First molecular detection of porcine bocavirus in Malaysia

Several strains of porcine bocaviruses have been reported worldwide since their first detection in Sweden in 2009. Subsequently, the virus has been reported to be associated with gastrointestinal and respiratory signs in weaner and grower pigs. Although Malaysia is host to a self-sufficient swine livestock industry, there is no study that describes porcine bocavirus in the country. This report is the first to describe porcine bocavirus (PBoV) in Malaysian swine herds. PBoV was identified in various tissues from sick and runt pigs using the conventional PCR method with primers targeting conserved regions encoding for the nonstructural protein (NS1) gene. Out of 103 samples tested from 17 pigs, 32 samples from 15 pigs were positive for porcine bocavirus. In addition, a higher detection rate was identified from mesenteric lymph nodes (52.9%), followed by tonsil (37.0%), and lungs (33.3%). Pairwise comparison and phylogenetic analyses based on a 658-bp fragment of NS1 gene revealed that the Malaysian PBoV strains are highly similar to PBoV3 isolated in Minnesota, USA. The presence of porcine bocavirus in Malaysia and their phylogenetic bond was marked for the first time by this study. Further studies will establish the molecular epidemiology of PBoV in Malaysia and clarify pathogenicity of the local isolates.     

Construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant economic losses in the swine industry worldwide. Two novel gene-deleted viruses were constructed and evaluated as vaccine candidates. Using the full-length infectious cDNA clone of North American PRRS isolate P129, the ORF2 and ORF4 genes (which encoded minor structural glycoproteins GP2a/2b and GP4, respectively) were individually deleted from the viral genome. Both deletion mutants were non-viable in MARC-145 cells and porcine alveolar macrophages, indicating that both genes are essential for virus replication. To rescue the replication-defective PRRSV, two complementing cell lines, MARC-2000 and MARC-400, were established to stably express the PRRSV GP2 and GP4 proteins, respectively. These cells were able to complement the deleted gene function of PRRSV in trans and supported production of the replication-defective DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses. Both DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses were propagated for 40-50 generations in the corresponding complementing cells and remained replication-defective in MARC-145 cells. To examine the immunogenic potential of the replication-defective PRRSV as vaccine candidates, four groups of pigs, 20 pigs per group, were immunized twice with DeltaORF2-PRRSV or DeltaORF4-PRRSV and challenged with the homologous virulent virus at 3 weeks post-immunization. In spite of the fact one group showed significant reduction in virus load, we could not demonstrate improvement from clinical diseases in this vaccination/challenge study. However, we did show that the cDNA clone of PRRSV can be a useful tool to genetically engineer PRRSV vaccine candidates and to study pathogenesis and viral gene functions.     

Detection of St-Valerien-like viruses in swine, Italy

St-Valerien-like viruses are newly recognized porcine caliciviruses recently detected in Canadian swine farms. These viruses that appeared genetically closest to the Tulane non-human primate virus and noroviruses (NoVs), may represent members of a potential new genera within the Caliciviridae family. To date, there are no information on the epidemiology of these novel caliciviruses in other continents. In this study, 264 faecal samples collected from asymptomatic adult pigs were screened by RT-PCR using St-Valerien-like specific primers. Porcine caliciviruses, resembling St-Valerien-like viruses were identified in five animals. The 3' end of the genome of one of these strains, 25A/ITA, was determined, revealing close genetic relatedness to the newly discovered St-Valerien-like caliciviruses, identified in swine in Canada. The findings of this investigation demonstrate that St-Valerien-like viruses are not geographically restricted, since they were identified outside the North American continent where there were first signalled.     

Adaptation and limitations of established hemagglutination inhibition assays for the detection of porcine anti-swine influenza virus H1N2 antibodies.

Hemagglutination inhibition (HI) has been a reliable method for determining porcine antibody levels to the well-characterized swine influenza virus (SIV) H1N1 and H3N2 subtypes. However, the recent emergence of the novel H1N2 serotype of SIV and the persistence of 2 other serotypes (H1N1 and H3N2) in the United States swine population represents a significant challenge to diagnostics. Both standardized and modified HI protocols were used in a blinded study to examine a collection of 50 control sera representing a total of 12 swine that were experimentally inoculated with one of the 3 SIV subtypes. Using these control sera data, a statistical basis for analysis was established in an attempt to classify 30 field sera with known case histories or seroprevalance into SIV serotype categories. By this approach 57% of the field sera could be classified into specific categories. The remaining samples that could not be classified reliably were most likely composed of heterogeneous anti-SIV antibody populations. These results indicate that although serological differentiation might be possible in a controlled environment, applications of these methods to field samples are currently problematic. Approaches other than HI will be required to fulfill the current need for SIV diagnostics and surveillance when specific serotype identification is required.     

