Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.

West Nile (WN) virus, a mosquito-transmitted virus native to Africa, Asia, and Europe, was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans, and from brain tissues of 28 American crows, Corvus brachyrhynchos, and one Cooper's hawk, Accipiter cooperii, in Connecticut. A portion of the genome of virus isolates from four different hosts was sequenced and analyzed by comparative phylogenetic analysis. Our isolates from Connecticut were similar to one another and most closely related to two WN isolates from Romania (2.8 and 3.6 percent difference). If established in North America, WN virus will likely have severe effects on human health and on the health of populations of birds.

Fluorescent antibody test for rapid detection of West Nile virus antigen in avian tissues.

An indirect fluorescent antibody test (IFA) was developed to detect West Nile virus (WNV) antigens in tissues from avian species. The test samples used in the study consisted of 100 sets of tissues from dead crows that had been collected during the 2001 surveillance in Connecticut. The test tissues were punctured with a fine point Dacron cotton-tipped applicator and smeared in duplicate on 10-well diagnostic printed glass slides. Among several fixatives tested, 4% paraformaldehyde was the best. Reagent calibration for the IFA test was done in WNV-infected Vero cells and control uninfected Vero cells. Optimized antibody and fluorescent conjugate concentrations were then applied for the detection of WNV antigen on fixed tissue impression smears. Several tissues, including brain, heart, liver, kidney, and spleen were tested by the IFA test. The brain and heart seemed to be unsuitable for the test because of excessive background. Both virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) were used for validation, with the latter technique having a higher sensitivity. Therefore, IFA results were compared with RT-PCR results. The diagnostic sensitivity was 96.8% for liver, 96.4% for kidney, and 100% for spleen. The diagnostic specificity was 69% for liver, 95.3% for kidney and 95.8 for spleen. The IFA test performed best with spleen and kidney. The IFA test described here is a useful, practical, and rapid test for screening for WNV.

Positive inductive effect of IL-2 on virus-specific cellular responses elicited by a PRRSV-ORF7 DNA vaccine in swine.

This study investigated the effect of swine interleukin 2 (IL-2) and swine interleukin 4 (IL-4) on the development of immune responses induced by a PRRSV-ORF7 DNA vaccine (phCMV-ORF7). The two cytokines were cloned separately in the eukaryotic expression vector phCMV, and delivered via gene gun as adjuvants for the DNA vaccine. Groups of 3-week-old certified PRRSV-free, castrated male, Yorkshire crossbred pigs, were vaccinated with or without the IL-2 or IL-4. The ensuing humoral and cellular immune responses were analyzed by a PRRSV-specific ELISA, and by an in vitro blastogenic response of peripheral blood mononuclear cells (PBMC) stimulated by viral antigen, respectively. The animals were boosted 21 days post-vaccination and challenged 28 days afterward. The virus loads post-challenge were measured by real time PCR. The group of swine receiving the vaccine plus IL-2 had significant virus-specific blastogenic responses 3 weeks after the vaccine-cytokine boost, when compared to those of the experimental pigs that received the vaccine plus IL-4, vaccine alone, unvaccinated controls or the pigs vaccinated with the DNA vaccine cloned in the reverse orientation (phCMV-ORF7(Rev)). None of the experimental swine had detectable specific antibodies against the virus during the vaccination phase. The virus load peak in vaccinated animals was delayed by about 72h as compared to that of the control pigs (unvaccinated and vaccinated with the phCMV-ORF7(Rev) construct). Interestingly, animals that received the phCMV-ORF7 vaccine alone consistently had low virus loads throughout the study. These results demonstrate that IL-2 has a positive inductive effect on the activation of vaccine-induced virus-specific cellular immunity, while IL-4 appeared to have a suppressive effect. Our data also suggest that ORF7 may play a role in reducing the virus load in PRRSV infected animals.

The West Nile virus: its recent emergence in North America.

West Nile fever emerged in New York in the summer of 1999 when seven people, several horses and thousands of wild birds died. It was soon established that the human disease and the mortality of birds were related. Continued surveillance detected West Nile virus in mosquitoes, birds, horses, small mammals, bats and humans, and has shown its spread to several northeastern states. These events confirm the establishment of West Nile virus endemically in the United States.

Detection of lymphoproliferative responses against pseudorabies virus immediate early protein (IE180) in swine immunized with a modified live virus vaccine.

