Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and fetal bovine serum lots originating in Mexico.

In this communication, we report the presence of RNA of bovine viral diarrhea virus (BVDV) as a contaminant of different biological products used in Mexico for veterinary vaccine production. For this purpose, six batches of monovalent vaccines, eight cell line batches used for vaccine production, and 10 fetal bovine serum lots (FBS) commercially available in Mexico from different suppliers were tested by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was detected in 62.5% of the samples analyzed. Phylogenetic analysis revealed the presence of the subgenotypes BVDV-1a, 1b, and BVDV-2a in the tested samples. Collectively, these findings indicate that contamination by BVDV RNA occurs in commercial vaccines and reagents used in research and production of biological products. The ramifications of this contamination are discussed.

Identification and genotyping of a new subtype of bovine viral diarrhea virus 1 isolated from cattle with diarrhea.

In 2019, diarrhea cases occurred on cattle farms in Qionglai and Guang'an, Sichuan Province. Two out of 20 (10%) serum and nasal swab samples were positive when tested using a bovine viral diarrhea virus (BVDV) antigen-capture ELISA kit. Two non-cytopathic strains of BVDV were isolated and named QL1903 and GA190608, respectively. The nucleotide sequences of the genomes of the two isolates were 89.52% identical. Phylogenetic analysis based on the 5'-UTR sequence revealed that the BVDV isolate QL1903 belonged to BVDV subtype 1b, whereas isolate GA190608 clustered with strains HN1814, EN-19, and BJ09_26 in a separate branch, which has tentatively been classified as a new genetic subtype, "1v".

Experimental infection of Korean native goats (Capra aegagrus hircus) with bovine viral diarrhea virus 1b.

BACKGROUND: Bovine viral diarrhea virus (BVDV) infects various ungulates and causes reproductive failure in infected goats. BVDV has been detected among goats in the Republic of Korea, but the route of transmission remains unclear. Here, we aimed to investigate whether BVDV-1b circulating among Korean cattle can be transmitted to Korean native goats (Capra aegagrus hircus) and characterize the outcomes of BVDV infection in these goats. RESULTS: Four goats were inoculated intranasally with the Korean noncytopathic (ncp) BVDV-1b strain. Two goats exhibited clinical signs of illness, including coughing and nasal discharge. Nasal swabs and blood were collected to screen for viral RNA and BVDV antibodies. Using the 5'-untranslated region (UTR), viral RNA was detected in the nasal swabs of two goats (Goat 1 and 3) on 12 day post-inoculation (dpi) and in the blood sample of one goat (Goat 1) on 7 and 19 dpi. Using the N-terminal protease (Npro) region, viral RNA was detected in the blood sample of Goat 1 on 7 and 12 dpi. Antibodies to BVDV were detected in Goats 1 and 3 on 16-21 dpi using enzyme-linked immunosorbent assay. Sequence analysis of the virus from nasal swabs and blood samples, which was detected via RT-PCR, using the 5'-UTR and Npro regions led to the identification of the strain as ncp BVDV-1b and revealed changes in the nucleotide sequence of these goats. CONCLUSIONS: Our results indicate that changes in the nucleotide sequence are associated with the establishment of BVDV infection in Korean native goats; these changes may be owing to a process required for the establishment of infection in a new host reservoir. Broadly, these findings highlight the importance of BVDV surveillance in ungulates other than cattle.

Prevalence of bovine viral diarrhea virus in dairy cattle herds in eastern China.

Bovine viral diarrhea virus (BVDV) is a worldwide spreading pestivirus affecting cattle and other ruminants; however, there have been few reports on epidemiologic investigation of BVDV in eastern China. In this study, bulk tank milk from 36 herds of dairy cattle in eastern China was submitted to serological investigations, 77.8% of herds was BVDV antibody positive. Individual animal status in two herds was further investigated collecting blood samples, the positive ratio was 49.74% and 24.64%, and the average positive ratio of calves, heifers, and lactating cows was 15.94%, 40.16%, and 41.7%, respectively. Moreover, clinical survey was carried out among 8170 dairy cattle from 36 herds, for diarrhea syndrome, respiratory problems and reproductive failure, and pathogens of all clinical cattle were further investigated. The results showed that BVDV was one of the main pathogen, which infected animals combining with various other viruses. Then, nine BVDV strains were isolated; phylogenetic analysis showed that BVDV subtypes currently circulating in eastern China were BVDV 1a and BVDV 1c. In addition, out of 377 cows tested, the 1.86% detected positive to the BVDV antigen. This study provided the foundation of further study on vaccination and control strategies of BVDV in eastern China.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Detection of persistent pestivirus infection in pudú (Pudu puda) in a captive population of artiodactyls in Chile.

