Swine Serum Samples Research Articles

Antigenic and Genetic Characterization of Swine Influenza Viruses Identified in the European Region of Russia, 2014-2020.

Pigs have long been recognized as “mixing vessels” in which new viruses are formed by reassortment involving various influenza virus lineages (avian, animal, human). However, surveillance of swine influenza viruses only gained real significance after the 2009 pandemic. A fundamentally important point is the fact that there is still no regular surveillance of swine flu in Russia, and the role of swine viruses is underestimated since, as a rule, they do not cause serious disease in animals. Since the pig population in Russia is large, it is obvious that the lack of monitoring and insufficient study of swine influenza evolution constitutes a gap in animal influenza surveillance, not only for Russia, but globally. A 6 year joint effort enabled identification of SIV subtypes that circulate in the pig population of Russia’s European geographic region. The swine influenza viruses isolated were antigenically and genetically diverse. Some were similar to human influenza viruses of A(H1N1)pdm09 and A(H3N2) subtype, while others were reassortant A(H1pdm09N2) and A(H1avN2) and were antigenically distinct from human H1N1 and H1N1pdm09 strains. Analysis of swine serum samples collected throughout the seasons showed that the number of sera positive for influenza viruses has increased in recent years. This indicates that swine populations are highly susceptible to infection with human influenza viruses. It also stresses the need for regular SIV surveillance, monitoring of viral evolution, and strengthening of pandemic preparedness.

Isolation and characterization of novel reassortant mammalian orthoreovirus from pigs in the United States

Mammalian orthoreovirus (MRV) infects multiple mammalian species including humans. A United States Midwest swine farm with approximately one thousand 3-month-old pigs experienced an event, in which more than 300 pigs showed neurological signs, like “down and peddling”, with approximately 40% mortality. A novel MRV was isolated from the diseased pigs. Sequence and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three MRV serotypes that infect human, bovine and swine. The M2 and S1 segment of the isolate showed 94% and 92% nucleotide similarity to the M2 of the MRV2 D5/Jones and the S1 of the MRV1 C/bovine/Indiana/MRV00304/2014, respectively; the remaining eight segments displayed 93%-94% nucleotide similarity to those of the MRV3 FS-03/Porcine/USA/2014. Pig studies showed that both MRV-infected and native contact pigs displayed fever, diarrhea and nasal discharge. MRV RNA was detected in different intestinal locations of both infected and contact pigs, indicating that the MRV isolate is pathogenic and transmissible in pigs. Seroconversion was also observed in experimentally infected pigs. A prevalence study on more than 180 swine serum samples collected from two states without disease revealed 40-52% positive to MRV. All results warrant the necessity to monitor MRV epidemiology and reassortment as the MRV could be an important pathogen for the swine industry and a novel MRV might emerge to threaten animal and public health.

Serological evidence of West Nile viral infection in archived swine serum samples from Peninsular Malaysia

West Nile virus (WNV), a neurotropic arbovirus, has been detected in mosquitos, birds, wildlife, horses, and humans in Malaysia, but limited information is available on WNV infection in Malaysian pigs. We tested 80 archived swine serum samples for the presence of WNV antibody and West Nile (WN) viral RNA using ID Screen West Nile Competition Multi-species enzyme-linked immunosorbent assay kits and WNV-specific primers in reverse transcription polymerase chain reaction assays, respectively. A WNV seroprevalence of 62.5% (50/80) at 95% confidence interval (51.6%–72.3%) was recorded, with a significantly higher seroprevalence among young pigs (weaner and grower) and pigs from south Malaysia. One sample was positive for Japanese encephalitis virus antibodies; WN viral RNA was not detected in any of the serum samples.

Lower seroprevalence of Toxoplasma gondii in swine from central China after an outbreak of African swine fever.