The immediate early protein (IE180) of pseudorabies virus (PrV) was generated by cycloheximide (CHX) reversal procedures in PrV-infected swine skin cells. Using this IE180 preparation as antigen, specific proliferation was detected in mononuclear cells (PBMNCs) from swine vaccinated with a modified live virus (MLV) PrV vaccine. Two sets of data support this conclusion. (a) PBMNCs of c/cSLA inbred swine vaccinated with an MLV vaccine a year before the test exhibited significant responses against structural virion proteins and IE180. (b) Vaccination of PrV-negative swine with the same MLV vaccine induced a conversion from an unresponsive state against IE180 to one of specific antigen-driven responsiveness. The responses of vaccinated swine against IE180 were significantly higher than their preimmune responses (p < or = 0.003) or to the response of control swine (p < or = 0.045). Moreover, IE180 antigens obtained from lysates of CHX-reversed, PrV-infected cells by heparin/agarose affinity separation also stimulated specific proliferation of PBMNCs from MLV-vaccinated swine, as their proliferative responses were significantly higher than those of unvaccinated swine (p < or = 0.05). These data suggest that PrV IE180 contributes at least in part to the overall cellular response to PrV.

Characterization of anti-idiotypic antibodies generated against foot-and-mouth disease virus neutralizing monoclonal antibodies.

A series of seven neutralizing monoclonal antibodies (nMAbs) against type A12 foot-and-mouth disease virus (FMDV) was used to induce polyclonal anti-idiotypic antibodies (anti-ids) in rabbits. The anti-ids were semi-purified through isotype affinity columns and assayed by solid-phase radioimmunoassay for cross-reactivity. nMAbs which map to the same epitope on the virion appear to contain a common idiotype, and the corresponding anti-ids competitively inhibited the virus-nMAb reaction. Using a modified ELISA assay, it was possible to demonstrate binding of purified anti-ids to FMDV susceptible tissue culture cells. Such antibodies however, did not interfere with the binding of virus to cells, and the binding of anti-ids to FMDV receptor-negative cells could also be demonstrated. Mice were inoculated with purified anti-ids, and two elicited anti-viral antibodies, although these antibodies were non-neutralizing. Thus anti-ids to anti-FMDV nMAbs failed to react with cellular receptors for the virus, but were able to induce anti-viral antibody and thus should be examined as an alternative vaccine strategy for this virus.

A vector DNA vaccine encoding pseudorabies virus immediate early protein demonstrates partial protection in mice against lethal virus challenge.

An earlier study in our laboratory provided evidence that pseudorabies virus (PrV) immediate early protein (IE180) may contribute to the overall immune response against PrV. To examine the response by IE180 more closely, we initiated a vaccine trial in mice with a vector DNA construct that contains the gene encoding for IE180, designated pcDNAIE180. The DNA vaccine was delivered in gold microcarriers using a Helios Gene Gun, and 70% of BALB/c mice given the DNA vaccine (2 microg/mouse) seroconverted within 2 weeks. The remaining negative mice seroconverted after a single vaccine booster. Essentially similar results were obtained on vaccination of C57BL/6 mice, whereas C3H/HeJ mice remained negative after the first vaccination, but responded after a booster. Seven months after immunization with pcDNAIE180, an overall 25% of BALB/c, C3H/HeJ, and C57BL/6 mice receiving a lethal PrV challenge were protected. In addition, a significant passive transfer of IE180-specific antibodies to the offspring from pregnant mice vaccinated with pcDNAIE180 was observed. Interestingly, a moderate level of protection (27.6%) was also observed when these offspring received a lethal PrV challenge. Moreover, an enhancement of immune responses and a twofold increase in the level of protection were observed in mice that received a second vaccine booster by gene gun 8 months after the first vaccination. Together, these data support the theory that IE180 may indeed play a role in the overall protective immunity against PrV.

Discrete cleavage patterns of pseudorabies virus immediate early protein (IE180) seen in some cell lines upon extraction after cycloheximide reversal.