BACKGROUND: Bovine Viral Diarrhea Virus (BVDV) is the viral agent causing the most important economic losses in livestock throughout the world. Infection of fetuses before their immunological maturity causes the birth of animals persistently infected with BVDV (PI), which are the main source of infection and maintenance of this pathogen in a herd. There is evidence of susceptibility to infection with BVDV in more than 50 species of the order Artiodactyla, and the ability to establish persistent infection in wild cervid species of South America could represent an important risk in control and eradication programs of BVDV in cattle, and a threat to conservation of these wild species. In this study, a serological and virological study was performed to detect BVDV infection in a captive population of non-bovine artiodactyl species in a Chilean zoo with antecedents of abortions whose pathology suggests an infectious etiology. RESULTS: Detection of neutralizing antibodies against BVDV was performed in 112 artiodactyl animals from a zoo in Chile. Three alpacas (Vicugna pacos), one guanaco (Lama guanicoe) and seven pudús (Pudu puda) resulted seropositive, and the only seronegative pudú was suspected to be persistently infected with BVDV. Then two blood samples nine months apart were analyzed by a viral neutralization test and RT-PCR. Non-cytopathogenic BVDVs were isolated in both samples. A phylogenetic analysis showed that the virus was highly related to BVDV-1b strains circulating among Chilean cattle. CONCLUSIONS: This is the first report of a South American deer persistently infected with Bovine Viral Diarrhea Virus. Further studies are needed to determine the possible role of BVDV as a pathogen in pudús and as a threat to their conservation.

Bovine viral diarrhea virus subgenotype 1b in water buffalos (Bubalus bubalis) from Brazil.

Serological studies have characterized the presence of the bovine viral diarrhea virus (BVDV) infection in water buffalo herds worldwide. However, the molecular characterization of BVDV strains circulating in this animal species is uncommon. The aim of this study was to identify young water buffalo with acute infection and characterize the subgenotype of the infecting wild-type BVDV strain. Two dairy water buffalo herds from Northeastern Brazil were selected based on the results of virus neutralization test which showed high titers of anti-BVDV antibodies. To identify viremic animals, the BVDV RNA was assessed by RT-PCR assay in serum samples from 44 asymptomatic young water buffalos, where 31 serum samples from herd A and 13 from herd B. Amplicons with 288 bp of BVDV 5'UTR region were obtained in 7 (15.9%) serum samples (herd A, n = 5; herd B, n = 2). One good-quality amplicon from each herd was selected for nucleotide sequencing. The phylogenetic analysis demonstrated that the two BVDV wild-type strains clustered with BVDV strains of the subgenotype 1b. This study identified for the first time the active infection by BVDV subgenotype 1b in two dairy water buffalo herds from Brazil. These results highlight the importance of that, as well as in cattle herds, also in water buffalo herds prophylaxis measures to control BVDV infection should be intensified, mainly because these species clearly coexist in buffalo farms within Brazil.

Prevention of fetal infection in heifers challenged with bovine viral diarrhoea virus type 1a by vaccination with a type 1c or type 1a vaccine.

