The impact of maternally derived immunity on influenza A virus transmission in neonatal pig populations.

The commonality of influenza A virus (IAV) exposure and vaccination on swine farms in the United States ensures that the majority of neonatal pigs will have some degree of maternal immunity to IAV. The influence of maternal immunity on IAV transmission in neonatal pig populations will impact virus prevalence and infection dynamics across pig populations. The main objective of this study was to assess the impact of maternally derived immunity on IAV transmission in an experimental setting. Neonatal pigs suckled colostrum and derived maternal (passive) immunity from sows in one of three treatment groups: (a) non-vaccinated control (CTRL) or vaccinated with (b) homologous (PASSV-HOM) or (c) heterologous (PASSV-HET) inactivated experimental IAV vaccines. Sentinel neonatal pigs derived from the groups above were challenged with IAV via direct contact with an experimentally infected pig (seeder pig) and monitored for IAV infection daily via nasal swab sampling. A susceptible-infectious-recovered (SIR) experimental model was used to obtain and estimate transmission parameters in each treatment group via a generalized linear model. All sentinel pigs in the CTRL (30/30) and PASSV-HET (30/30) groups were infected with IAV following contact with the seeder pigs and the reproduction ratio estimates (95% confidence interval) were 10.4 (6.6-15.8) and 7.1 (4.2-11.3), respectively. In contrast, 1/20 sentinel pigs in the PASSV-HOM group was infected following contact with the seeder pigs and the reproduction ratio estimate was significantly lower compared to the CTRL and PASSV-HET groups at 0.8 (0.1-3.7). Under the conditions of this study, IAV transmission was reduced in neonatal pigs with homologous maternal immunity compared to seronegative neonatal pigs and pigs with heterologous maternal immunity as defined in this study. This study provides estimates for IAV transmission in pigs with differing types of maternal immunity which may describe the influence of maternal immunity on IAV prevalence and infection dynamics in pig populations.

Infectivity of PRRS virus in pig manure at different temperatures.

PRRSv is an economically important swine pathogen which can be disseminated from infected pig herds via movement of contaminated manure. The process of manure handling and inadequate cleaning of transport vehicles are commonly implicated as sources of PRRSv transmission. Stability of PRRSv in pig manure at different temperatures is unknown. The objective of this study was to determine PRRSv-infectivity half-life in manure and in a cell culture medium at 4, 20, 60 and 80°C. To assure sample consistency across the study, all samples were prepared from common homogenized solutions (MEM and manure) and frozen at -20°C. Samples were thawed, transferred to a water bath set at a specific temperature, inoculated with 100 µl of PRRSv at designated time points and then tested for virus infectivity. Regression models were created to estimate PRRSv half-life based on incubation temperature. There was an exponential decrease in PRRSv infectivity with increasing temperature. At every temperature tested, PRRSv had shorter half-life when incubated in manure compared to MEM. PRRSv half-life in MEM and manure was estimated at 112.6 and 120.5 h at 4°C, 14.6 and 24.5 h at 20°C, 1.6 and 1.7 h at 40°C, 2.9 and 8.5 min at 60°C, and 0.36-0.59 min at 80°C, respectively. Results of this study can be used as basis for developing strategies to inactivate PRRSv present in manure-contaminated environments using heating treatments. For example, these data suggest that submitting transport trailers to temperature of 50°C for 8h would decrease PRRSv from 10(6) TCID(50)/ml to less than 10(1) TCID(50)/ml.

Comparison of Porcine circovirus type 2 (PCV2) infection in light and heavy pigs of market age on farms with routine PCV2 vaccination.