Pseudorabies virus (PrV) encodes for a single and essential immediate early phosphoprotein designated IE180. In this study, IE180 was examined in lysates from various cell lines infected at high multiplicities under cycloheximide inhibition of protein synthesis and subsequent reversal. Three distinct protein patterns of IE180 which were cell-specific and dependant on the extraction procedure were revealed. Detergent lysates of PrV infected MDBK cells yielded almost exclusively wild type IE molecule (180 kDa). In contrast, SSG/94 cells, VERO or CV-1 cells did not yield 180 kDa molecules but predominantly a shorter variant of approximately 60 kDa in molecular mass. Additional bands of about 50/55 kDa were also detected in lysates of SSG/94 and VERO cells by immunoprecipitation. Lysates of CV-1 and MDBK cells also yielded a 120 kDa molecule. The smaller molecular mass bands occurred in the presence of PMSF and aprotinin however, cleavage was blocked completely by addition of N alpha-p-Tosyl-L-lysine chloromethyl ketone (TLCK) into the lysis buffer. Moreover, an ability of the shorter IE180 variants to bind heparin was also revealed in the study. These data provide useful insights on protease profiles encountered among different PrV susceptible cells and indicates the use of appropriate protease inhibitors such as TLCK to protect IE180 under these experimental conditions.

Co-delivery of IL-2 or liposomes augment the responses of mice to a DNA vaccine for pseudorabies virus IE180.

Recently we have demonstrated, with a DNA vaccine, that the immediate early protein (IE180) of pseudorabies virus provides a moderate level of protection in mice. In order to improve its immunogenicity and protective capacity, this IE180 DNA vaccine was delivered to C3H/HeJ mice either in combination with an IL-2 expressing plasmid or complexed with cationic liposomes. Co-delivery of the vaccine and IL-2 DNA by gene gun resulted in seroconversion in 5/5 of the vaccinated mice after a single administration, whereas two intramuscular (i.m.) injections were required to achieve seroconversion in all mice. Antibody and delayed-type hypersensitivity responses were augmented in mice, which received the DNA vaccine and the IL-2 gene compared to those of mice receiving the DNA vaccine alone. In addition, the time of death after challenge was significantly delayed in mice, which received the IL-2 gene. The proportion of surviving mice (40%), however, was similar to that obtained in mice which received the vaccine alone by gene gun. Liposome-mediated vaccine delivery also resulted in a higher rate of seroconversion when compared with that induced by the naked DNA vaccine. Thus, all vaccinated mice seroconverted after either two i.v. or three i.m. injections of the liposome/DNA complex, with 40 and 25% of these mice being protected against challenge, respectively. These data support that co-administration of the IE180 DNA vaccine with the IL-2 gene or delivery in liposomes are two effective approaches to increase its immunogenicity.

Porcine lymphocyte gamma interferon responses to mitogenic stimuli monitored by a direct immunoassay.

Gamma interferon produced by porcine lymphocytes (nPoIFN gamma) in response to stimulation with phorbol-myristate-acetate (PMA) and phytohemagglutinin (PHA) was monitored by a radioimmunoassay (RIA). The RIA was developed with antibodies raised in rabbits against recombinant porcine IFN gamma (rPoIFN gamma) and made monospecific on a rPoIFN gamma/Sepharose matrix. This anti-PoIFN gamma antibody was shown to bind and neutralize rPoIFN gamma specifically and to cross-react with bovine IFN gamma but not with murine IFN gamma or porcine IFN alpha. The nPoIFN gamma levels produced by lymphocytes in response to PMA and PHA were at least two-fold higher than control lymphocytes as measured by the RIA in culture fluids. These culture fluids were fractionated on Concanavalin A Sepharose (Con A/Seph) and anti-rPoIFN gamma/Seph in attempts to evaluate the induced nPoIFN gamma further. The separation was monitored by RIA and showed that nPoIFN gamma was retained on Con A/Seph suggesting the presence of sugar residues on the molecules. Pools of Con A/Seph fractions, positive in RIA, were separated further on the anti-rPoIFN gamma/Seph matrix where a total adsorption of nPoIFN gamma occurred. On a weight basis, the eluates from the anti-rPoIFN gamma/Seph had a reactivity in RIA at least four times higher than the fractions derived from Con A/Seph. This indicated that the nPoIFN gamma remained immunochemically reactive after being eluted from Con A/Seph and that the separation on anti-rPoIFN gamma/Seph chromatography was specific. Purified nPoIFN gamma exhibited a major band with the same migration characteristics of rPoIFN gamma in PAGE-SDS electrophoresis and several minor bands when reacted with 125I anti-rPoIFN gamma antibody in Western blots. A total loss of reactivity to antibody after radioiodination of nPoIFN gamma, however, prevented the confirmation of these results by immunoprecipitation. A direct, rapid and sensitive immunoassay for measurement of the relative levels of nPoIFN gamma present in biological fluids is presented and this method could be a useful complement to bioassays for IFN gamma.

