Antibody dependent enhancement and its potential as a contributing factor to the pathogenesis of porcine reproductive and respiratory syndrome virus infection

Antibody dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection as a contributing factor to viral pathogenesis was studied. Virus yields in porcine alveolar macrophages (PAM) culture and infection rates of PAM by PRRSV were significantly enhanced in the presence of subneutralizing levels of virus-specific antibody. Increased yields and infection rates were directly correlated (r = 0.95). Enhancement of virus yield in the presence of antibody was blocked by Protein A, indicating that the increased yield was due to enhanced uptake of virus by PAM through Fc receptors. However, the yield of progeny virions from individual PAM was also increased, suggesting that an additional mechanism contributed to yield enhancement. A similar enhancement of PRRSV infection in the presence of antibody was also observed in pigs. The mean level and duration of viremia was significantly greater in pigs that were injected with subneutralizing amounts of virus-specific IgG prior to virus challenge than in control pigs which were injected with normal serum globulin. Conversely, virus replication was significantly suppressed in pigs with a high level of neutralizing antibody. The period of time that subneutralizing levels of passively supplied antibody can persist and contribute to ADE was estimated. Results indicated that passively immunized pigs are susceptible to the potential enhancing effect of ADE for a period of 5 to 6 weeks following the disappearance of SVN antibody. A similar period of potential susceptibility to ADE was demonstrated in a similar manner in pigs that were infected with PRRSV by the nasal route. Western immunoblot analysis of sera collected during the period that ADE was expressed indicated that the 26kD viral envelope protein is primarily responsible for inducing ADE-associated antibodies. Field isolates of PRRSV were shown to vary in their susceptibility to ADE of infection. This variability was dramatically demonstrated by the neutralization of homologous virus by a concentration of antibody that enhanced the replication of heterologous isolates. Collectively, these observations suggest that ADE has the potential to contribute to the pathogenesis of PRRSV infection and to interfere with immunization strategies

Application of PCR Assay to Differentiate Two Subtypes of Swine Influenza Viruses

A reverse transcription-polymerase chain reaction (RTPCR) test was developed to differentiate between H1 and H3 subtypes of swine influenza virus (SIV). The sensitivity and specificity of this test was evaluated by comparing the results of the PCR test with subtyping results of an immunological assay performed at the National Veterinary Services Laboratories (NVSL). The test was performed on 68 eggderived SIV isolates, as well as directly on 30 lung homogenates. The data suggest that a PCR-based assay may be a reliable screening test for SIV both from egg fluid and directly from lung tissue

Characterization of H3N2 Swine Influenza Viruses in Iowa Swine

Since December 1998 when swine influenza virus (SIV) with H3N2 was first identified in Iowa swine, prospective and retrospective studies were conducted to monitor and evaluate H3N2 SIV infections in swine population in Iowa until February 2000. A serological survey revealed that H3 SIV had been widely spread in Iowa swine within first 6 months after initial identification and that both H1 and H3 SIV coexited. Many herds tested had the evidence that animals were exposed to both subtypes of SIV. In some cases, both subtypes were isolated from the same animal. All circumstantial evidences strongly suggested the emergence of new subtype due to reassortment of H1N1 and H3N2 strains; however, no evidence of new reassortant was yet found in Iowa during this study period. A one-way hemagglutination inhibition (HI) assay on banked field isolates of H3N2 SIV demonstrated that the majority of field isolates were antigenically conserve. These observations suggest that diagnostic assays using an initial Midwest H3N2 SIV isolate should be reliable for diagnosing infection of H3N2 SIV. This observation also suggests that a vaccine using an initial Midwest H3N2 SIV may provide reliable cross protection against a variety of H3N2 strains. However, a few isolates were found resistant to HI activity conferred by antiserum raised against a H3N2 Midwest isolate, warranting a continuous surveillance for antigenic drift among isolates

Characterization of Immune Ontogeny of Young Swine to Porcine Circovirus Type 2 Infection

