Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter.

A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV.

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses.

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.

Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice.

Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.

Induction of humoral and cellular immune responses in mice by a recombinant fowlpox virus expressing the E2 protein of bovine viral diarrhea virus.

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.

Prevalence of coronavirus antibodies in Iowa swine.

Three hundred and forty-seven serum samples from 22 Iowa swine herds were screened for TGEV/PRCV neutralizing antibody. Ninety-one percent of the sera and all 22 herds were positive. These sera were then tested by the blocking ELISA test to distinguish TGEV and PRCV antibody. The ELISA test confirmed the high percentage of TGEV/PRCV positive sera. By the blocking ELISA test, 12 herds were PRCV positive, 6 herds were TGEV positive and 4 herds were mixed with sera either positive for TGEV or PRCV antibody. The results suggest a recent increase in TGEV/PRCV seroprevalence in Iowa swine most likely due to subclinical PRCV infections.

Maternal immunization of pregnant cattle with recombinant VP8* protein of bovine rotavirus elicits neutralizing antibodies to multiple serotypes. Colostral neutralizing antibody by rotavirus VP8*.

Neonatal calf diarrhoea caused by bovine rotavirus is one of the most common diseases in cattle. VP8 portion of the rotavirus VP4 protein is known to contain neutralizing epitopes and hemagglutination activity. We expressed the VP8* portion of bovine rotavirus strain C486 (G6P1 serotype) in E. coli, and examined potential of the recombinant VP8* protein for induction of neutralizing antibody responses in host animals. One hundred twenty pregnant beef cows were selected and immunized eight weeks prior to parturition with the recombinant VP8* protein. Colostral and milk samples were collected 12 hours and 10 days post-calving, respectively, and the virus neutralizing titers elicited by the recombinant subunit antigen were determined by plaque reduction assay. Colostral antibody titres of the vaccinated group were significantly higher than those of the unvaccinated control group, and these titers were equivalent to the titers elicited by a commercial vaccine. While titers of the control group rapidly decreased to 220 after 10 days of calving, neutralizing titers in the milk of the vaccinees remained 510. Rabbit and bovine antibodies induced by the recombinant VP8* protein were also able to neutralize bovine rotavirus P5 serotype (B641) at significant level and P11 serotype (B223) moderately. Our results suggest that the E. coli-produced recombinant VP8* protein can be an useful subunit vaccine candidate to prevent rotavirus infection in new-born calves.

Immune response to vaccination with DNA encoding the bovine viral diarrhea virus major glycoprotein gp53 (E2).

Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle which has not been controlled by classical vaccination. The region encoding the BVDV major glycoprotein gp53 (E2) known to possess virus-neutralizing activity was cloned into a mammalian expression vector under the human cytomegalovirus (CMV) intermediate early promoter. Intramuscular and intradermal administration of the recombinant plasmid DNA into BALB/c mice induced BVDV gp53-specific antibody responses to both biotypes (cytopathic and noncytopathic) of BVDV genotype 1, and to cytopathic BVDV genotype 2. BVDV-neutralizing antibodies were generated against BVDV genotype 1 strains and they also persisted 6 months after the last injection.

Antigenic variation among bovine viral diarrhea virus (BVDV) strains and the role of different cell fixation methods in immunoassays.

Antigenic variation among 13 Quebec isolates of bovine viral diarrhea virus (BVDV), 4 reference strains and 2 American isolates were studied by peroxidase-linked antibody assay (PLA assay) and neutralization test (NT). The Quebec strains consisted of 3 isolates before 1993 and 10 isolates from 1993. In the PLA assay, we compared 2 different fixatives, acetone and formalin. Acetone-fixation allowed us to identify 6 groups from amongst the viruses tested. All the Quebec isolates were different from the reference strains. In addition, antigenic variation was detected between Quebec isolates obtained before and during 1993. However, PLA assays performed after formalin fixation did not detect these antigenic variations. Neutralization tests were carried out with 2 polyclonal antibodies (PAb) and 6 monoclonal antibodies (MAb). They were used to classify BVDV strains and isolates into 4 groups and 7 subgroups respectively. In conclusion, we demonstrated that the BVDV isolates from the 1993 outbreak in Quebec are antigenically different from reference strains and from isolates existing in Quebec before 1993. In addition, we have shown that 2 internationally used fixation-methods in PLA assay give different results. The usefulness of each method is discussed.

Restriction endonucleases whose sites are predictable from the amino acid sequence offer an improved strategy for typing bovine rotaviruses.