Functional characterization of a swine CD4(+)/CD8(+) double positive lymphoblastoid T-cell line with a CD25(+)/CD45RA(-) phenotype generated in vitro with interleukin-2.

A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2.

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland.

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.

Mechanism and isotypes involved in passive immunoglobulin transfer to the newborn alpaca (Lama pacos).

Crias, newborn alpacas (Lama pacos), that were almost agammaglobulinemic at birth had a 70% increase in total serum proteins within 24 hours largely because of absorption of gamma globulins from colostrum. Immunoglobulin G was the isotype in highest concentration in colostrum and in serum from 24-hour-old crias. The serum IgG concentration of 10 crias increased linearly (r = 0.97) from a mean of 0.3 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 30.1 mg/ml (+/- 8.1 SD) at 24 hours. The 24-hour concentration decreased by half in 10 days. Immunoglobulin M also was absorbed from colostrum and increased linearly (r = 0.99) from a mean of 0.5 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 4.2 mg/ml (+/- 2.2 SD) 24 hours after birth. The 24-hour serum concentration of IgM decreased by half in 7 days. Therefore, on a weight basis, 7 times more IgG than IgM was transferred to crias; IgG accounted for greater than 85% of the passively transferred proteins in serum of 24-hour-old crias. Absorption of functional antibodies of IgG and IgM isotypes from colostrum of immunized dams by crias also was demonstrated. Immunoglobulin G and IgM antibody titers to chicken RBC increased linearly to maximal geometric mean titers of 1,139 and 843, respectively, 24 hours after birth. The 24-hour IgG and IgM antibody titers decreased by half in 6 and 3.8 days, respectively. Purified alpaca IgG had a molecular mass of 166 kilodaltons, a predominant gamma mobility, and an extinction coefficient of 14.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of passive immunoglobulin transfer: a major determinant of mortality in newborn alpacas (Lama pacos).

Failure of passive transfer (FPT) of immunoglobulin from colostrum was demonstrated as a major determinant of mortality in newborn alpacas (Lama pacos; crias). Serum IgG concentrations of dying crias were significantly (P less than 0.0001) lower than were serum IgG concentrations of crias that lived. Of 82 crias, 10 (12%) died within 1 month of age, and 7 of these had 0 to 9 mg of IgG/ml of serum at 48 hours after birth; 5 of the 7 had evidence of infectious diseases. The serum IgG concentrations of the remaining dead crias were 12, 13, and 20 mg/ml. On the basis of serum IgG concentrations of crias that died in the first month, FPT was defined as a 48-hour serum IgG concentration less than 9 mg/ml, which was greater than 2 SD below the 48-hour mean of clinically normal crias. Using this definition, the prevalence of FPT in the 82 crias studied was 9%. Corroborative evidence of the relationship between FPT and mortality was obtained from a retrospective study of 21 dead crias. The postmortem serum IgG concentration of 5 crias that died 2 to 10 days after birth ranged from less than 1 to 3 mg/ml; all were greater than 2 SD below the mean of age matched clinically normal crias. The range of serum IgG concentration was 2.2 to 21 mg/ml in 8 crias that died 11 to 20 days after birth; serum IgG concentration in 1 cria was greater than 2 SD below the normal mean, and 6 were greater than 1 SD below the normal mean.(ABSTRACT TRUNCATED AT 250 WORDS)

Brief characterization of muskrat (Ondatra zibethicus) immunoglobulin G (IgG) separated from serum on protein A.

Muskrat (Ondatra zibethicus) immunoglobulin fraction was separated from whole serum by Protein A Sepharose chromatography. In serum electrophoresis, this fraction had a gamma motility; when electrophoresed on a polyacrylamide gel with sodium dodecyl sulfate it resolved into two protein bands of approximately 52 and 25 kilodaltons, respectively. These bands were consistent with molecular weights of known heavy and light chains of immunoglobulin G (IgG) in other closely related species. Furthermore, the putative muskrat immunoglobulins had a strong cross-reactivity with mouse IgG1, IgG3, and kappa chain in an enzyme-linked immunosorbent assay. We propose, that the proteins bound to the Protein A Sepharose represent muskrat immunoglobulins of the IgG class.

