Bovine herpesvirus-1: Genetic diversity of field strains from cattle with respiratory disease, genital, fetal disease and systemic neonatal disease and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes disease in cattle with varied clinical forms. In the U.S. there are two BoHV1 subtypes, BoHV-1.1 and BoHV-1.2b. Control programs in North America incorporate modified live (MLV) or killed (KV) viral vaccines. However, BoHV-1 strains continue to be isolated from diseased animals or fetuses after vaccination. It is possible to differentiate BoHV-1 wild-type from MLV vaccine strains by determining their single nucleotide polymorphism (SNP) patterns through either whole-genome sequencing or PCR sequencing of genomic regions containing vaccine-defining SNPs. To determine the BoHV-1 subtype in clinical isolates and their relationship to MLV strains, 8 isolates from varied clinical disease at three different laboratories in the U.S. were sequenced and phylogenetically analyzed. Five samples were isolated within the past 5 years from New York and 3 were archived samples recovered 35 years prior from Oklahoma and Louisiana. Based on phylogenetic analysis, four of the cases appeared to be due to an MLV vaccine: 3 cases of aborted fetuses and one neonate with systemic BoHV-1 disease. One aborted fetus was from a herd with no reported history of MLV vaccination in two years. The remaining four isolates did not group with any MLV vaccines: two were associated with bovine respiratory disease, one with vulvovaginitis, and a fourth was determined to be a BoHV-1.2b respiratory isolate. Recovery of BoHV-1.1 that is very closely related to an MLV vaccine virus from a herd not receiving vaccines in an extended period prior to its isolation suggests that MLV viruses may remain latent or circulate within herds for long periods.

Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease.

This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected from 114 cattle on initial BRD treatment. Processing included modified live virus (MLV) vaccination. Seven BRD necropsy cases were included for 121 total cases. Mean number of days on feed before first sample was 14.9 days. Swabs and tissue homogenates were tested by gel based PCR (G-PCR), quantitative-PCR (qPCR) and quantitative real time reverse transcriptase PCR (qRT-PCR) and viral culture. There were 87/114 (76.3%) swabs positive for at least one virus by at least one test. All necropsy cases were positive for at least one virus. Of 121 cases, positives included 18/121 (14.9%) BoHV-1; 19/121 (15.7%) BVDV; 76/121 (62.8%) BoCV; 11/121 (9.1%) BRSV; and 10/121 (8.3%) PI3V. For nasal swabs, G-PCR (5 viruses) detected 44/114 (38.6%); q-PCR and qRT-PCR (4 viruses) detected 81/114 (71.6%); and virus isolation detected 40/114 (35.1%). Most were positive for only one or two tests, but not all three tests. Necropsy cases had positives: 5/7 G-PCR, 5/7 q-PCR and qRT-PCR, and all were positive by cell culture. In some cases, G-PCR and both real time PCR were negative for BoHV-1, BVDV, and PI3V in samples positive by culture. PCR did not differentiate field from vaccines strains of BoHV-1, BVDV, and PI3V. However based on sequencing and analysis, field and vaccine strains of culture positive BoHV-1, BoCV, BVDV, and PI3V, 11/18 (61.1%) of BoHV-1 isolates, 6/17 (35.3%) BVDV isolates, and 1/10 (10.0%) PI3V identified as vaccine. BRSV was only identified by PCR testing. Interpretation of laboratory tests is appropriate as molecular based tests and virus isolation cannot separate field from vaccine strains. Additional testing using sequencing appears appropriate for identifying vaccine strains.