Variation in the third base of a codon hampers genotypic characterization, particularly of RNA viruses. Some restriction endonucleases, however, have a recognition site with a variable base at the third position and will always cleave when a certain amino acid pair occurs (such as glycine-proline for Sau96I and glutamic or aspartic acid followed by serine usually for HinfI). We developed a restriction fragment length polymorphism (RFLP) procedure based on these enzymes for P-typing bovine group A rotaviruses (BRV). Employing this procedure 20 BRV local strains, isolated in tissue culture as well as the original faecal sample, could be typed in one of three patterns. More variability was observed when restriction endonucleases were employed whose cleavage sites cannot be predicted from the amino acid sequence (TaqI and Tsp509I). These RFLP results agreed with the PCR-VP4 typing assay, neutralization tests, and nucleotide sequence analysis. RFLP with Sau96I and HinfI provided quick and objective P-typing of strains and could detect multiple genotypes in the same sample.

Evaluation of a protective immunity induced by an inactivated influenza H3N2 vaccine after an intratracheal challenge of pigs.

A challenge study was conducted to evaluate the safety and efficacy of an inactivated influenza H3N2 virus vaccine combined with Quil A/Alhydrogel mixture under controlled conditions in piglets. Twenty-four piglets from 12 sows were allocated to 2 groups; injected intramuscularly with 2 doses of the tested vaccine or with PBS at 2 wk intervals and challenged intratracheally with 105TCID50 of the H3N2 swine influenza virus 6 d after the 2nd immunization. Clinical and virological parameters were recorded for 4 d after the challenge. The use of the tested vaccine produced high serum hemagglutination-inhibition titers against the swine H3N2 strain virus. This strong immune response suppressed all clinical signs and viral shedding and reduced pulmonary lesions due to the challenge in the vaccinated group, without causing any secondary effects. Our results suggest that the serum HI titers correlated with the degree of protection induced by an inactivated swine influenza H3N2 vaccine.

Immunological response to recombinant VP8* subunit protein of bovine roravirus in pregnant cattle.

Bovine rotavirus VP8*, N-terminal trypsin cleavage product of VP4, was produced in Escherichia coli. To examine if this antigen could induce neutralizing antibody responses, different species of animals were immunized with the recombinant VP8* protein. The VP8* antigen was found to stimulate a neutralizing immune response in rabbits. When VP8*-immunized mice were exposed to bovine rotavirus strain C486, significantly higher antibody responses were observed than if they were only exposed to C486. To stimulate a current vaccination protocol in the field with livestock, mice were exposed to live C486 virus first and then to VP8*. These mice had the elevated immune responses indicating that VP8* could boost immunity in primed mice. The immune response to VP8* was also tested in pregnant cows. The efficacy of VP8* in stimulating milk antibody was compared with a commercial inactivated vaccine. Differences in colostral antibody titres between VP8*-vaccinated and unvaccinated cows were statistically significant (P < 0.05) and equivalent to the commercial vaccine (P = 0.0569). The milk antibody titres on day 10 were comparable between VP8*- and commercial vaccine-vaccinated animals and were significantly higher (P < 0.05) than in unvaccinated controls. Furthermore, rabbit and bovine antibodies induced by VP8* were able to neutralize different P types of bovine rotaviruses to varying degrees, suggesting that serotype-specific and cross-reactive epitope(s) are present on the VP8* protein of rotavirus. Taken together, E. coli-expressed VP8* may be useful as a subunit vaccine candidate for bovine rotavirus.

Persistence of a 1930 swine influenza A (H1N1) virus in Quebec.

Two antigenically distinct H1N1 influenza A viruses were isolated during an outbreak of respiratory disease in Quebec swine in 1990/91. Analysis of haemagglutinin and partial nucleoprotein sequences indicated that one was a variant of the swine H1N1 influenza virus circulating in the American Midwest whereas the other was very similar to virus isolated from swine in 1930. The existence of this latter isolate supports the concept that influenza viruses can be maintained for long periods in swine, perhaps in geographically limited pockets. Serological evidence indicates that these distinct strains continued to circulate widely in south-central Quebec until at least 1993.

Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein genes similar to 1975 human strains.

Of the four pandemic strains of human influenza A virus observed this century, the 1977 virus strain was very similar in all genes to a 1950 isolate. Since mammalian influenza A viruses change annually by genetic drift, this reappearance could only be attributed at that time to conservation of the virus in a frozen state. We report here the isolation of swine influenza A viruses with haemagglutinin and nucleoprotein genes which are virtually identical to those of the human virus that circulated in 1975. We have also found serological evidence that this virus is circulating extensively in Quebec swine herds. We propose that human-like H3N2 influenza A strains may remain invariant for long periods in swine, which may serve as a reservoir for human pandemics.

