Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells.

Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle.

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.

Antigenic differences in the H proteins of canine distemper viruses.

Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.

Prevalence of American trypanosomiasis (Chagas disease) among dogs in Oklahoma.

OBJECTIVE: To determine the prevalence of Trypanosoma cruzi infection among dogs in Oklahoma. DESIGN: Cross-sectional study. ANIMALS: 301 owned or impounded dogs related by ownership or general geographic location to 3 dogs determined to have trypanosomiasis. PROCEDURES: Blood samples were obtained from dogs between November 1996 and September 1997. Infection status was determined by use of a radioimmunoprecipitation assay. Second blood samples were obtained from some of the seropositive dogs for study by hemoculture and polymerase chain reaction (PCR) assay. Sites where infected dogs were found were inspected for triatomine insects, and light traps were used for vector trapping. RESULTS: 11(3.6%) dogs were seropositive for T. cruzi infection. Ten of the 11 were owned rural hunting dogs. Protozoal organisms isolated from the blood of 1 seropositive dog were identified as T. cruzi by PCR testing. Only 1 adult Triatoma sanguisuga was captured in a light trap at a site near infected dogs; this insect was not infected. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that T. cruzi is enzootic in eastern Oklahoma. Measures that would reduce the risk of dogs acquiring T. cruzi infection are unlikely to be acceptable to their owners, and no effective drugs are available for treatment. The presence of T. cruzi-infected dogs poses a threat of transmission to persons at risk of exposure to contaminated blood Veterinarians who practice in the southern United States should be cognizant of this blood borne zoonosis and educate all personnel about appropriate precautions.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed pseudorabies virus glycoprotein gE and gI complex.

A direct competition enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed complex of pseudorabies virus (PRV) glycoproteins E (gE) and I (gI) has been developed. For that purpose gE and gI genes of PRV were co-expressed in insect cells. Complex formation was confirmed by radioimmunoprecipitation assay. The specificity and sensitivity of the test were evaluated and compared with an ELISA using only gE as an antigen and a commercially available test. For validation, 245 negative sera and 165 positive sera have been tested. The gE/gI ELISA had a higher sensitivity and specificity when compared with the ELISA using only gE as the antigen. Both sensitivity and specificity were comparable with the commercially available test. Moreover, the test based on the baculovirus gE/gI complex allows the detection of anti-gE antibodies in pig serum as early as two weeks after infection. The gE/gI ELISA test is easy to perform; its additional advantage is that the gE/gI antigen can be produced in baculovirus system in large quantities without handling live pseudorabies virus.

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens.

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.

Comparative characteristics of the Israeli Newcastle disease virus field strains by means of a wide panel of monoclonal antibodies against hemagglutinin-neuraminidase glycoprotein.

Fourteen mouse monoclonal antibodies (MAB) were tested for their ability to react with 15 reference and 52 local Newcastle disease virus (NDV) strains isolated in Israel during the last decade from feral birds. All the field isolates had no antigenic difference when examined by classic serological tests. However, MAB-mediated analysis revealed wide antigenic heterogeneity amongst the studied viruses. By the pattern of the MAB reactivity, all the isolates could be distributed into 13 groups.

Expression of the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) elicits virus-type specific neutralising antibodies.

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.

Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

Loss of glycosylation at Asn144 alters the substrate preference of the N8 influenza A virus neuraminidase.

Role of asparagine-linked (N-linked) oligosaccharide side chains in the maturation and the function of influenza virus neuraminidase (NA) subtype N8 was examined by site-directed mutagenesis and vaccinia virus expression system. Mutations in the consensus sequence for N-linked glycosylation at Asn 84 or 398 prevent the proper maturation of mutant NAs. On the contrary, mutation at Asn 144, that is conserved in all except two strains of influenza virus NA ever sequenced, did not affect the proper maturation and the transport of the mutant NA to the cell surface. Furthermore, this mutation led the alternation of substrate preference of this enzyme. These observations indicate that N-glycosylation at Asn 144 of N8 NA may be conserved from the functional requirement, but not from the structural necessity.

Comparative antibody response in harbour and grey seals naturally infected by a morbillivirus.