AIMS: To evaluate a vaccine containing type 1c bovine viral diarrhoea (BVD) virus for prevention of fetal infection in pregnant heifers when challenged with New Zealand BVD virus type 1a 6 months after vaccination, compared to unvaccinated heifers and heifers vaccinated with a vaccine containing type 1a BVD virus. METHODS: Fifty five crossbred Friesian heifers, free from BVD virus and antibody, were randomly allocated to three groups. Twenty five heifers were vaccinated twice with a vaccine containing type 1c BVD virus (T1c group), and 10 heifers with a vaccine containing type 1a BVD virus (T1a group), and 20 heifers were unvaccinated (NC group). After oestrus synchronisation the heifers were bred by artificial insemination followed by natural bull mating. Six months after booster vaccination 15 heifers from the T1c group, eight from the T1a group, and 15 from the NC group, were exposed to four calves that were persistently infected with type 1a BVD virus, for 4 weeks. At the beginning of the challenge phase 36/38 heifers were 72-74 days pregnant and 2/38 heifers were approximately 53 days pregnant. Approximately 52 days after the start of the challenge the heifers were subjected to euthanasia and fetal tissues were collected for the detection of BVD virus by ELISA in fetal heart blood and PCR in fetal tissues. RESULTS: Based on PCR results, BVD virus was detected in 15/15 fetuses in the NC group, compared to 4/14 fetuses in the T1c group and 3/8 fetuses in the T1a group. The proportion of BVD virus-positive fetuses was lower in both vaccinated groups compared to the NC group (p<0.002), but there was no difference in proportions between the vaccinated groups (p=1.00). Fetal protection, expressed as the prevented fraction, was 71.4 (95% CI=41.9-91.6)% and 62.5 (95% CI=24.5-91.5)% for the T1c and T1a groups, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The vaccines containing killed type 1c and type 1a BVD viruses significantly reduced fetal infection following challenge with a New Zealand type 1a BVD virus. Prevention of fetal infection by vaccination may not be 100%, and the risk of persistently infected calves being born to some vaccinated cattle should be acknowledged and managed as part of a BVD control programme.

Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self.

Bovine viral diarrhea virus (BVDV) infection in dairy cattle herds in northeast Thailand.

Bovine viral diarrhea virus causes a wide range of clinical manifestation with subsequent economic losses in dairy production worldwide. Our study of a population of dairy cattle in Thailand based on 933 bulk tank milk samples from nine public milk collection centers aimed to monitor infective status and to evaluate the effect of the infection in cows as well as to examine the reproductive performance of heifers to provide effective recommendations for disease control in Thailand. The results showed a moderate antibody-positive prevalence in the herd (62.5 %), with the proportion of class-3 herd, actively infected stage, being 17.3 %. Fourteen persistently infected (PI) animals were identified among 1196 young animals from the class-3 herds. Most of the identified PI animals, 11/14, were born in one sub-area where bovine viral diarrhea virus (BVDV) investigation has not been performed to date. With respect to reproductive performance, class-3 herds also showed higher median values of reproductive indices than those of class-0 herds. Cows and heifers in class-3 herds had higher odds ratio of calving interval (CI) and age at first service (AFS) above the median, respectively, compared to class-0 herds (OR = 1.29; P = 0.02 and OR = 1.63; P = 0.02). Our study showed that PI animals were still in the area that was previously studied. Furthermore, a newly studied area had a high prevalence of BVDV infection and the infection affected the reproductive performance of cows and heifers. Although 37.5 % of the population was free of BVDV, the lack of official disease prevention and less awareness of herd biosecurity may have resulted in continuing viral spread and silent economic losses have potentially occurred due to BVDV. We found that BVDV is still circulating in the region and, hence, a national control program is required.

Role of thymic epithelial cells in lymphoid depletion after experimental infection with the noncytopathogenic BVDV1 strain 7443.

Thymic epithelial cells could play an important role in lymphoid depletion during bovine viral diarrhea virus (BVDV) infection. To evaluate this hypothesis, we examined proliferation of lymphocytes, expression of cytokeratins by thymic epithelial cells, and ultrastructural features at sequential time points after experimental infection of colostrum-deprived calves with the noncytopathogenic BVDV1 strain 7443. Ten clinically healthy Friesian calves were used. Eight were inoculated with the virus, and 2 were used as uninfected controls. Calves were sedated and euthanized in batches between 3 and 14 days postinoculation. At necropsy, thymus samples were collected for structural, immunohistochemical, and ultrastructural study. Thymic lymphoid depletion was accompanied by a decrease in lymphocyte proliferation and immunohistochemical and ultrastructural changes in thymic epithelial cells. Immunohistochemical and ultrastructural results reflect a disturbance of the thymic epithelial cell network, which may explain the decrease in lymphocyte proliferation by defective thymocyte-epithelial cell interactions.