Bovine herpesvirus-1: evaluation of genetic diversity of subtypes derived from field strains of varied clinical syndromes and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Bovine herpesvirus-1: comparison and differentiation of vaccine and field strains based on genomic sequence variation.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle including respiratory, fetal diseases, and reproductive tract infections. Control programs usually include vaccination with a modified live viral (MLV) vaccine. On occasion BoHV-1 strains are isolated from diseased animals or fetuses postvaccination. Currently there are no markers for differentiating MLV strains from field strains of BoHV-1. In this study several BoHV-1 strains were sequenced using whole-genome sequencing technologies and the data analyzed to identify single nucleotide polymorphisms (SNPs). Strains sequenced included the reference BoHV-1 Cooper strain (GenBank Accession JX898220), eight commercial MLV vaccine strains, and 14 field strains from cases presented for diagnosis. Based on SNP analyses, the viruses could be classified into groups having similar SNP patterns. The eight MLV strains could be differentiated from one another although some were closely related to each other. A number of field strains isolated from animals with a history of prior vaccination had SNP patterns similar to specific MLV viruses, while other field isolates were very distinct from all vaccine strains. The results indicate that some BoHV-1 isolates from clinically ill cattle/fetuses can be associated with a prior MLV vaccination history, but more information is needed on the rate of BoHV-1 genome sequence change before irrefutable associations can be drawn.

Bovine viral diarrhea virus cytopathic and noncytopathic biotypes and type 1 and 2 genotypes in diagnostic laboratory accessions: clinical and necropsy samples from cattle.

One hundred three bovine samples submitted to the Oklahoma Animal Disease Diagnostic Laboratory (OADDL) that were positive for bovine viral diarrhea virus (BVDV) were typed by a nested reverse transcription-polymerase chain reaction for BVDV genotypes. These BVDV samples included supernatants from virus isolation (79), serums (17), and buffy coats (7). The biotype, cytopathic (CP) or noncytopathic (NCP), was determined by cell culture virus isolation. Twenty-eight of 103 samples were submitted for herd screening for BVDV, 32 from OADDL necropsy cases, and 43 from live cattle with varied clinical conditions. Two samples contained 2 bands indicating presence of both BVDV types 1 and 2. Of the 105 BVDV samples, 26 were type 1 CP strains (24.8%), 38 were type 1 NCP strains (36.2%), 10 were type 2 CP strains (9.5%), and 31 were type 2 NCP strains (29.5%). From the 105 BVDV isolates, NCP biotypes were isolated more frequently (69, 65.7%) than CP biotypes (36, 34.3%), and type 1 genotypes were more frequently isolated (64, 61.00%) than type 2 genotypes (41, 39.0%). The NCP strains were more common than CP in herd screening samples. Cattle with respiratory disease history at time of sampling had more NCP than CP biotypes and more type 1 than type 2 genotypes. Of the necropsy cases, more were type 1 than type 2 genotypes for the respiratory cases with fibrinous pneumonia, more were type 1 than type 2 genotypes in cattle with enteritis/colitis without systemic lesions, and more were CP than NCP biotypes in cattle with enteritis/colitis with systemic lesions. No CP biotype was isolated from serum samples.

Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus.

A nested reverse transcription (RT) polymerase chain reaction (PCR) assay was evaluated for differentiating reference bovine viral diarrhea virus (BVDV) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BDV). The detection level of this assay was compared to viral infection in cell culture. The PCR assay was used to distinguish 3 ruminant pestiviruses, types 1 and 2 BVDV, and type 3 BDV. The consensus (first) PCR assay detected all 3 ruminant pestiviruses, a result of the shared sequence homology. The consensus PCR product was subjected to a second (nested) PCR which used type-specific primers. The nested PCR was able to differentiate the 3 ruminant pestiviruses. Viral stocks of BVDV were diluted 10-fold and processed for the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compared to viral infectivity in cell culture based on identical volumes of the system tested (cell culture assay and processing for RNA). The RT-PCR type-specific assay differentiated BVDV laboratory reference strains (12), diagnostic laboratory isolates (15), 2 MLV BVDV vaccine strains, and a BDV strain. The 30 ruminant pestiviruses typed included: (1) 27 reference strains and diagnostic laboratory isolates; 18 cytopathic (CP) type 1 strains, 3 CP type 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strains; (2) 2 MLV strains, type 1; and (3) 1 CP BDV type 3. The PCR assay had a detection limit of 10 TCID50/0.025 mL of virus when 3 separate BVDV were tested. This 2 step RT-PCR assay would be useful for the typing of ruminant pestiviruses, particularly BVDV isolates from the diagnostic laboratory.

