Characterization of bovine respiratory syncytial virus isolated in Brazil

This paper presents the first isolation of bovine respiratory syncytial virus in Brazil and its physicochemical, morphological and molecular characterization. The virus was isolated from 33 samples of nasotracheal secretions, successively inoculated into a Madin-Darby bovine kidney cell culture, which was characterized by physicochemical tests and morphological observation by electron microscopy. The Brazilian sample is an RNA pleomorphic, enveloped, thermolabile and non-hemagglutinating spicular virus. Reverse transcription, followed by nested polymerase chain reaction (nRT-PCR) assay was carried out using oligonucleotides B1, B2A, B3 and B4 for the fusion proteins (F) and B5A, B6A, B7A and B8 for the attachment protein (G). The nRT-PCR-F amplified a fragment of 481 bp corresponding to part of the gene that codes for protein F, whereas nRT-PCR-G amplified a fragment of 371 bp, in agreement with part of the G gene. The virus isolated from Brazilian samples in this study corresponded to the bovine respiratory syncytial virus, and RT-PCR proved to be useful for the diagnosis of bovine clinical samples.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-la Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-la NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model. (C) 2011 Elsevier Ltd. All rights reserved.

Detection of antibodies against bovine respiratory syncytial virus (BRSV) in dairy cattle with different prevalences of bovine herpesvirus type 1 (BoHV-1) in São Paulo State, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Productive and reproductive performance in cattle infected with bovine leukosis virus

Economic losses caused by enzootic bovine leukosis (EBL) have been of interest since World War II, when the neoplastic form of EEL increased dramatically in Europe. Olson (1974) and House et al. (1975) ed that animals with lymphosarcoma caused by the bovine leukosis virus (BLV) had reduced milk yields. a less efficient reproductive performance and high veterinary costs and mortality rates, while many carcasses were rejected at slaughter. However, the actual impact of BLV infection in cattle without lymphosarcoma is not; clear. The purpose of the study reported here was to compare some productive and reproductive responses of cattle that were antibody-positive (BLV+) or negative (BLV-) for BLV.Holstein dairy cows in commercial dairy farms were used in this study. Blood samples were collected and subjected to BLV serological examination by the agar gel immunodiffusion test of Miller & van der Maaten (1976). Animals were then grouped as BLV+ or BLV- according to their serological response to the BLV antigen. Productive and reproductive histories were obtained from individual animal records and the following factors were considered: milk production, calving interval and birth rate. For milk production, we had the daily milk yields of 547 animals, and for calving interval the time between two successive parturitions for 444 cows. These values were examined by ANOVA and when this was significant a Student's t test was carried out for each age group. Birth rates, the percentage of animals that calved in 1 gear, were available for 557 animals and were examined with the Z-two proportion test. For all analyses, P < 0.05 was considered significant.

Detection of an untyped strain of bovine respiratory syncytial virus in a dairy herd

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of serum antibodies to parainfluenza type 3 virus, respiratory syncytial virus, bovine viral diarrhea virus, and herpes virus type 1 in sheep in the region of Botucatu, São Paulo - Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intramammary coinfection by vaccinia virus and staphylococcus aureus in a bovine vaccinia outbreak

Introduction: Bovine vaccinia virus (VACV) is a well-known zoonotic agent related to exanthemous lesions in skin and mucous membranes of dairy cattle and humans, characterized by the formation of vesicles, pustules and ulcers. Mastitis is one of the most common infectious diseases of dairy herds. Bovine mammary infections are caused mainly by bacterial microorganisms, especially staphylococci. To the best of our knowledge, intramammary coinfection with VACV and Staphylococcus aureus in cows has not been reported previously. Case presentation: During an outbreak of exanthematic bovine VACV infection with animals showing vesicles, pustules and haemorrhagic ulcers on the teats, milk samples were collected for mastitis detection. Conclusion: The present report describes a case of intramammary coinfection by VACV and S. aureus in a bovine VACV outbreak.

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.

Intracellular Reactive Oxygen Species Production by Polymorphonuclear Leukocytes in Bovine Leukemia Virus-Infected Dairy Cows

The present study assesses the oxidative burst activity from polymorphonuclear leukocytes (PMNLs) from bovine leukemia virus (BLV)-infected cows. Fifteen clinically healthy cows were divided into serologically positive cows without any hematological alteration, serologically positive animals with persistent lymphocytosis (PL) and healthy serologically negative cows. The oxidative burst activity from the PMNLs was evaluated by now cytometry using 2',7'-dichlorofluorescein diacetate as a probe. PMNLs from each cow were incubated with heat-killed Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to stimulate oxidative burst activity. The results of the present work showed no significant difference in the oxidative burst activity without any stimulus and elicited by S. caucus. Conversely, a decrease in the oxidative burst index induced by E. coli in PMNLs was observed in BLV-infected cows.

Quantification of B cells and T lymphocyte subsets in bovine leukemia virus infected dairy cows

The aim of the present trial was to determine the frequencies and absolute number of B and T lymphocytes subpopulations in bovine leukemia virus (BLV)-infected dairy cows with distinct lymphocyte profile known as non-leukemic (AL) and persistent lymphocytosis (PL). Thus, 15 animals were selected and divided uniformly in three groups (negative, AL, PL). The BLV infection was detected by agar gel immunodiffusion and enzyme-linked immunosorbent-assay. The lymphocytes subsets were evaluated using monoclonal antibodies by flow cytometry. The results of the present study pointed out to an increase in B lymphocytes, and also an augment in CD5(+) and CD11b(+) cells in animals showing PL. Consequently, it can be observed a decrease in the percentage of T cells subsets in these animals. Conversely, no significant alterations in the absolute number of the T lymphocytes, T CD4(+) cells and T CD8(+) lymphocytes were found in BLV-infected dairy cows with PL. Therefore, the correlation between the absolute numbers of B- and T cell subsets in the peripheral blood applied to each group showed a significant and positive strong correlation between numbers of B cells and T cells or T CD8(+) cells in the PL animals, although the same cannot be predicted for T CD4(+) lymphocytes. No such correlation was encountered for the AL and negative-control animals.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

[How the bovine viral diarrhea virus outwits the immune system]

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.