Commercial vaccines against Porcine circovirus type 2 (PCV2) are widely used on swine farms. Marked body weight variation at marketing age is a problem on conventional pig farms using all-in/all-out barn management. The aim of this study was to investigate whether PCV2 infection could be a factor influencing body weight variation. Seven conventional farms that routinely used PCV2 vaccination were selected, and 60 serum samples from light and heavy pigs at each site were tested for PCV2 antibody titers and viremia. At 3 farms the mean antibody titer, proportion of viremic pigs, and virus load differed significantly between the light and heavy groups. These preliminary results suggest that PCV2 infection may be a factor contributing to weight variation in vaccinated market-age hogs.

Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs.

BACKGROUND/OBJECTIVE: We evaluated the sensitivity of PCR on oral fluids in detecting influenza virus in vaccinated and non-vaccinated pigs. METHODS: Three-week-old influenza-free pigs were divided into three groups: (i) control, non-vaccinated, (ii) vaccinated with a commercial, heterologous vaccine, and (iii) vaccinated with an experimental, homologous vaccine. After vaccination, an influenza-infected pig was placed in contact with each of the groups. Individual nasal swabs and pen oral fluids were collected daily. Viral RNA was tested for the presence of influenza by RRT-PCR and virus isolation attempted from oral fluids. A pen was considered positive if at least one nasal swab was positive. RESULTS: Based on nasal swab results, 43·8% of pens were detected positive but only 35% based on oral fluids. Overall sensitivity of oral fluids was 80%, and virus was isolated from 51% of RRT-PCR-positive oral fluids. The kappa coefficient for agreement (ĸ) between oral fluids and nasal swabs was 0·82. Among groups, ĸ was 1 (95% CI, 1-1), 0·74 (95% CI, 0·55-0·92), and 0·76 (95% CI, 0·5-1) for control, heterologous, and homologous-vaccinated groups, respectively. There was less agreement when within pen prevalence was 10% or less. Probability of detecting influenza virus in oral fluids was 99% when within pen prevalence was higher than 18% and decreased to 69% when prevalence was 9%. CONCLUSIONS: Results indicated that pen-based collection of oral fluids is a sensitive method to detect influenza even when within pen prevalence is low and when pigs have been vaccinated and highlight the potential use of oral fluids for influenza surveillance.

Vaccination of influenza a virus decreases transmission rates in pigs.

Limited information is available on the transmission and spread of influenza virus in pig populations with differing immune statuses. In this study we assessed differences in transmission patterns and quantified the spread of a triple reassortant H1N1 influenza virus in naïve and vaccinated pig populations by estimating the reproduction ratio (R) of infection (i.e. the number of secondary infections caused by an infectious individual) using a deterministic Susceptible-Infectious-Recovered (SIR) model, fitted on experimental data. One hundred and ten pigs were distributed in ten isolated rooms as follows: (i) non-vaccinated (NV), (ii) vaccinated with a heterologous vaccine (HE), and (iii) vaccinated with a homologous inactivated vaccine (HO). The study was run with multiple replicates and for each replicate, an infected non-vaccinated pig was placed with 10 contact pigs for two weeks and transmission of influenza evaluated daily by analyzing individual nasal swabs by RT-PCR. A statistically significant difference between R estimates was observed between vaccinated and non-vaccinated pigs (p < 0.05). A statistically significant reduction in transmission was observed in the vaccinated groups where R (95%CI) was 1 (0.39-2.09) and 0 for the HE and the HO groups respectively, compared to an Ro value of 10.66 (6.57-16.46) in NV pigs (p < 0.05). Transmission in the HE group was delayed and variable when compared to the NV group and transmission could not be detected in the HO group. Results from this study indicate that influenza vaccines can be used to decrease susceptibility to influenza infection and decrease influenza transmission.

Evaluation of a nested polymerase chain reaction assay to differentiate between two genotypes of Porcine circovirus-2.