The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus.

The 5' untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5' UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.

Detection of porcine respiratory coronavirus and transmissible gastroenteritis virus by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was developed. A bovine TGEV-specific polyclonal antibody was purified by affinity chromatography with the TRIO Bioprocessing System and was used as the capture antibody, at a concentration of 1.5 micrograms/well. The F5.39 monoclonal antibody was obtained by the fusion of spleen lymphocytes from TGEV immunized mice with NS-1 myeloma cells. This mAb was used as a second antibody for the ELISA. The ELISA detected 40 ng of TGEV and 407 ng of PRCV. To study the ability of ELISA to detect TGEV in field cases, 53 intestinal samples were taken from pigs exhibiting clinical signs of transmissible gastroenteritis. All the positive samples detected by the ELISA were confirmed as positive by immunofluorescence or cell culture immunofluorescence. To study the ability of this ELISA to detect PRCV in nasal swabs and lung samples, 20 seven-day-old piglets were inoculated with a Quebec strain of PRCV. The ELISA was able to detect PRCV in both kinds of samples.

Antigenic characterization of an H3N2 swine influenza virus isolated from pigs with proliferative and necrotizing pneumonia in Quebec.

A new strain of swine influenza A virus, designated A/Swine/Saint-Hyacinthe/150/90 has been isolated from pigs with severe proliferative and necrotizing pneumonia in Quebec. The antigenic characterization of the hemagglutinin was performed by hemagglutination inhibition test, immunoblot and indirect immunoprecipitation using polyclonal antisera. Only the last test was able to detect an antigenic relationship between the hemagglutinin of this isolate and an H3 subtype influenza virus. The immunoprecipitation test was a useful alternative for determining the hemagglutinin of influenza A virus subtypes. The neuraminidase inhibition test demonstrated a reactivity between the A/Swine/Saint-Hyacinthe/150/90 and antiserum against a N2 subtype influenza virus. Our results indicate that this new strain isolated for the first time in the porcine population of Canada is related to A/Sw/Hong Kong/76 H3N2 swine influenza virus.

Pathogenicity of porcine respiratory coronavirus isolated in Québec.

Porcine respiratory coronavirus (PRCV) is present in many countries, including Canada, but controversy still exists concerning its pathogenicity. Eight-week-old piglets were inoculated intratracheally with a Quebec PRCV isolate (1Q90). Two contact piglets were kept with the inoculated animals. Three animals served as control. Polypnea and dyspnea were the main clinical signs observed. Diffuse bronchioloalveolar damage occurred 24 hours postinoculation. Changes compatible with bronchointerstitial pneumonia were present six days postinoculation. The inoculated virus was recovered from the respiratory tract and mesenteric lymph nodes, but not from the digestive tract, of the inoculated as well as the contact piglets. No virus was isolated from the control piglets. The development of clinical signs and histopathological changes in inoculated as well as in contact piglets and the reisolation of the inoculated virus demonstrated that PRCV can be an important respiratory pathogen.

[Bovine serum albumin inhibits the adsorption of respiratory syncytial virus on MDBK cells]. / La sérum albumine bovine inhibe l'adsorption du virus respiratoire syncytial sur les cellules MDBK.

In order to obtain optimal bovine respiratory syncytial virus adsorption to host cells, the effect of several products (fetal bovine serum, bovine serum albumin, ovalbumin and cytochrome c) was studied. The adsorption of the virus to MDBK cells was higher in the presence of 2% than in the presence of 5% fetal bovine serum. Adsorption was inhibited in the presence of bovine serum albumin at concentrations > 0.2% when added before or at the same time as the virus. Ovalbumin and cytochrome c did not inhibit adsorption. These results will allow the study of virus adsorption on cell receptors.

Bovine rotavirus type detection by neutralizing monoclonal antibodies.

A series of monoclonal antibodies were developed against bovine rotavirus Q17. Among the five high affinity antibodies characterized, two (RQ 31 and RQ 4) were able to neutralize type G 6 viruses and may be specific for PB 1 type virus. Seventy seven feces from diarrheic calves were tested by "double sandwich" ELISA using four monoclonal and one polyclonal anti-rotavirus antibodies. The combination of mono- and polyclonal antibodies thus appears to be a more efficient strategy for detection and typing of bovine rotaviruses.