Toxoplasma gondii is widespread worldwide and can infect swine. This study evaluated the seroprevalence of T. gondii in swine from central China after an outbreak of African swine fever (ASF). A total of 2683 swine serum samples were collected from farms in four provinces. Of the serum samples, 1.42% (38/2683) (95% CI, 1.03–1.94) tested positive for T. gondii IgG antibody by a modified agglutination test (MAT) (cut-off: 1:25). Comparing with the results of previous studies, specifically our survey from before the outbreak, the seroprevalence of T. gondii in swine from central China was significantly decreased after the occurrence of ASF (OR = 7.679, 2015–2017 vs. 2019–2020). In general, the proportion of seropositive animals increased with the age of the swine, indicating post-natal transmission of T. gondii. Furthermore, there was a significant difference in seroprevalence between suckling pigs and weaned pigs (p < 0.05). This is the first large-scale investigation of T. gondii infection in swine after an ASF outbreak in China. The lower seroprevalence of T. gondii in swine after ASF may be due to stricter biosecurity measures on the farms, but results indicated swine exposure to zoonotic parasites despite these measures. This highlights that pigs must be considered a potential source of human T. gondii infections.

Molecular detection and clinical aspects of porcine circovirus 3 infection in pigs from Brazil

ABSTRACT Porcine circovirus 3 (PCV-3) DNA has been detected in serum samples from apparently healthy pigs as well as pigs with different clinical conditions. Molecular detection of PCV-3 was observed in swine serum samples from Southeastern - Brazil using a nested PCR designed specifically for this study. The epidemiology and clinical aspects of PCV-3 infection were evaluated. The samples originated from 154 pigs of both genders from different production phases and with different clinical presentations, sampled from 31 pig farms visited between 2013 and 2018. In this study, PCV-3 was detected in 26.7% of samples from all populations across varying ages. Statistical association (P=0.0285) was observed only between animals with respiratory signs and PCV-3; no PCV-3-positive animal had diarrhea. No statistical association was observed between PCV-3 and age, or gender of the pigs. Because PCV-3 is a newly discovered virus, there is very little information about its epidemiology. We hope that these data can help in future studies investigating PCV-3 epidemiology.

Detection of anti-HEV antibodies and RNA of HEV in pigs from a hyperendemic Italian region with high human seroprevalence.

Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products. Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR. The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk.

Development of dipstick enzyme linked immunosorbent assay for on-site sero-diagnosis of Japanese encephalitis in swine

Japanese encephalitis (JE) is an important viral zoonotic disease in Asia, especially in rural and suburban areas where rice cultivation and pig farming coexist. Pigs serve as a suitable sentinel model, the surveillance of which could predict a potential JE outbreak in human population in the immediate vicinity. However, existing diagnostics like ELISA and VNT require sophisticated laboratory facilities which are more often not available in field conditions. In the present study, we aimed at developing recombinant non-structural (NS1) protein-based dipstick IgG ELISA as an on-site assay for sero-diagnosis of JE in swine. The assay was standardized by optimizing various parameters and the following conditions were found to be ideal including 1 μg of rNS1 protein in carbonate buffer per strip of nitrocellulose membrane comb; bovine serum albumin as blocking agent at 4 °C overnight; serum dilution of 1:10 and conjugate dilution of 1:5000 in skimmed milk powder. Relative diagnostic sensitivity and specificity of dipstick IgG ELISA was 100% and 92.9%, respectively. The dipstick assay was validated in three laboratories as per OIE guidelines. The storage life of dipstick was up to 7 months at 4 °C. The assay is easy to perform and the results can be interpreted with visual observation that precludes the need for absorbance reading equipment. The standardized dipstick assay was found promising for screening swine serum samples in field conditions. Timely detection of JE virus in swine will aid in predicting the outbreak in humans and thus in taking suitable preventive and control measures.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay.

The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h. We further evaluated the performance of CASLFA in a nonlaboratory environment and successfully confirmed 27 ASFV-infected samples from 110 suspected swine serum samples, with an accuracy of 100% when compared to real-time PCR (RT-PCR) assay. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for gene analysis in resource-poor or nonlaboratory environments.

Self-assembly into virus\u2013like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