Effects of Preinfection With Bovine Viral Diarrhea Virus on Immune Cells From the Lungs of Calves Inoculated With Bovine Herpesvirus 1.1.

The aim of this work was to study the interstitial aggregates of immune cells observed in pulmonary parenchyma of calves preinfected with bovine viral diarrhea virus and challenged later with bovine herpesvirus 1. In addition, the intent of this research was to clarify the role of bovine viral diarrhea virus in local cell-mediated immunity and potentially in predisposing animals to bovine respiratory disease complex. Twelve Friesian calves, aged 8 to 9 months, were inoculated with noncytopathic bovine viral diarrhea virus genotype 1. Ten were subsequently challenged with bovine herpesvirus 1 and euthanized at 1, 2, 4, 7, or 14 days postinoculation. The other 2 calves were euthanized prior to the second inoculation. Another cohort of 10 calves was inoculated only with bovine herpesvirus 1 and then were euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Pulmonary lesions were evaluated in all animals, while quantitative and biosynthetic changes in immune cells were concurrently examined immunohistochemically to compare coinfected calves and calves challenged only with bovine herpesvirus 1. Calves preinfected with bovine viral diarrhea virus demonstrated moderate respiratory clinical signs and histopathologic evidence of interstitial pneumonia with aggregates of mononuclear cells, which predominated at 4 days postinoculation. Furthermore, this group of animals was noted to have a suppression of interleukin-10 and associated alterations in the Th1-driven cytokine response in the lungs, as well as inhibition of the response of CD8+ and CD4+ T lymphocytes against bovine herpesvirus 1. These findings suggest that bovine viral diarrhea virus preinfection could affect the regulation of the immune response as modulated by regulatory T cells, as well as impair local cell-mediated immunity to secondary respiratory pathogens.

Comparison of reproductive performance of primiparous dairy cattle following revaccination with either modified-live or killed multivalent viral vaccines in early lactation.

The objective of this randomized clinical trial was to compare the effect of revaccination in primiparous dairy cows with modified live viral (MLV) or killed viral (KV) vaccines containing bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) on (1) pregnancy rate following estrus synchronization-timed artificial insemination (TAI), (2) serum progesterone concentrations, and (3) serum neutralizing antibody titers at revaccination and at TAI. Primiparous dairy cows (n=692) that had been previously vaccinated with 4 doses of MLV vaccine as calves or heifers were randomized to receive either an MLV or a KV vaccine between 21 and 28 d in milk and 17 d before initiation of a double-Ovsynch-TAI protocol. Serum was collected within the double-Ovsynch protocol for determination of progesterone concentrations, and at vaccination and TAI for serum neutralizing antibody titers. Ultrasound pregnancy determinations were made at 30 and 60 d after TAI. No differences in pregnancy rates were observed between cows receiving MLV vaccine (44%; n=326) or KV vaccine (43%; n=336). No differences were observed in serum progesterone concentrations during a double-Ovsynch-TAI protocol between cows receiving MLV and KV vaccines. No differences were observed in BVDV 1 or BVDV 2 antibody titers at vaccination and TAI between cows receiving MLV or KV vaccine; however, BoHV-1 antibody titers were greater at TAI in cows receiving KV vaccine. Overall response to vaccination-defined as the percent of all individual cows that had any detectable increase in antibody titer from vaccination to TAI-was 39% for BVDV 1, 45% for BVDV 2, and 61% for BoHV-1. In this research, use of an MLV vaccine did not impede reproduction when revaccination was performed between 21 and 28 DIM and just before enrollment in an estrus synchronization-TAI program in primiparous dairy cows; however, response to vaccination as defined by increases in virus-specific antibody titers could be considered less than ideal for this population of cattle.

Molecular analyses detect natural coinfection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) in serologically negative animals.

Infection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) has been confirmed in several studies by serological and molecular techniques. In order to determine the presence of persistently infected animals and circulating species and subtypes of BVDV we conducted this study on a buffalo herd, whose habitat was shared with bovine cattle (Bossp.). Our serological results showed a high level of positivity for BVDV-1 and BVDV-2 within the buffalo herd. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in the buffaloes. Cloning and sequencing of the 5' UTR of some of these samples revealed the presence of naturally mix-infected buffaloes with at least two different subtypes (1a and 1b), and also with both BVDV species (BVDV-1 and BVDV-2).