A longitudinal study was conducted to characterize the humoral immune response of pigs to porcine circovirus type 2 (PCV2), the postulated causative agent for postweaning multisystemic wasting syndrome (PMWS). Eight-week-old cesarean-derived, colostrumdeprived (CD/CD) pigs were inoculated with a purified isolate of PCV2 and kept for 35 days post-inoculation (PI). Serum samples were collected from all pigs on day 0 and, thereafter, every 7 days until termination of the study. Naïve young pigs were shown to be susceptible to PCV2. PCV2-specific antibodies were detected by an indirect fluorescent antibody test at day 7 PI and after, whereas neutralizing antibodies were not detected until day 28 PI. Western immunoblot analysis of the sera demonstrated three virus-specific proteins with molecular mass of 28, 28.5 and 35 kD. By comparing the appearance of antibody with protein specificity of antibody response, the 28-kD protein was highly immunogenic and specific for PCV2, suggesting that the 28-kD protein may provide the antigenic basis for the development of diagnostic tests for the detection of PCV2-specific antibody. Other two proteins may be associated with virus neutralization

Development of Monoclonal Antibodies for Universal Use in the Diagnosis of PRRS

A panel of seven different groups of monoclonal antibodies (Mabs) specific for the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus (PRRSV) was generated from mice that were immunized with antigens derived from PRRSV isolate ATCC VR-2402 (ISU-P). These groups, designated A through B, were determined by their reactivity in the IFA to 67 different field isolates representing 13 states,Canada,the European Lelystad virus and the current North American vaccine strain, ATCC VR-2332. The field isolates were collected during 1989 through 1995. Group A Mab’s reacted with all field isolates and the Lelystad virus and as such should be considered for use as universal diagnostic Mab’s for PRRSV. The field isolates and the Lelystad virus were able to be categorized into seven distinct groups (I-VII) based on their reactivity to the panel of Mab’s. Group I represented 56 isolates. The six remaining groups were represented by one to four field isolates each. These observations demonstrate that there is a wider degree of diversity among North American PRRSV isolates then was previously believed

Susceptibility of Swine to Hepatitis E virus and its Significance to Human Health

Previous reports indicate that swine can be experimentally infected with Asian isolates of human hepatitis E virus (HEV), which supports epidemiological data indicating that domestic swine can serve as a reservoir for the virus in parts of Asia and as such have the potential to transmit the virus to humans by the fecal-oral route or through contact with pork products. The increasing incidence of human HEV infections in the western hemisphere raises the question of whether or not pigs can play a role in the transmission of this virus in the Americas. Accordingly the susceptibility of swine to a New World isolate of the human hepatitis E virus, Mexico 14, was evaluated. No evidence of infection was detected in experimental pigs. However a high herd and individual prevalence rate for seroreactivity to recombinant HEV antigen was detected in Iowa swine during the selection of experimental pigs. These observations suggests that swine vary in their susceptibility to human HEV isolates. Whether or not swine are susceptible to other New World isolates of HEV and can serve as a reservoir for human infection remains to be determined. The significance of the high rate of seroreactivity of swine to recombinant antigen with respect to human and swine health is not known. An epidemiological study currently in progress should help answer this important question

Emergence of H3N2 Subtype Swine Influenza Viruses in Midwest Swine

A prospective study was conducted to monitor the pig population in Iowa for the emergence of new subtype(s) of swine influenza virus (SIV) other than classic H1N1 subtype. During the study, an apparently new subtype of SIV, H3N2, was isolated in association with severe reproductive and respiratory clinical disease in swine operations in Iowa and southern Minnesota. At the same time, influenza outbreaks of this subtype also were reported in other states such as North Carolina, Illinois, and Texas. To date, the new subtype of SIV has been determined to be widely spread in swine operations throughout Iowa and is expected to coexist with H1N1 subtype in swine populations in the United States. A serological survey on 6-month-old finishing pigs (N=1064) from 129 herds throughout 29 counties in Iowa demonstrated that 64% of the pigs and 92.2% of the herds had serological evidence of exposure to H3N2 SIV as of June, 1999. The isolation of H3N2 subtype SIV in association with severe clinical disease brings a new perspective to the diagnosis and control of swine influenza in this country. New vaccines will be needed for effective control of H3N2 because field reports have indicated no cross protection against H3N2 subtype SIV by vaccines currently available for H1N1 strains. Consequently it is critical for diagnosticians to have rapid and accurate methods for the diagnosis and differentiation of SIV subtypes so that effective control measure can be suggested in timely manner