Porcine circovirus-2 (PCV-2) is associated with several diseases in pigs, including postweaning multisystemic wasting syndrome (PMWS). A new genotype of PCV-2 was isolated from swine farms with and without clinical PMWS in North America. The new genotype was differentiated in a separate cluster by phylogenetic analyses and is now named PCV-2b compared with PCV-2a for the previously known genotype. The purpose of this study was to develop and evaluate a nested polymerase chain reaction (nPCR) assay to detect and differentiate between PCV-2a and PCV-2b. Genotype-specific primer sets were designed by using sequence data published for different PCV-2 strains. Specificity and sensitivity of the nPCR were examined by using PCV-2 isolates with known genotype. Nested PCR was found to be highly specific and sensitive for detecting and differentiating between the PCV-2 genotypes compared with the conventional 1-step PCR assay. Nested PCR was applied to detect PCV-2 and to identify the genotype in serum samples from swine farms with and without a clinical history of PMWS. Of 60 serum samples collected from 4 farms during clinical PMWS outbreaks, PCV-2a and PCV-2b were detected in 6 and 49 samples, respectively. Six of the 10 samples from one of the 4 farms had both PCV-2a and PCV-2b. Of 20 serum samples from 2 farms without PMWS, 11 were positive for PCV-2a only. These results suggest that the differential nPCR can be used to detect PCV-2 and to differentiate the 2 genotypes from field samples.

Serologic and genetic characterization of North American H3N2 swine influenza A viruses.

The H3N2 subtype of influenza A viruses isolated from pigs in the United States and Canada has shown both genetic and antigenic diversity. The objective of this study was to determine the serologic and genetic characteristics of contemporary strains of these viruses. Genetic analysis of 18 reference strains and 8 selected strains demonstrated differences in 1% to 9% of the nucleotides of the hemagglutinin (HA) gene. Phylogenetic analysis of the HA gene revealed 3 genetic clusters, as well as divergence of cluster III viruses from a cluster III prototype virus (A/Swine/Illinois/21587/99). By means of 1-way cross-hemagglutination inhibition with antiserum against 5 field isolates and 3 vaccine viruses, most of 97 isolates tested could be placed in 1 of 3 serogroups. The several isolates that did not react with any antiserum were in genetic cluster III, which suggests that continuous antigenic drift in cluster III may have resulted in virus variants. The efficacy of commercial vaccines against these virus variants should be evaluated with vaccination and challenge studies.

Efficacy of swine influenza A virus vaccines against an H3N2 virus variant.

We compared the efficacy of 3 commercial vaccines against swine influenza A virus (SIV) and an experimental homologous vaccine in young pigs that were subsequently challenged with a variant H3N2 SIV, A/Swine/Colorado/00294/2004, selected from a repository of serologically and genetically characterized H3N2 SIV isolates obtained from recent cases of swine respiratory disease. The experimental vaccine was prepared from the challenge virus. Four groups of 8 pigs each were vaccinated intramuscularly at both 4 and 6 wk of age with commercial or homologous vaccine. Two weeks after the 2nd vaccination, those 32 pigs and 8 nonvaccinated pigs were inoculated with the challenge virus by the deep intranasal route. Another 4 pigs served as nonvaccinated, nonchallenged controls. The serum antibody responses differed markedly between groups. After the 1st vaccination, the recipients of the homologous vaccine had hemagglutination inhibition (HI) titers of 1:640 to 1:2560 against the challenge (homologous) virus. In contrast, even after 2nd vaccination, the commercial-vaccine recipients had low titers or no detectable antibody against the challenge (heterologous) virus. After the 2nd vaccination, all the groups had high titers of antibody to the reference H3N2 virus A/Swine/Texas/4199-2/98. Vaccination reduced clinical signs and lung lesion scores; however, virus was isolated 1 to 5 d after challenge from the nasal swabs of most of the pigs vaccinated with a commercial product but from none of the pigs vaccinated with the experimental product. The efficacy of the commercial vaccines may need to be improved to provide sufficient protection against emerging H3N2 variants.

The impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs.

