 68–69% identity to strain VR‐2332 and 58–59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re‐isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re‐isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.     

Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV‐A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT‐PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV‐A, and the polyprotein genes of PKV and PSaV. This multiplex RT‐PCR could specifically detect TGEV, PEDV, PDCoV, PRV‐A, PKV and PSaV without cross‐reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 10⁰–10¹ ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT‐PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV‐A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV‐A, PRA‐V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV‐A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT‐PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China.     

Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus

BACKGROUND: An important issue with respect to virus safety in xenotransplantation is the search for human analogues of porcine viruses, because transmission of a porcine virus followed by recombination with a related human virus may lead to a new emerging virus of unknown pathogenicity, host range and virulence. In case of circoviruses, two types of porcine circovirus (PCV1 and PCV2) are described, but the existence of an analogous human circovirus has not yet been investigated. METHODS: This study describes the analysis of human samples with a consensus primer-PCR approach designed to amplify conserved regions from the rep gene of circoviruses from the genus Circovirus. DNA from human sera, lymph nodes, blood and urine was extracted and investigated with this method that has led previously to the identification of a new avian circovirus. RESULTS: By screening 1101 samples (there of 168 from immunocompromised patients), no evidence for the existence of a human circovirus related to the genus Circovirus was obtained. CONCLUSIONS: This result renders the existence of a human circovirus related to the porcine circoviruses more unlikely, nevertheless the presence of such a virus cannot be ruled out.