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea viruses: detection by inhibition of viral cytopathology and infectivity by immunoperoxidase assay.

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea virus (BVDV) strains were measured by a microtiter virus neutralization test (MVNT) in cell culture. Antibodies (neutralizing) were detected by inhibition of viral infectivity, by the absence of viral cytopathology for cytopathic strains, or by immunoperoxidase staining for noncytopathic strains. The immunoperoxidase-stained monolayers could be detected without the aid of light microscopy. Twenty BVDV strains were used as challenge viruses in the in vitro MVNT, including 14 type 1 and 6 type 2 strains. Representative noncytopathic and cytopathic strains of both types were used. Positive control serum samples available for diagnostic testing contained both type 1 and type 2 BVDV antibodies. There did not appear to be major differences in antibody titers among the respective type strains, regardless of biotype (cytopathic or noncytopathic). In a study with sera from calves receiving a modified live virus or inactivated BVDV vaccine, the calves receiving type 1 strains responded with higher antibody titers to type 1 strains than to type 2 strains.

Effects of B vitamin injection on bovine herpesvirus-1 infection and immunity in feed-restricted beef calves.

Because feed and water deprivation during marketing and transport of feedlot calves may reduce ruminal B vitamin synthesis at a time when calves are most susceptible to infectious agents, we studied the effect of B vitamin injections on infection and immunity in 12 6-mo-old beef steer calves (153 +/- 8 kg) that were weaned, limit-fed, and deprived of feed. Six calves were injected with B vitamins and ascorbic acid every 48 h for 28 d starting 2 wk before virus inoculation. All calves were infected with an attenuated strain of bovine herpesvirus type 1 (BHV-1) on d 0. From time of arrival (d -20) until the end of a 3-d period without food (d -6), calves lost 13.1% of their initial weight. However, they regained weight after re-feeding so that net weight loss was 7.7% for the 20-d period prior to infection. The stress/BHV-1 model resulted in a mild respiratory infection in all calves with no difference observed between treatment groups. Vitamin injections did not significantly affect virus and interferon titers in nasal secretions, or lymphocyte blastogenesis. However, the B vitamin treatment tended to increase serum IgG titers to BHV-1 on both d 14 (1,120 vs 550, P = .115) and d 28 (2,400 vs 1,830, P = .37) after infection. Averaged across d 14 and d 28, IgG titers tended to be higher (P < .09) for the calves receiving B vitamin injections, indicating that the humoral immune response was enhanced by B vitamin treatment. B vitamin status in stressed calves at the time of vaccination or disease challenge may affect the success of the immune response.

Antibody responses by cattle after vaccination with commercial viral vaccines containing bovine herpesvirus-1, bovine viral diarrhea virus, parainfluenza-3 virus, and bovine respiratory syncytial virus immunogens and subsequent revaccination at day 140.

Calves were vaccinated with four different commercial viral vaccines containing bovine herpesvirus-1 (BHV-1), bovine viral diarrhea (BVDV), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) immunogens. For the initial vaccination certain vaccines were given twice (days 0 and 28), whereas other vaccines were given on day 0. The calves received another injection on day 140 with the vaccine originally given on day 0. The sera were collected at days 0, 7, 14, 21, 28, 42, 56, 84, 112, 140, 154, 168, and 196 and assayed for viral neutralizing antibodies. The calves were seronegative to BHV-1, BVDV, and BRSV at the onset of the experiment; however, the calves were PI-3V antibody positive due to prior active infection. The commercial vaccines were: (I) inactivated; (II) modified live virus (MLV); (III) combination of chemically altered live virus, MLV, and inactivated virus; and (IV) combination of inactivated and MLV. Among the vaccine groups there were differences in onset and duration of antibodies as measured by geometric mean titers to each immunogen in postvaccination collection dates compared to day 0 titers; and likewise compared to day 140 titers after revaccination at day 140. There were also differences in antibody titers to the various viruses among the vaccine groups on specific collection dates. All four vaccines induced increased BHV-1 antibodies by day 14 after the initial injection. The antibody titers induced by MLV BHV-1 and the chemically altered BHV-1 vaccines had greater duration than those induced by the inactivated vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of encephalitic bovine herpesvirus type 1 isolates from cattle in North America.

