Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G.

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3-86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26-506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

Molecular characterization of a porcine sapelovirus strain isolated in China.

Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV.

Structural basis for hijacking of the host ACBD3 protein by bovine and porcine enteroviruses and kobuviruses.

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.

Molecular survey of porcine teschovirus, porcine sapelovirus, and enterovirus G in captive wild boars (Sus scrofa scrofa) of Paraná state, Brazil / Estudo molecular de teschovírus suíno, sapelovírus suíno e enterovírus G em javalis (Sus scrofa scrofa) de cativeiro no Paraná

Porcine teschovirus (PTV), porcine sapelovirus (PSV), and enterovirus G (EV-G) are infectious agents specific to pig host species that are endemically spread worldwide. This study aimed to investigate the natural infection by these porcine enteric picornaviruses in wild boars (Sus scrofa scrofa) of Paraná state, Brazil, and to evaluate peccaries (Pecari tajacu and Tayassu pecari) as alternative host species for these viruses. Fecal samples (n=36) from asymptomatic wild boars (n=22) with ages ranging from 2 to 7 months old (young, n=14) and 2 to 4 years old (adult, n=8) and from peccaries (6 to 8 months old, n=14) were collected from a farm and a zoo, respectively, both located in Paraná state. Reverse transcription-polymerase chain reaction (RT-PCR) and nested-PCR (n-PCR) assays targeting the 5'non-translated region of the virus genome were used for screening the viruses. Porcine enteric picornaviruses were detected in 12 out of the 22 wild boar fecal samples. According to each of the viruses, EV-G was most frequently (11/22, 50%) detected, followed by PTV (10/22, 45.5%) and PSV (4/22, 18.2%). Regarding the age groups, young wild boars were more frequently (9/14, 64.3%) infected with PTV, PSV, and EV-G than adult animals (3/8, 37.4%). One n-PCR amplified product for each of the viruses was submitted to sequencing analysis and the nucleotide sequences were compared with the related viruses, which showed similarities varying from 97.7% to 100% for PTV, 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Peccaries tested negative for the viruses and in this study they did not represent infection reservoirs. This study is the first to report the molecular detection of PTV, PSV, and EV-G from captive wild boars in a South American country and the first to screen peccaries as alternative host species for porcine enteric picornavirus.

Teschovírus suíno (PTV), sapelovírus suíno (PSV) e enterovírus G(EV-G) são agentes infecciosos específicos da espécie suína que estão endemicamente disseminados em todo o mundo. O objetivo deste estudo foi investigar a infecção natural por estes picornavírus entéricos suínos em javalis (Sus scrofa scrofa) do estado do Paraná, Brasil e avaliar pecaris (Pecari tajacu e Tayassu pecari) como hospedeiros alternativos para estes vírus. Amostras fecais (n=36) de javalis assintomáticos (n=22) com idades de 2 a 7 meses (jovens, n=14) e 2 a 4 anos (adultos, n=8) e de pecaris (6 a 8 meses de idade, n=14) foram coletadas em um cativeiro e zoológico, respectivamente, ambos localizados no estado do Paraná. A transcrição reversa seguida por reações da polimerase em cadeia (RT-PCR) e nested-PCR com alvo na região 5'-não traduzida do genoma viral foram utilizadas para a identificação dos vírus. Picornavírus entéricos suínos foram detectados em 12 das 22 amostras fecais de javalis. De acordo com cada um dos vírus, EV-G foi mais frequentemente (11/22, 50%) detectado, seguido pelo PTV (10/22; 45,5%) e PSV (4/22; 18,2%). Considerando os grupos de idade, javalis jovens foram mais frequentemente (9/14; 64,3%) infectados com PTV, PSV e EV-G do que os javalis adultos (3/8; 37,4%). Um produto amplificado na nested-PCR para cada um dos vírus foi submetido à análise de sequenciamento e as sequências de nucleotídeos foram comparadas com vírus relacionados, o que mostrou que as similaridades variaram entre 97,7% a 100% para o PTV, 92,4% a 96,2% para o PSV e 87,1% a 100% para o EV-G. Os pecaris foram negativos para as viroses investigadas e neste estudo não se apresentaram como hospedeiros alternativos para as infecções. Este estudo é o primeiro a relatar a detecção molecular de PTV, PSV e EV-G em javalis de cativeiro de um país da América Latina e o primeiro a avaliar pecaris como espécie hospedeira alternativa para picornavírus entéricos suínos.

Lack of cross-species transmission of porcine endogenous retrovirus (PERV) to transplant recipients and abattoir workers in contact with pigs.

This study investigated the potential transmission of porcine endogenous retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.

John Burns Brooksby CBE: 25 December 1914 - 17 December 1998.