Field-Based Assessment of the Role of Porcine Cytomegalovirus in Respiratory Disease of Nursery Pigs

Porcine cytomegalovirus (PCMV) is an ubiquitous infectious agent in swine population throughout the world. Field and some experimental observations have suggested that PCMV plays an important role in causing or enhancing respiratory and/or reproductive disease of swine. However, no actual measure of this has been documented. As the first step in assessing the economic significance of PCMV infection for swine herds in the United States, a field-based case-control study was conducted to evaluate the potential role of the virus in respiratory disease of young swine. The data in this study, thus far, suggest that there may be an association between PCMV infection and increased risk of respiratory disease development in nursery pig populations and that, as was expected, PCMV infection is a common finding among nursery pigs. In an era in which multifactorial respiratory disease and associated decrease in production efficiency is such a large concern, it may be prudent to consider PCMV when developing and implementing strategies for production management and pig flow

Overview of PCR Methods Applied for the Identification of Freshwater Toxigenic Cyanobacteria

Although cyanobacteria are essential microorganisms on earth, some cyanobacteria produce toxins known as cyanotoxins, threatening humans and animals’ health. Hence, it is imperative to rapidly and accurately identify those toxic cyanobacteria. Unfortunately, traditional microscopic methods have limitations for accurate identification due to the lack of discernable morphological difference between toxic and non-toxic strains within the same cyanobacterial species or genus. In contrast, their genetic profiles are inherently conserved; therefore, nucleic acid-based assays can be more reliable for precise identification. Furthermore, molecular assays can provide high throughput and significantly reduce the turnaround time of test results. Such advantages make those assays a preferred method for rapid detection and early warning of potential toxicity. Toxigenic cyanobacterial species have synthetase genes (DNAs) for toxin production, which can be excellent marker genes. Numerous molecular assays targeting cyanotoxin synthetase genes have been developed for the identification of toxigenic cyanobacteria at various taxonomic levels. Polymerase chain reaction (PCR)-based assays are the most prevailing. Among different versions of PCR assays, the real-time quantitative PCR can be utilized to quantify the genes of interest in samples, fulfilling the purpose of both taxonomic recognition and biomass estimation. Reverse transcription (RT)-PCR assays can be used to detect transcripts (i.e., mRNAs) from toxin synthetase genes, probably enhancing the predictive value of PCR detection for toxin production from observed cyanobacterial species. Nevertheless, the utility of toxin synthetase gene- or its transcript-based PCR assays for routine cyanotoxin monitoring needs to be further evaluated on a large scale

Field and Experimental Assessment of PRRSV Evolution

The genetic and antigenic stability of porcine reproductive and respiratory syndrome (PRRS) virus during infection in pigs was assessed using field and experimental data. Among field isolates, we found that PRRS virus varied both genetically and antigenically. Even within the same farm, various phenotypic strains of PRRS virus were present simultaneously. Experimentally, we demonstrated that PRRS virus “quasi-species” appeared over time as the virus replicated in animals. Although the degree of genotypic changes in the gene coding for the major envelope protein (i.e., open reading frame 5) was less than expected based on field observations; some mutants appear to have been changed significantly. Overall, our observations suggest that persistence of PRRS virus in pigs may be attributed to viral mutation, although the actual role of viral mutation in persistent PRRS virus infection remains to be determined. The presence of various phenotypic strains within the same farm or herd may account for the apparent ineffectiveness of PRRS control by monovalent vaccine