Nine CNS bovine herpesvirus type 1 (BHV-1) isolates, recovered from bovine brain samples submitted to the Texas Veterinary Medical Diagnostic Laboratories from 1974-1989, were compared by analyzing their DNA restriction endonuclease (RE) fragment migration pattern. Seven had pattern similar to that of the respiratory BHV-1 Cooper strain. The remaining 2 isolates, however, had variant patterns, similar to that of each other, but completely different from patients for the other 7. The RE patterns of these 2 variants were similar to published RE patterns for 2 encephalitic or neuropathogenic BHV-1 strains--the Australian N-569 strain and the Argentine A-663 strain. One of the Texas encephalitic variants (No. 30326) was isolated from the CNS of a calf that died during an epizootic of encephalitis in 1974. The other, designated TX-89, was isolated in 1989 from the CNS of a 7-month-old feedlot steer with acute fatal encephalitis. Microscopic lesions of encephalitis with neuronal degeneration and intranuclear inclusions were observed for 3 of the 9 isolates, the 2 variant isolates (No. 30326 and TX-89), and a respiratory isolate. The remaining 6 CNS isolates, all respiratory subtypes, were recovered from cattle that did not have clinical CNS disease or gross or microscopic CNS lesions; in 5 of these cattle, virus was recovered from at least 1 other organ (lungs) besides the CNS. We conclude that the CNS of calves can be naturally infected with 2 distinct BHV-1 subtypes, the respiratory and the encephalitic, and that the encephalitic subtype (subtype 3 or BHV-1.3) has been present in Texas cattle since at least 1974.

Combined effects of fasting and diet on interferon production and virus replication in calves infected with a vaccine strain of infectious bovine rhinotracheitis virus.

A study was undertaken to investigate the combined effects of fasting and different diets on interferon (IFN) production and virus replication measured in nasal secretions of calves inoculated with a vaccine strain of infectious bovine rhinotracheitis virus. Four groups of calves were inoculated intranasally with infectious bovine rhinotracheitis virus. Two groups were inoculated 24 hours after onset of a 3-day fast; upon refeeding, 1 group was fed a maintenance diet (M diet) of hay, and the other was fed a higher energy diet (HE diet) of hay and concentrate. Nonfasted control groups were fed the M diet or the HE diet. Overall IFN production was highest (P less than 0.01) in nonfasted calves fed the M diet throughout the study and lowest in nonfasted calves fed the HE diet. Fasted calves refed the HE diet produced consistently and significantly more IFN than did nonfasted calves fed this diet. Fasted calves refed the M diet, however, produced significantly less IFN, compared with control calves fed the M diet throughout the study. Overall mean virus excretion was similar in all groups; therefore, the amount of virus replication per se did not account for the differences in IFN production, nor did greater IFN production result in less virus excretion. Serum cortisol concentrations and immune responses were not significantly affected by fasting or diet.

Interferon production and replication of infectious bovine rhinotracheitis virus in fasted calves.

Three groups of calves were inoculated intranasally with infectious bovine rhinotracheitis (IBR) virus, to determine any effects of a 3-day fast on virus replication and interferon (IFN) production measured in nasal secretions (NS). One group ("fasted") was inoculated 24 h after onset of the fast and another ("refed") at the end of fasting, immediately before refeeding. A third ("control") group was inoculated but not fasted. In fasted calves, overall mean virus excretion (during the first 5 postinoculation days) did not differ from that in control calves, though average virus excretion was higher on days 3 and 4, 24 and 48 h after refeeding. In refed calves, overall mean virus excretion was lower (p less than 0.05), yet on day 5 these calves secreted two times more IFN than nonfasted calves. Analysis of the overall data (all 5 postinoculation sampling days) showed that fasted calves produced more IFN (p less than 0.05), with IFN titers sometimes exceeding 1000, than either control nonfasted calves or refed calves. We conclude that fasting enhanced the ability of calves to produce IFN, and this did not result from increased IBR virus replication.