John Brooksby was an outstanding veterinary virologist, who worked at the Animal Virus Disease Research Institute, Pirbright, for 40 years, for 16 of which he was Director of the Institute. He will be remembered for his contributions to the diagnosis of foot-and-mouth disease, for his discovery of four new types, for the classification of subtypes and for fundamental studies of the virus. As Deputy Director and Director he was responsible for programmes on fundamental investigations of foot-and-mouth disease virus and other viruses exotic to the UK and for the application of the results both in the UK and worldwide. His advice on the distribution and the control of foot-and-mouth disease was sought by international organizations and by individual countries and was responsible for reducing the risk of spread of disease.

Hepatitis C virus-related internal ribosome entry sites are found in multiple genera of the family Picornaviridae.

The internal ribosome entry site (IRES) elements from porcine enterovirus 8 and simian virus 2, two members of a proposed new genus within the family Picornaviridae, were characterized. These IRES elements, in common with the porcine teschovirus 1 IRES, were found to be related functionally and structurally to the IRES element from Hepatitis C virus, a member of the family Flaviviridae. Partial secondary structure predictions were derived and functional assays demonstrated that these IRES elements continued to be active when eIF4G was cleaved and when the activity of eIF4A was blocked.

Detection of porcine teschovirus and enterovirus type II by reverse transcription-polymerase chain reaction.

Porcine enteroviruses (PEVs) have been recognized as the causative agents of various clinical manifestations such as fertility disorders, neurological defects, and dermal lesions in pigs. Currently, the diagnosis of PEV infection is carried out by virus isolation, which although useful, is labor- and time-intensive. The present investigation describes the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for the rapid and sensitive detection of PEVs of cytopathic effect groups I (now known as porcine teschoviruses [PTVs]) and II. The assay described not only detects the PTVs and CPE group II of PEVs but also allows them to be differentiated on the basis of the size of the amplification product, using the same set of oligonucleotide primers. The availability of specific and sensitive molecular diagnostic tools such as the RT-PCR assay described herein should facilitate efficient diagnosis of PTV and CPE group II infections in pigs.

The complete nucleotide sequence of a pathogenic swine vesicular disease virus.

The nucleotide sequence of a swine vesicular disease virus (SVDV) strain that is pathogenic for pigs has been determined and compared with that of a non-pathogenic strain of SVDV, as well as a number of other enteroviruses. It shows only 98 base changes in comparison with a non-pathogenic strain of SVDV (Inoue et al., 1989, J. Gen. Virol. 70, 919-934). Fourteen of these nucleotide differences between the pathogenic and the non-pathogenic SVDV strains occur in the 5' non-coding region which, by analogy with the other picornaviruses, has been implicated in the efficiency with which the RNA is employed as mRNA. Additional differences found throughout the coding regions are largely conservative in nature. A number of residues are discussed as candidates for determinants of pathogenicity. This sequence has been submitted to the PIR database and has accession number A30061.

Single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: clinical signs and microscopic lesions.

Neonatal colostrum-deprived pigs were inoculated with cell-culture preparations of three rotaviruses and three enteroviruses, singly or in combination. The three enteroviruses established intestinal and systemic infection but did not induce diarrhea or intestinal lesions. The three rotaviruses produced severe enteric disease characterized by profuse watery diarrhea, dehydration and death. Villi were severely stunted. All three isolates were equally virulent. Inoculation with three different rotavirus-enterovirus combinations resulted in disease less severe than that produced by the rotaviruses alone. Intestinal lesions were less extensive and fewer pigs became moribund or died.

Plaque morphology and pathogenicity for newborn mice of swine vesicular disease virus. I. Wild strains and their clones.

The population of wild swine vesicular disease virus (SVDV) strains was found non-homogeneous as manifested by varying plaque size and different pathogenicity of the clones obtained. The clones derived from large plaques (5-9 mm)--dominating among wild strains--were more virulent for newborn mice than those obtained from smaller plaques (1-2 mm). To evaluate the pathogenicity of wild SVDV strains the dose index was calculated; the clones were compared by dose index and theoretical pathogenicity index, respectively.

Plaque morphology and pathogenicity for newborn mice of swine vesicular disease virus. II. Temperature-dependent mutants and their clones.

From wild swine vesicular disease virus (SVDV) strains temperature-dependent (td) mutants td 27 degrees C, td 32 degrees C and td 42 degrees C were derived. Differences were noted in their pathogenicity for newborn mice. The non-homogeneity of the td populations was manifested by formation of plaques of various sizes and confirmed by differential pathogenicity of the clones derived from them. A higher pathogenicity of the td mutants and their clones was associated not only with larger plaques, but also with higher temperature permissive for the given mutant. The td 27 degrees C mutants were not virulent for newborn mice, similarly to the attenuated SVDV strain and its clones.

The pathogenesis of vesicular exanthema of swine virus and San Miguel sea lion virus in swine.

Vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2, when inoculated intradermally into swine, resulted in fluid-filled vesicles at the sites of inoculation in the snout, coronary band, and tongue. Pigs that developed vesicles also had fevers. Secondary vesicle formation varied, depending on virus serotype. Viremia was found in one pig infected with San Miguel sea lion virus five days after infection. Virus was recovered from nasal-oral passages for up to five days after infection in both groups of pigs and from the gastrointestinal and urinary tracts of pigs infected with San Miguel sea lion virus. Neutralizing antibodies began to increase three days after inoculation and reached peak titers in seven to ten days. In the absence of secondary bacterial infection, healing was well advanced by ten days after inoculation. Lesions usually were limited to nonhaired portions of the integument and tongue. Individual epithelial cells became infected when a break in the skin allowed virus access to susceptible epithelial cells from either exogenous or endogenous sources. Individual infected cells ruptured and adjacent cells were infected, resulting in the formation of multiple microvesicles. Centrifugal coalescence of microvesicles led to formation of grossly visible macrovesicles. Lesions rarely developed from viral contamination of intact hair follicles. A mild virus-induced encephalitis was seen in pigs infected with vesicular exanthema of swine virus, and virus was recovered from brain tissue of pigs infected with San Miguel sea lion virus.

Vesicular exanthema of swine and San Miguel sea lion virus: experimental and field studies in otarid seals, feeding trials in swine.

The naturally occurring disease caused by San Miguel sea lion virus in fur seals was characterized by small fluid-filled vesicles 1 to 25 mm in diameter on the nonhaired portions of the flippers. Early epithelial lesions contained multifocal sites of cell lysis. The resultant microvesicles enlarged and coalesced, forming grossly visible macrovesicles. Mature vesicles progressed to involve all layers of the epithelium but did not involve the underlying dermis. Intradermal inoculation of vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2 into otarid (fur) seal pups caused plaque-like lesions around inoculated coronary bands. These swellings regressed without rupture by 96 hours postinoculation. One seal inoculated with San Miguel sea lion virus had a linear lingual erosion at ten days postinoculation. Virus was isolated from this site and from two uninoculated sites, the tonsil and testicle. Contact controls showed no evidence of infection. Virus was isolated in low titers from some sites of inoculation and draining lymph nodes from seals infected with vesicular exanthema of swine virus. Virus was recovered more easily, in higher titers, and from more tissues, from seals infected with San Miguel sea lion virus. Inoculated seals tested after four to ten days seroconverted. Feeding swine seal tissues from the inoculation experiments resulted in seroconversion in swine which were fed tissues from seals infected with vesicular exanthema of swine virus but not in those which were fed tissues from seals infected with San Miguel sea lion virus.

The characterisation and pathogenicity of porcine enteroviruses isolated in Victoria.

Approximately 23 viruses were isolated from healthy pigs, pigs with encephalitis, and in cases of reproductive failure. Five viruses were identified as enteroviruses and a total of 10 isolates were shown to cross-react serologically to varying degrees. Twenty viruses were neutralised by a reference antiserum of serotype 8 porcine enterovirus. Intracerebral inoculaton of colostrum-deprived piglets with 2 of the characterised viruses caused lesions of encephalomyelitis which were not induced by oral infection. Intrafoetal inoculation of 2 sows with one characterised faecal isolate caused foetal death and abortion, but no adverse effects followed oral dosage.

Comparison of proliferation and cytopathogenicity of swine vesicular virus and coxsackievirus B5.

Sequential appearance of both swine vesicular disease virus and Coxsackievirus B5 antigens in a pig kidney cell line was studied by immunofluorescence and electron microscopy. The replication cycle of each virus was approximately 3-4 h. Viral antigens were demonstrable in the cytoplasm 2 h after inoculation. A compact mass of fluorescence was seen when cells showed cytopathogenic effect at 5.5 h. After 3 h, a few viral particles, seen by electron microscopy, were in the cytoplasm. Morphological changes of cells occurred at the same time. Cytoplasmic crystalline arrays of virus were first detected at 7 h.

In vivo and in vitro studies on temperature-sensitive mutants of swine vesicular disease virus.

Two temperature-sensitive mutants of the Ukg 27/72 strain of swine vesicular disease virus were isolated in tissue culture and a third was derived following adaptation in mice. All three were found to have similar growth restrictive temperatures, but varied considerably in their virulence when administered to pigs. The route of inoculation appeared to exert a considerable influence on the apparent degree of attenuation, the antibody titre engendered and the transmission of disease to pigs held in contact with inoculated animals. One strain appeared almost totally attenuated when inoculated animals. One strain appeared almost totally attenuated when inoculated into pigs but spread to animals in contact causing severe disease. Virus re-isolated from one such animal was found to have retained its temperature sensitive phenotype, suggesting that virulence in this case was not directly related to temperature sensitivity. Pigs with high antibody titres were found to be susceptible when placed in contact with challenge animals, although the lesions which developed were mild.