猪五种繁殖障碍性疫病病原体多重PCR的建立及初步应用

为了建立能够同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)的多重PCR,并用于猪场感染情况的动态监控以及临床诊断,根据GenBank中已发表的5种病毒的基因序列,针对CSFV的E2、PRRSV的Nsp2、PRV的gB、PCV2的ORF2和PPV的VP2基因,分别设计了特异性引物。在建立的单项PCR基础上,通过优化反应条件,建立了能同时检测5种病毒的多重PCR,并具有较高的灵敏性和良好的特异性。采用建立的多重PCR对127份疑似病猪的扁桃体活体组织进行检测,检出了5种病毒的存在,其中感染2种及以上病毒的样品比例为38.6%(49/127),表明该方法是一种快速、灵敏、高效的病原学检测手段。     

Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) was isolated from tissues of aborted fetuses and weaned and suckling piglets from 4 different pig farms in Quebec. The farms were experiencing reproductive failure in sows of different parities concomitant to respiratory problems in suckling and postweaning piglets. At necropsy, gross lesions were confined to the lung and consisted of pulmonary congestion and edema of various degrees. Lesions of multifocal interstitial to proliferative pneumonia were found in the lungs of these piglets. Bacteriologic examination of various tissues from necropsied pigs yielded no pathogens in most cases. No significant antibody titers against 3 swine viruses (transmissible gastroenteritis virus, porcine parvovirus, and swine influenza virus) and two bovine viruses (bovine viral diarrhea and infectious bovine rhinotracheitis viruses) were detected in the sera of convalescent pigs. The Quebec EMCV isolates were antigenically related to the reference ATCC-VR129 strain of EMCV, as demonstrated by indirect immunofluorescence, serum neutralization (SN), and Western immunoblotting. However, one of the Quebec isolates could be distinguish by SN. EMCV-specific SN antibody titers up to 1:12,800 were detected in thoracic and ascitis fluids of aborted fetuses and in sera of convalescent pigs. A possible pneumotropic EMCV variant in swine may exist.     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

Coincidental detection of genomes of porcine parvoviruses and porcine circovirus type 2 infecting pigs in Japan

The infection status of 15 viruses in 120 pigs aged about 6 months was investigated based on tonsil specimens collected from a slaughterhouse. Only 5 species of porcine parvoviruses and porcine circovirus type 2 (PCV2) were detected at high frequencies; 67% for porcine parvovirus (PPV) (PPV-Kr or -NADL2 as the new abbreviation), 58% for PPV2 (CnP-PARV4), 39% for PPV3 (P-PARV4), 33% for PPV4 (PPV4), 55% for PBo-likeV (PBoV7) and 80% for PCV2. A phylogenetic analysis of PPV3 suggested that Japanese PPV3s showed a slight variation, and possibly, there were farms harboring homogeneous or heterogeneous PPV3s. Statistical analyses indicated that the detection of PCV2 was significantly coincidental with each detection of PPV, PPV2 and PPV3, and PPV and PPV4 were also coincidentally detected. The concurrent infection with PCV2 and porcine parvoviruses in the subclinically infected pigs may resemble the infection status of pigs with the clinical manifestations of porcine circovirus associated disease which occurs in 3–5 months old pigs and is thought to be primarily caused by the PCV2 infection.     

Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.     

Antigenic Detection of Human Strain of Influenza Virus A (H3N2) in Swine Populations at Three Locations in Nigeria and Ghana during the Dry Early Months of 2014