PCV3 capsid protein (Cap) is an important antigen for diagnosis and vaccine development. To achieve high-level expression of recombinant PCV3 Cap in Escherichia coli (E. coli), the gene of wild-type entire Cap (wt-eCap) was amplified from clinical samples, and three optimized entire Cap (opti-eCap) and one optimized Cap deleted nuclear location signal (NLS) (opti-dCap) gene fragments encoding the same amino acid sequence with wt-eCap were synthesized based on the codon bias of E. coli. Those gene fragments were inserted into the pET30a expression vector. One recombinant strain with the highest expressed soluble eCap from four entire Cap (one wt-eCap and three opti-eCap) and one recombinant strain expressed opti-dCap were selected for further purification. The purified eCap and dCap were identified by transmission electron microscopy (TEM), a large number of round hollow particles with a diameter of 10 nm virus-like particles (VLPs) were observed in eCap, whereas irregular aggregation of proteins observed in dCap. After formation the VLPs were applied as a coating antigen to establish an indirect ELISA (I-ELISA) for detection of PCV3-specific antibody in swine serum. 373 clinical swine serum samples from China collected in 2019 were tested utilizing the VLP-based I-ELISA method under optimized conditions. To the best of our knowledge, this is the first report of self-assembly into VLPs of PCV3 recombinant Cap. Our results demonstrated that the VLP-based I-ELISA will be a valuable tool for detecting the presence of PCV3 antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes.

Proactive preparedness for Cat Que virus: An Orthobunyavirus existing in India

Background & objectives:The presence of Cat Que virus (CQV) in Culex mosquitoes and pigs has been reported in China and Vietnam. Due to the spread of similar species of the Culex mosquitoes in India, there is a need to understand the replication kinetics of this virus in mosquito models. As a part of preparedness and to identify the presence of this CQV in humans and swine, this study was carried out to develop diagnostic tests.Methods:Serological and molecular diagnostic assays were developed for testing the mosquito population, human and swine serum samples. In this line, RNA-dependent RNA polymerase (L), glycoprotein (M) and nucleocapsid (S) genes-based reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for CQV. Real-time RT-PCR was used for screening of retrospectively collected human serum samples (n=1020) with acute febrile illness during 2014-2017. Simultaneously, an in-house anti-CQV swine and human IgG ELISAs were also developed to detect anti-CQV IgG antibody. Human serum samples (n=883) with post-onset of disease (POD) >4 days and swine serum samples (n=459) were tested for the presence of anti-CQV IgG antibodies. CQV NIV 612,045 isolate was used for susceptibility and replication kinetics experiment using three different species of mosquitoes to understand its behaviour in Indian mosquitoes.Results:All human serum samples (n=1020) screened for the presence of CQV using real-time RT-PCR were found to be negative. Anti-CQV IgG antibody positivity was recorded in two of 883 human serum samples tested. Virus susceptibility experiments indicated that three species of mosquito, namely Aedes aegypti, Culex quinquefasciatus and Cx. tritaeniorhynchus supported multiplication of CQV by intrathoracic as well as artificial membrane/oral feeding routes.Interpretation & conclusions:Anti-CQV IgG antibody positivity in human serum samples tested and the replication capability of CQV in mosquitoes indicated a possible disease causing potential of CQV in Indian scenario. Screening of more human and swine serum samples using these assays is required as a proactive measure for understanding the prevalence of this neglected tropical virus.

Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.

BackgroundEnzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity.MethodsPhage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated.ResultsSix SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79–4.57% and 5.54–9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot.ConclusionA specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.

Similar frequency of Porcine circovirus 3 (PCV-3) detection in serum samples of pigs affected by digestive or respiratory disorders and age-matched clinically healthy pigs.

Porcine circovirus 3 (PCV-3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV-3 infection in serum samples from animals suffering from post-weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV-3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4week- to 4month-old pigs clinically diagnosed with respiratory (n=129) or digestive (n=126) disorders. Serum samples of age-matched healthy animals (n=60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous-necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV-3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV-3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV-3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV-3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.

Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine

Japanese encephalitis virus (JEV) causes severe neurological disease in humans, especially among children. The disease is endemic in several South Asian countries including India. Swine play a major role as amplifier host for JEV and act as a source of infection to humans through mosquito bite. Early detection of either virus or antibodies in swine will aid to undertake control measures to prevent virus spread to humans. Swine seldom show symptoms of JEV infection and the viraemic phase lasts for a short period of 3 to 4 days indicating the potential of detection of antibodies, which remain for relatively longer period, as a suitable alternative. Cost effective and sensitive assays for the detection of JEV antibodies in swine are not available indigenously. Hence, we have developed a recombinant nonstructural protein 1 (rNS1) based enzyme linked immunosorbent assay for the detection of IgG antibodies against JEV in swine. The test is robust, highly sensitive (91%), specific (97%), reproducible and affordable. Field validation of the assay was done by screening 3628 swine Serum samples collected from different parts of India. The overall sero-positivity was found to be 32.22%. The developed ELISA can be readily incorporated into surveillance programs for detection of Japanese encephalitis virus activity in swine population thereby aiding in prediction of outbreaks in humans.