The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV.

BACKGROUND: Dendritic cells (DC) are important antigen presentation cells that monitor, process, and present antigen to T cells. Viruses that infect DC can have a devastating impact on the immune system. In this study, the ability of bovine viral diarrhea virus (BVDV) to replicate and produce infectious virus in monocyte-derived dendritic cells (Mo-DC) and monocytes was studied. The study also examined the effect of BVDV infection on Mo-DC expression of cell surface markers, including MHCI, MHCII, and CD86, which are critical for DC function in immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from bovine blood through gradient centrifugation. The adherent monocytes were isolated from PBMCs and differentiated into Mo-DC using bovine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GMCSF). To determine the effect of BVDV on Mo-DC, four strains of BVDV were used including the severe acute non-cytopathic (ncp) BVDV2a-1373; moderate acute ncp BVDV2a 28508-5; and a homologous virus pair [i.e., cytopathic (cp) BVDV1b TGAC and ncp BVDV1b TGAN]. The Cooper strain of bovine herpesvirus 1 (BHV1) was used as the control virus. Mo-DC were infected with one of the BVDV strains or BHV-1 and were subsequently examined for virus replication, virus production, and the effect on MHCI, MHCII, and CD86 expression. RESULTS: The ability of monocytes to produce infectious virus reduced as monocytes differentiated to Mo-DC, and was completely lost at 120 hours of maturation. Interestingly, viral RNA increased throughout the course of infection in Mo-DC, and the viral non-structural (NS5A) and envelope (E2) proteins were expressed. The ncp strains of BVDV down-regulated while cp strain up-regulated the expression of the MHCI, MHCII, and CD86 on Mo-DC. CONCLUSIONS: The study revealed that the ability of Mo-DC to produce infectious virus was reduced with its differentiation from monocytes to Mo-DC. The inability to produce infectious virus may be due to a hindrance of virus packaging or release mechanisms. Additionally, the study demonstrated that ncp BVDV down-regulated and cp BVDV up-regulated the expression of Mo-DC cell surface markers MHCI, MHCII, and CD86, which are important in the mounting of immune responses.

Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

Outbreak of persistently infected heifer calves with bovine viral diarrhea virus subgenotypes 1b and 1d in a BVDV-vaccinated open dairy herd.

Bovine viral diarrhea virus (BVDV) infection has a significant economic impact on beef and dairy industries worldwide. Fetal infection with a non-cytopathic strain may lead to the birth of persistently infected (PI) offspring, which is the main event in the epidemiological chain of BVDV infection. This report describes the birth of 99 BVDV-PI heifer calves within 52 days of birth in a regular BVDV-vaccinated Brazilian dairy cattle herd and the subgenotypes of the infecting field strains. This study was conducted in a high-yielding open dairy cattle herd that frequently acquired heifers from neighboring areas for replacement. The farm monitors the birth of PI calves by screening all calves born using an ELISA (IDEXX) for BVDV antigen detection. All calves aged 1-7 days were evaluated. For positive and suspected results, the ELISA was repeated when the calves were close to one month old. A total of 294 heifer calves were evaluated between February and March 2021. Of these, 99 (33.7 %) had positive ELISA results and were considered PI calves. To evaluate the predominant BVDV species and subgenotypes in this outbreak, whole blood samples were collected from 31 calves born during the study period. All samples were submitted to the RT-PCR assay for the partial amplification of the BVDV 5'-UTR region, and these amplicons were subjected to nucleotide sequencing. Phylogenetic analysis identified BVDV-1b and BVDV-1d in 16 and 13 heifer calves, respectively. In two calves, it was not possible to determine the BVDV-1 subgenotype. Detection of PI animals and monitoring of circulating BVDV subgenotype strains are central to disease control. This study shows that regular BVDV vaccination alone may be insufficient to prevent BVDV infection in high-yielding open dairy cattle herds. Other biosecurity measures must be adopted to avoid the purchase of cattle with acute infections by BVDV or BVDV-PI, which can cause a break in the health profile of the herd and economic losses.

Influence of bovine pestivirus heterogeneity on serological responses to 10 different commercial vaccine formulation.

Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system's ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6-9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H.