The antibody response of free-ranging harbour and grey seals, naturally infected by a morbillivirus, was assessed using a virus neutralizing test and a radio-immunoprecipitation assay. The prevalence of antibody was similar between species, however, grey seals had significantly higher virus neutralizing titers. Serum from clinically healthy grey seals precipitated the nucleocapsid (N) protein along with the hemagglutinin (H) and fusion (F) glycoproteins. By contrast, significantly fewer harbour seal sera precipitated the envelope glycoproteins and responses were weaker than those of grey seals. One harbour seal with acute morbillivirus pneumonia, and two with encephalitis precipitated only the N protein. Serum from four harbour seals with encephalitis weakly recognized the envelope glycoproteins. Thus, the antibody response of grey seals appears more competent than that of harbour seals with respect to morbillivirus antigens. We speculate that this difference between the species may be an important determinant of morbillivirus susceptibility.

Epizootiology of morbillivirus infection in harp, hooded, and ringed seals from the Canadian Arctic and western Atlantic.

Using a virus neutralization technique, we found phocine distemper virus (PDV) antibody in 130 (83% of 157) harp seals (Phoca groenlandica) from the western North Atlantic sampled between 1988 and 1993 inclusive. In contrast, only 44 (24% of 185) hooded seals (Cystophora cristata) had antibodies against PDV even though they were sympatric with harp seals and were sampled over a similar period, from 1989 to 1994 inclusive. Antibodies occurred in 106 (41%) of 259 ringed seals (Phoca hispida); this prevalence was higher than expected given the solitary behavior and territoriality characteristic of this species. Seropositive ringed seals were found at each of seven locations across Arctic Canada from Baffin Bay to Amundsen Gulf at which samples were collected between 1992 and 1994. However, the prevalence of infection was highest where ringed seals are sympatric with harp seals in the eastern Canadian Arctic.

Identification of native foot-and-mouth disease virus non-structural protein 2C as a serological indicator to differentiate infected from vaccinated livestock.

Cattle and pigs which have been vaccinated against foot-and-mouth disease can be distinguished from convalescent animals by radio-immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis of the virus-induced proteins reacting with the respective sera. Baby hamster kidney cells infected with foot-and-mouth disease virus (FMDV) (serotype A24) were labelled with 35S-methionine and the virus-induced proteins were precipitated with sera from vaccinated and subsequently challenged animals, convalescent animals retained for over 300 days, animals vaccinated or infected with viruses belonging to all serotypes of FMDV, and animals infected with encephalomyocarditis (EMC) or porcine or bovine enteroviruses. In addition to the structural proteins of the virus, the non-structural proteins 2C, 3ABC, 3C, 3CD and 3D were precipitated by convalescent sera, but only 3D was precipitated by serum from vaccinated animals. Proteins L, 2C and 3C were precipitated only after challenge with a heterotypic virus (serotype O1 Tunisia), indicating that virus replication of the challenge virus had taken place. No precipitation was detected with sera from EMC or enterovirus-infected animals. The results indicate that protein 2C, and to a lesser extent the polypeptide 3ABC, could be used to differentiate potential carrier convalescent animals from vaccinated livestock.

A proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing.

Sixty-six pestiviruses from ruminant and porcine hosts were analysed with a panel of 76 monoclonal antibodies raised against 9 different viruses. Reactivity was used to construct epitope similarity maps for all of the viruses. Four principal virus subgroups were demonstrated. One subgroup equated to classical swine fever virus (CSFV) and included most porcine pestiviruses but none from ruminants. A second subgroup contained mainly viruses of bovine origin, including reference bovine viral diarrhoea viruses (BVDV) such as NADL; however viruses from pigs and sheep were also represented. A third subgroup represented by reference strains of border disease virus (BDV) comprised mainly ovine isolates, but also viruses from pigs. The fourth and most recently defined subgroup contained no reference strains of CSFV, BVDV or BDV, but included atypical viruses from cattle, sheep and pigs. The subgrouping scheme was supported by genetic comparisons between representative viruses from the 4 subgroups and by virus neutralisation with polyclonal sera.

Virulence-associated antigenic and genetic characteristics of bluetongue virus-17 isolates.