Seroprevalence of Hepatitis E Virus Infection in Pigs from Southern Bulgaria.

Hepatitis E virus (HEV) has been isolated from humans and several animals' species. During the last years, the knowledge of HEV infection dramatically changed and enriched. The aim of this study was to estimate the seroprevalence of HEV in industrial pigs in different districts of Southern Bulgaria. Three hundred sixty swine serum samples were tested for anti-HEV IgG antibodies. The samples were collected from four industrial farms from three districts of Southern Bulgaria. HEV-specific antibodies in porcine serum were detected by enzyme-linked immunosorbent assay (PrioCHECK HEV Ab porcine). The overall HEV seroprevalence was 60.3%. The seropositivity varied widely depending on age groups and investigated farms. The overall prevalence in weaners was 25%, in fattening pigs 75.8%, and in group of sows was found the highest HEV positivity of 80%. The occurrence of HEV positivity in sows and fattening pigs presented odds ratio (OR) = 17.200 (95% confidence interval [CI]: 8.8-33.7) and OR = 11.342 (95% CI: 6.1-21.0), respectively, compared to weaners. The study indicated that HEV is widespread in industrial farms in Bulgaria and presented high seroprevalence in pigs. The results found that HEV seropositivity showed age dependency. The National Health Authorities should raise awareness of HEV and its zoonotic potential.

Development and evaluation of lateral flow assay for sero-diagnosis of Japanese encephalitis in swine

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic flaviviral disease. Swine sero-convert 2–3 weeks before infection occurs in humans and thus serves as a suitable sentinel for JE surveillance and outbreak prediction in human population. The present study was conducted with the objective of developing a lateral flow assay (LFA) for detecting JEV antibodies in swine sera. Three different formats were tried using recombinant NS1 protein as antigen in order to select the best format. In format I, gold nanoparticles were conjugated with antigen followed by spotting of antigen on NCM as test line and anti-antigen IgG on NCM as control line. In format II, gold nanoparticles were conjugated with antigen followed by spotting of staphylococcal protein A as test line and anti-antigen IgG as control line. Format III used gold nanoparticles conjugated with goat anti-pig IgG followed by spotting of antigen as test line and pig IgG as control line. Amongst the three formats, format II was found to be superior with 100% relative diagnostic sensitivity and 100% relative diagnostic specificity during monsoon and post-monsoon period. A panel of 500 field swine serum samples was tested using format II which revealed sero-positivity of 15.6%, and the format was found suitable to screen swine serum samples during monsoon and post-monsoon period.

Wine Sero-Status of Japanese Encephalitis among Kathmandu and Morang Districts of Nepal

Japanese Encephalitis (JE) a vector borne zoonoitc disease caused by arbovirus of Flavivirus and transmitted by Culex tritaeniorhynchus additionally pig acts as amplifying host for the virus. A total 115 swine serum samples 100 pig farmers from each district were tested against Japanese encephalitis virus (JEV) infection. The collected samples were analyzed by using “Porcine Encephalitis B Virus Antibody Rapid Test Kit”. Among them 17% samples were positive for JE while 83% samples were negative for JE. Total 18% were positive for JE in Morang district while 115.4% were positive for Kathmandu district. There was no significant difference in prevalence of JE in these two districts (p&gt;0.05). In Kathmandu, the prevalence was 15.4% while in Morang it was slightly higher, 18%. 9.5% younger pigs of 3-9 months of age were positive for JE and 20.6% of age above 9 months were positive of JE but were no significant (p&gt;0.05). Similarly, 17.9% female pigs and 14.6% male pigs were positive for JE however difference was not significantly different. In case of breed, no association of pigs with seropositivity (p&gt;0.05), 7 (13.5%) out of 52 local breed pigs and 12 (19.1%) out of 63 were JE positive. The prevalence rate of JEV was higher in Morang district among study area which might be due to several factors like lack of education in pig farmers, non vaccinated pig population and lack of awareness regarding risk factor of JEV.&#x0D; Int. J. Appl. Sci. Biotechnol. Vol 6(4): 373-378

Influenza B viruses in pigs, Taiwan.