Since the first detection of human H3N2 influenza virus in Taiwanese pigs in 1970, infection of pigs with wholly human viruses has been known to occur in other parts of the world. These viruses, referred to as human‐like H3N2 viruses, have been known to cause clinical and subclinical infections of swine populations. Due to the paucity and complete unavailability of information on transmission of influenza viruses from other species, especially humans, to swine in Nigeria and Ghana, respectively, this study was designed to investigate the presence and prevalence of a human strain of influenza A (H3N2) in swine populations at three locations in two cities within these two West African countries in January and February, 2014. Using stratified random technique, nasal swab specimens were collected from seventy‐five (75) pigs at two locations in Ibadan, Nigeria and from fifty (50) pigs in Kumasi, Ghana. These specimens were tested directly by a sensitive Quantitative Solid Phase Antigen‐detection Sandwich ELISA using anti‐A/Brisbane/10/2007 haemagglutinin monoclonal antibody. Influenza virus A/Brisbane/10/2007 (H3N2) was detected among pigs at the three study locations, with an aggregate prevalence of 4.0% for the two locations in Ibadan, Nigeria and also 4.0% for Kumasi, Ghana. Transmission of influenza viruses from other species to swine portends serious sinister prospects for genetic reassortment and evolvement of novel viruses. We therefore recommend that further studies should be carried out to investigate the presence of other circulating human and avian influenza viruses in swine populations in West Africa and also determine the extent of genetic reassortment of strains circulating among these pigs. This would provide an early warning system for detection of novel influenza viruses, which could have pandemic potentials.     

Highly cited article published in the Veterinary Quarterly in 1991

In early 1991, the Dutch pig industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the aetiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hysonoviae, M. hyopneumoniae, and Acheloplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterized as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly affected by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

A serological survey of selected pathogens in wild boar in Slovenia

Serum samples collected from 178 shot wild boars (Sus scrofa) were tested for the presence of antibodies against classical swine fever virus, Aujeszky's disease virus (ADV), porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, swine influenza virus, porcine parvovirus (PPV), swine vesicular disease virus, Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella spp., Brucella spp. and Haemophilus parasuis (HPS) throughout Slovenia during the hunting season 2003/2004. The number of samples corresponds to 3% of the total hunting bag. By enzyme-linked immunosorbent assay (ELISA) antibodies against ADV were detected in 55 sera (31%), against PRCV in five sera (3%), PPV in 87 sera (49%), APP in 93 sera (52%), M. hyopneumoniae in 38 sera (21%), Salmonella spp. in 85 sera (47%) and HPS in 33 sera (18%).     

Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.     

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.     

Influenza A viruses: epidemiologic study in fatteners in Spain (1987-89).

2,979 sera were collected from slaughtered swine in two geographic areas of Spain from 1987 to 1989. They were tested for antibodies against an H1N1- and H3N2-influenza virus by haemagglutination-inhibition tests (HI). The percentage of positive sera was higher in area I (78%-69.2%) than in area II (63.1%-60.4%) for both viruses respectively. The coexistence of high titres to both H1N1- and H3N2-influenza virus became apparent in cold months simultaneously in each area, although influenza viruses circulated in the Spanish swine population for two years. Also this study suggests the possible circulation of A/Texas/1/77-like strains in Spain, results which have not been reported before.     

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.

This study reports the antigenic relatedness of isolates of Lelystad virus collected in the Netherlands, Germany, and the United States. The binding of antibodies directed against these isolates was tested in a set of field sera collected during outbreaks of porcine epidemic abortion and respiratory syndrome in Europe and outbreaks of swine infertility and respiratory syndrome (SIRS) in North America. Two sets of sera from pigs experimentally infected with Lelystad virus or SIRS virus were also tested. Although all 7 isolates reacted with anti-Lelystad virus sera, antigenic variation was considerable. The 4 European isolates resembled each other closely, but differed from the American isolates, and the 3 American isolates differed antigenically from each other. To reliably diagnose Lelystad virus infection, a common antigen must first be identified.     

Novel swine influenza virus subtype H3N1 in Italy

To date, three subtypes of swine influenza viruses, H1N1, H1N2, and H3N2 have been isolated in Italy. In 2006, a novel swine influenza virus subtype (H3N1) was isolated from coughing pigs. RT-PCR performed on lung tissues, experimental infection in pigs with the novel isolate, and cloning the virus by plaque assay confirmed this unique H and N combination. The novel isolate was also antigenically and genetically characterized. Genetic and phylogenetic analysis showed that the complete HA gene of the H3N1 strain has the highest nucleotide identity to three Italian H3N2 strains, one isolated in 2001 and two in 2004, whereas the full length NA sequence is closely related to three H1N1 subtype viruses isolated in Italy in 2004. The remaining genes are also closely related to respective genes found in H1N1 and H3N2 SIVs currently circulating in Italy. This suggests that the novel SIV could be a reassortant between the H3N2 and H1N1 SIVs circulating in Italy.