The objective of this study was to evaluate the role of different variables (animal age, bacterial coinfection, and isolate pathogenicity) on the shedding of Porcine reproductive and respiratory syndrome virus (PRRSV) in aerosols. Animals were grouped according to age (2 versus 6 mo) and inoculated with a PRRSV isolate of either low (MN-30100) or high (MN-184) pathogenicity. Selected animals in each group were also inoculated with Mycoplasma hyopneumoniae. The pigs were anesthetized and aerosol samples (1000 breaths/sample) collected on alternating days from 1 to 21 after PRRSV inoculation. The results indicated that animal age (P = 0.09), M. hyopneumoniae coinfection (P = 0.09), and PRRSV isolate pathogenicity (P = 0.15) did not significantly influence the concentration of PRRSV in aerosols. However, inoculation with the PRRSV MN-184 isolate significantly increased the probability of aerosol shedding (P = 0.00005; odds ratio = 3.22). Therefore, the shedding of PRRSV in aerosols may be isolate-dependent.

Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs.

OBJECTIVE: To evaluate the influences of animal age, bacterial coinfection, and porcine reproductive and respiratory syndrome virus (PRRSV) isolate pathogenicity on virus concentration in pigs. ANIMALS: Twenty-one 2-month-old pigs and eighteen 6-month-old pigs. PROCEDURE: Pigs were grouped according to age and infected with mildly virulent or virulent isolates of PRRSV. The role of concurrent bacterial infection was assessed by infecting selected pigs with Mycoplasma hyopneumoniae 21 days prior to inoculation with PRRSV. On alternating days, blood and swab specimens of nasal secretions and oropharyngeal secretions were collected. On day 21 after inoculation with PRRSV, selected tissues were harvested. Concentrations of PRRSV were determined by use of quantitative real-time PCR and expressed in units of TCID(50) per milliliter (sera and swab specimens) or TCID(50) per gram (tissue specimens). RESULTS: Concentrations of virus were higher in blood and tonsils of pigs infected with virulent PRRSV. Pigs infected with virulent PRRSV and M hyopneumoniae had significantly higher concentrations of viral RNA in lymphoid and tonsillar tissue. Coinfection with M hyopneumoniae resulted in a higher viral load in oropharyngeal swab specimens and blood samples, independent of virulence of the PRRSV isolate. Two-month-old pigs had significantly higher viral loads in lymph nodes, lungs, and tracheal swab specimens than did 6-month-old pigs, independent of virulence of the PRRSV isolate. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple factors affect PRRSV concentration in pigs, including pathogenicity of the PRRSV isolate, age, and concurrent infection with M hyopneumoniae.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death.

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK- stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a prolonged active infection followed by a persistent infection in lymphoid tissues lasting for several months. Pigs develop both an antibody and cell-mediated immune response following PRRSV infection, but the specific role of each type in the development of protective immunity and clearance of the virus is not yet known. The aims of this study were to characterize the dynamics of PRRSV persistence from 0 to 135 d post infection (pi), characterize the kinetics of the antibody mediated immune response following PRRSV infection, and characterize the cell mediated immune responses to PRRSV infection. Eighty, 4-month-old PRRSV-free gilts were obtained from a source known to be negative for PRRSV. On day 0, gilts were infected intranasally with 10(2.4) TCID/50 MN 30-100 PRRSV. Following infection, animals were bled between days 0 to 135 pi. Viremia was detected up to day 30. Serum antibody response (by enzyme-linked immunosorbent assay [ELISA] and virus neutralization antibody) was detected from day 14 to 120 pi. Cell-mediated immune response represented by interferon gamma (IFN-gamma) was detected from day 14 to 120 pi. Persistence of PRRSV in tissues was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) between days 30 to 135. These results indicate that serum neutralizing antibodies and IFN-gamma play an important role in the clearance of PRRSV. Nevertheless none of the parameters measured (virus neutralizing antibodies), either alone or in combination, are solely responsible for the clearance of the virus from the host and the development of sterilizing immunity.