Clinicopathological features and histopathology of experimental hepatic capillariasis in muskrats (Ondatra zibethicus).

Ten muskrats (Ondatra zibethicus) each were infected with 17,000 eggs (long-term study) and eight muskrats each were infected with 8,000 eggs (short-term study) of Capillaria hepatica (Nematoda). Food intake, body weight, and selected clinicopathological parameters were measured every 2 days for 28 days in the short-term study and every 14 days for 184 days in the long-term study. Muskrats in the short-term study had moderate to severe necrotizing granulomatous hepatitis associated with mild anorexia and weight loss, varying degrees of leukocytosis with eosinophilia and elevation of serum alanine and aspartate aminotransferases. No significant changes in packed cell volume, hemoglobin, total plasma protein, albumin, blood urea nitrogen, bilirubin, lactate dehydrogenase or alkaline phosphatase were found among animals from the short-term study. Muskrats in the long-term study had severe necrotizing granulomatous hepatitis associated with marked anorexia, weight loss and 60% mortality over 39 days post-inoculation (PI); animals that survived for 184 days did not return to pre-inoculation body weights despite returning to normal food intake. Hepatic lesions at 184 days PI consisted of minimal to severe liver replacement by C. hepatica eggs. No statistically significant differences in values of clinical parameters between inoculated animals and a non-inoculated control group from the long term study were found.

Marked differences between MARC-145 cells and swine alveolar macrophages in IFNß-induced activation of antiviral state against PRRSV.

The activation of antiviral activity induced by recombinant swine interferon beta (rswIFNß) against PRRSV was comparatively examined in MARC-145 cells and porcine alveolar macrophages (PAMs). A dose-response analysis showed, in MARC-145 cells, that isolate Mo25544 was highly sensitive to rswIFNß while a vaccine strain and isolate PDV130-9301 were resistant to different extents. In contrast, all three viruses were equally sensitive to rswIFNß in PAMs even at the lowest dose of IFN utilized in the bioassays. To analyze potential differences in mechanisms of antiviral activation between these cells, treatment with 2-aminopurine (2-AP), an inhibitor of double-stranded RNA-dependent protein kinase (PKR), was performed in rswIFNß-treated cells. Addition of 2-AP to rswIFNß-primed MARC-145 cells restored replication of the Mo25544 isolate, and to some extent that of vaccine virus and PDV130-9301. In contrast, virus replication could not be rescued for any of the three viruses with 2-AP in rswIFNß-treated PAMs. The differences in sensitivity of PRRSV to rswIFNß as well as the effects of 2-AP strongly suggest that MARC-145 cells and PAMs utilize different rswIFNß-associated antiviral pathways. Therefore, studies to understand virus-host cell interactions performed in MARC-145 cells require additional scrutiny when utilized as a host cell model for immunologic responses to PRRSV.

Herpetiform genital lesions in a heifer with mucosal disease.

A 14-month-old heifer with a 17-day history of unresponsive bloody diarrhea was necropsied. There were focal, pink-red erosions of the nares and hard palate; ulcers and fissures of the tongue; and multiple ulcerative lesions of the alimentary canal. Interdigital skin of both rear limbs was ulcerated and bleeding; and the margins of the vulva contained punctiform red ulcers. The gross lesions were consistent with mucosal disease. Histopathology and laboratory testing ruled out rinderpest, foot-and-mouth disease, and vesicular stomatitis, and identified bovine virus diarrhea virus to be the cause of this disease. Lesions of the vulva similar to those seen in some stages of infectious pustular vulvovaginitis were negative for bovine herpesvirus-1 and tested positive for bovine viral diarrhea virus antigen by immunohistochemistry.

Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus.

Swine beta interferon (swIFN-beta) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-beta gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-beta or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-beta showed no CPE. To confirm the antiviral activity of swIFN-beta, culture fluids from Ad5-swIFN-beta-infected cells were affinity-purified on a Sepharose-anti-swIFN-beta matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-beta. Additional cultures of MARC-145 cells treated with swIFN-beta-containing supernatants or affinity-purified swIFN-beta were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-beta. swIFN-beta was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-beta or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-beta-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-beta protects both MARC-145 cells and PAMs from PRRSV infection.