Bluetongue virus (BLU), an orbivirus, is of importance to the sheep and cattle industries. We have obtained 5 United States BLU-17 isolates which have been tested for virulence in sheep and 16 BLU-17 field isolates from the Caribbean and Central America. Using a panel of 15 monoclonal antibodies (MAb) against an avirulent BLU-17, we observed that 6 MAbs had negligible or very low neutralization titers for the virulent isolates in contrast to moderate to high titers for the avirulent isolates. These MAbs also differentiated the field isolates into two groups--inadequate vs effective neutralization. All 6 MAbs immunoprecipitated the outer capsid protein, VP2. Electropherotyping of genomic RNA from all 21 viruses identified an increase in RNA segment 3 mobility for those isolates which were not neutralized by the 6 specific MAbs. RNA segment 3 codes for the inner core protein, VP3. There were no detectable electrophoretic differences for RNA segment 2, which encodes VP2. In summary, the virulent BLU-17 isolates differed from the avirulent isolates in both the antigenicity of the outer capsid protein, VP2, and the electrophoretic mobility of RNA segment 3, and we hypothesize that one or both of these changes may result in BLU virulence.

Vaccination of pigs against pseudorabies with highly attenuated vaccinia (NYVAC) recombinant viruses.

Poxvirus recombinants, based on the highly attenuated NYVAC strain of vaccinia virus (Tartaglia et al., 1992), containing single gene inserts encoding the pseudorabies virus (PRV) gII, gIII, or gp50 glycoproteins were tested for their immunogenicity in pigs. Twenty-four pigs were randomly divided into six groups of four. Groups 1-3 were inoculated with 10(7) CCID50 of NYVAC/PRV gII, NYVAC/PRV gIII, or NYVAC/PRV gp50, respectively, while groups 4 and 5 received the NYVAC parent virus or an inactivated PRV vaccine control, respectively. Group 6 represented the sham vaccinated control group. All inoculations were given by the intramuscular route on weeks 0 and 4. The candidate vaccines were shown to be safe with no local or systemic reactions. At 4 weeks following the second inoculation, all pigs were challenged by an oronasal administration of a virulent PRV strain. Pigs were monitored before and after challenge for clinical manifestations resulting from vaccination and challenge exposure, respectively. Sera were analyzed for PRV neutralizing activity. Virological analyses after challenge included assessment of virus shedding and the development of latent PRV infections. All but one animal developed latent PRV infection following challenge exposure; however, significant protection against PRV-induced signs was afforded by vaccination with either the NYVAC/PRV gp50 or NYVAC/PRV gII recombinant viruses, as well as with the inactivated PRV vaccine. The NYVAC/PRV gp50 also reduced overall virus shedding after challenge. The extent of protection against PRV-induced clinical signs, in general, was associated with the level of pre-challenge virus neutralizing activity.

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of the specificity and activity of serum antibodies.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and serum antibody responses were analyzed. Compared with preinculation values, at Day 14 after two biweekly immunizations with modified-live or inactivated vaccines there were significant increases in BRSV-specific titers in the sera of cattle that received both types of vaccines, as determined by a whole cell ELISA. Using a blocking ELISA and radioimmune precipitation it was determined that there was recognition of the fusion (F) protein by antibodies from cattle that received both types of BRSV antigens: however, virus neutralization assays revealed that only cattle that received modified live virus, either in monovalent or polyvalent vaccines, developed neutralizing antibodies to BRSV after two immunizations. These results indicate that inactivation of BRSV can lead to a dissociation between serological recognition of the F protein and virus neutralization in vaccinated cattle.

Bovine immunodeficiency virus: immunochemical characterization and serological survey.

Bovine immunodeficiency virus (BIV) was purified by isodensity centrifugation; viral activities were monitored in gradient fractions using the reverse transcriptase assay and a p26-specific monoclonal antibody ELISA. In the coincident peak fractions (density about 1.17 g/ml) proteins with Mr values of 26K, 17K, 53K, 14K and 100K (with decreasing intensity) were detected by Western blotting using serum of a calf after experimental BIV infection. When 957 randomly collected cattle sera from The Netherlands were tested by indirect immunofluorescence and confirmed using Western blot and/or radioimmunoprecipitation, 1.4% appeared seropositive. Thus BIV infection is not uncommon in one European cattle population.