Retrospective analysis of etiologic agents associated with respiratory diseases in pigs.

Twenty-eight hundred and seventy-two cases of respiratory disease in pigs were analyzed for their etiologic agents. Two or more pathogens were detected from 88.2% of the cases, indicating that porcine reproductive and respiratory syndrome virus (PRRSV) or swine influenza virus (SIV) combined with other bacterial agents was a common cause for porcine respiratory diseases in the mid-western USA.

Isolation and characterization of porcine circovirus type-2 from sera of stillborn fetuses.

In order to examine an association between porcine circovirus type-2 (PCV2) infection and reproductive failure in pigs, sera (n = 171) from stillborn fetuses were collected from 3 different farms with prolonged histories of reproductive problems. These sera were tested for the presence of antibodies to PCV2 using an immunoperoxidase monolayer assay. Of the 171 sera tested, 28 had PCV2 antibody titers of > or = 1:16. When these 28 samples were tested by a polymerase chain reaction assay,13 were found to contain PCV2 viral DNA. Of these 13 samples containing both PCV2 antibodies and viral DNA, 9 yielded PCV2 on virus isolation. Amino acid sequences comprising open reading frame 2 of PCV2 from 2 of these isolates were compared to PCV2 isolates from cases of post-weaning multi-systemic wasting syndrome (PMWS). The amino acid sequences of the 2 isolates from stillborn pigs were shown to be nearly identical to each other, as well as to other PCV2 isolates associated with reproductive failure. When compared with PMWS isolates, the isolates from the stillborn fetuses showed differences of at least 2 amino acids. These results confirm previous findings that transplacental infection of PCV2 occurs in the field and that stillbirths in pigs may be associated with PCV2 infections. At present, the significance of minor differences in amino acid sequences is not known.

Identification and characterization of cytopathogenic Mycoplasma hyorhinis from swine farms with a history of abortions.

A virus-like cytopathic agent isolated from swine farms with a history of recurrent abortion episodes was investigated. We employed a differential display reverse transcription-polymerase chain reaction (ddRT-PCR) to obtain genetic information of the cytopathic agent. Partial nucleotide sequence (527 bp) obtained from differentially displayed PCR fragments showed 88.7% similarity with the 23S rRNA gene of Mycoplasma hyopneumoniae. Unexpectedly, the 5' portion (1-333 bp) of the sequence shared 96.1% similarity with 5' untranslated region (UTR) of human prostate tumor inducing gene 1 (PTI-1). Cytopathic effects and extranuclear DNA fluorescence were no longer observed when BM-cyclin was added in the culture medium, suggesting that BM-cyclin sensitive mycoplasma-like organisms caused the cell death. Further evidence supporting the cytopathic agent as a mycoplasma-like organism was obtained by the capability of (3)H-thymidine and (3)H-uridine incorporation, a single peak in buoyant density gradient profile (1.20-1.24 g/ml), and ultrastructural morphology. Unlike M. hyopneumoniae, the organism was not propagated in Friis medium. Nucleotide sequence of 16S rRNA obtained from the cytopathic agent showed 0.8-1.0% divergences with other M. hyorhinis strains, suggesting that the newly isolated cytopathogenic swine mycoplasma was a variant form of M. hyorhinis. Striking homology between a portion of the 23S rRNA gene of M. hyorhinis and 5' UTR of human PTI-1 implicated that M. hyorhinis might potentially be related to the evolution of human PTI-1.

Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during warm weather.

Mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) throughout a coordinated sequence of events that replicated common farm worker behavior during warm weather (10 degrees C to 16 degrees C) was assessed using a field-based model. The model involved fomites (boots and containers), vehicle sanitation, transport, and personnel movement. In a previous study, the model successfully demonstrated mechanical transmission of PRRSV in 8 out of 10 replicates during cold weather. A field strain of PRRSV was inoculated into carriers consisting of soil samples, which were adhered to the undercarriage of a vehicle. The vehicle was driven approximately 50 km to a commercial truck washing facility where the driver's boots contacted the carriers during washing, introducing the virus to the vehicle interior. The vehicle was then driven 50 km to a simulated farm site, and the driver's boots mechanically spread virus into the farm anteroom. Types of containers frequently employed in swine farms contacted drippings from the footwear on the anteroom floor. The truck wash floor, vehicle cab floor mats, boot soles, anteroom floor, and the ventral surface of containers were sampled to track the virus throughout the model. Ten replicates were conducted, along with sham-inoculated controls, and control replicates. In 2 replicates, infectious PRRSV was detected on the anteroom floor and in 1 replicate, infectious PRRSV was detected on the surface of the container by swine bioassay. All sham-inoculated controls and protocol controls were negative. These results indicate that mechanical transmission of PRRSV throughout a coordinated sequence of events in warm weather can occur, but in contrast to data from studies conducted during cold weather, it appears to be a relatively infrequent event.

Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather.

Using a field-based model, mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) wa assessed throughout a coordinated sequence of events that replicated common farm worker behavior during cold weather (< 0 degrees C). The model involved fomites (boots and containers), vehicle sanitation, transport, and the movement of personnel. A field strain of PRRSV was inoculated into carriers consisting of snow and water, and carriers were adhered to the undercarriage of a vehicle. The vehicle was driven approximately 50 km to a commercial truck washing facility where the driver's boots contacted the carriers during washing, introducing the virus to the vehicle interior. The vehicle was then driven 50 km to a simulated farm site, and the driver's boots mechanically spread virus into the farm anteroom. Types of containers frequently employed in swine farms (styrofoam semen cooler, metal toolbox, plastic lunch pail, and cardboard animal health product shipping parcel) contacted drippings from footwear on the anteroom floor. The truck wash floor, vehicle cab floor mats, boot soles, anteroom floor, and the ventral surface of containers were sampled to track the virus throughout the model. Ten replicates were conducted, along with sham-inoculated controls. At multiple sampling points PRRSV nucleic acid was detected in 8 of 10 replicates. In each of the 8 PCR-positive replicates, infectious PRRSV was detected on the surfaces of containers by virus isolation or swine bioassay. All sham-inoculated controls were negative. These results indicate that mechanical transmission of PRRSV can occur during coordinated sequence of events in cold weather.

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States.

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.

Evaluation of a multiplex reverse transcription-polymerase chain reaction assay for subtyping hemagglutinin genes 1 and 3 of swine influenza type A virus in clinical samples.

A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect and identify subtypes of hemagglutinin (H) 1 and H3 swine influenza virus (SIV). Two oligonucleotide primer sets were prepared using published sequence data for H1N1 and H3N2. The PCR products with unique size characteristics of each subtype were sequenced, and the sequences were confirmed to be subtype specific for H gene 1 or 3. These primer sets did not amplify when RT-PCR assay was performed for genomic DNA or RNA from other common swine pathogens. The RT-PCR assay was able to detect viral RNA up to 1 tissue culture infective dose of reference SIV H1N1 or H3N2. The multiplex RT-PCR was applied to 30 SIV isolates subtyped by hemagglutination inhibition (HI) test. Forty-three positive and 20 negative swine field samples for SIV by virus isolation were also tested. Of these 73 SIV-positive samples tested, H1 and H3 were identified in 38 and 28 samples by the multiplex RT-PCR, respectively. The remaining 7 samples were positive for both H1 and H3 genes. No positive reaction was found for all 20 SIV-negative field samples. Subsequently, 235 random field samples from pigs with respiratory problems were tested by the multiplex RT-PCR, and 26 and 13 samples were found to be positive for H1 and H3, respectively. Results of these multiplex RT-PCR were comparable with those of the HI test. These results suggest that multiplex RT-PCR can be a useful test for detection and subtyping of SIV in clinical samples.