BackgroundInfluenza B viruses (IBVs) have never been isolated from natural‐infected pigs in clinical cases, although the susceptibility of domestic pigs to experimental IBV infections had been confirmed as well as IBV‐specific antibodies were detected from pigs under natural and experimental conditions.ObjectivesWe aimed to assess and investigate the activities for infection and circulation of IBVs in pigs.MethodsAnnual active surveys for influenza have been implemented on swine populations in Taiwan since July 1998. Nasal swabs, trachea, lungs, and blood from pigs were tested using virological and serological assays for influenza. Gene sequences of influenza viral isolates were determined and characterized. Preliminary sero‐epidemiological data for influenza virus were investigated.ResultsThree strains of IBV were isolated and identified from natural‐infected pigs in 2014. Genetic characterization revealed the highest identities (>99%) of molecular sequence with the contemporary IBVs belonged to the B/Brisbane/60/2008 genetic clade of Victoria lineage in the phylogenetic trees for all 8 genes. IBV‐specific antibodies were detected in 31 (0.2%; 95%CI: 0.1%‐0.2%) of 15 983 swine serum samples from 29 (2.8%; 95%CI: 1.9%‐3.9%) of 1039 farm visits under annual active surveys from 2007 through 2017. Seropositive cases have been found sparsely in 1‐5 of test prefectures every year except 2015 and 2017 as well as scattered loosely over 26 townships/districts of 11 prefectures in Taiwan cumulatively in 11 years.ConclusionsInfluenza B viruse infections from humans to pigs remained sporadic and accidental currently in Taiwan but might have paved potential avenues for newly emerging zoonotic influenza in the future.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was establishedbased on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and theIDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Molecular evolution of H1N1 swine influenza in Guangdong, China, 2016–2017

Swine are the main host of the H1N1 swine influenza virus (SIV), however, H1N1 can also infect humans and occasionally cause serious respiratory disease. To trace the evolution of the SIV in Guangdong, China, we performed an epidemic investigation during the period of 2016–2017. Nine H1N1 influenza viruses were isolated from swine nasal swabs. Antigenic analysis revealed that these viruses belonged to two distinct antigenic groups, represented by A/Swine/Guangdong/101/2016 and A/Swine/Guangdong/52/2017. Additionally, three genotypes, known as GD52/17-like, GD493/17-like and GD101/16-like, were identified by phylogenetic analysis. Importantly, the genotypes including a minimum of 4 pdm/09-origin internal genes have become prevalent in China in recent years. A total of 2966 swine serum samples were used to perform hemagglutination inhibition (HI) tests, and the results showed that the seroprevalence values of SW/GD/101/16 (32.2% in 2016, 32.1% in 2017) were significantly higher than the seroprevalence values of SW/GD/52/17 (18.0% in 2016, 16.7% in 2017). Our study showed that the three reassortant genotypes of H1N1 SIV currently circulating in China are stable, but H1N1pdm09 poses challenges to human health by the introduction of internal genes into these reassortant genotypes. Strengthening SIV surveillance is therefore critical for SIV control and minimizing its potential threat to public health.

Investigation on pseudorabies prevalence in Chinese swine breeding farms in 2013–2016

Pseudorabies (PR) has been prevalent in Chinese swine breeding farms since the outbreak at the end of 2011. For investigating current prevalence of PR, a nationwide surveillance has been performed in this study. The swine serum samples were collected from 93, 100, 92, and 91 swine farms in China during 2013-2016, respectively. Since the extensive use of gE-deleted pseudorabies virus (PRV) vaccine, we could apply the PRV-gE antibody for determining wild-type virus infection and the PRV-gB antibody for evaluating vaccine immunization. The results were concluded as follows: (1) Nationally, the positive rate of PRV-gB was maintained at a high level (> 90%), while the positive rate of PRV-gE continued to decrease (from 22.17 to 13.14%). (2) The positive rates of PRV-gE were greatly varied in different geographical regions and swine farms (0~100%), while the positive rate of PRV-gB was generally high (> 90%). (3) The number of imported PRV attenuated vaccines were about twice that of domestic PRV attenuated vaccines, while the positive rate of PRV-gB was not significantly different (P> 0.05). (4) The performance of PR eradication developing or developed farms was better than the performance of common farms, with higher positive rate of PRV-gB (> 90%) and much lower positive rate of PRV-gE (nearly 0%).