Mário Celso Sperotto Brum

Immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine

We have previously shown that swine inoculated with recombinant, replication-defective human adenovirus type 5 containing the porcine interferon alpha gene (Ad5-pIFNalpha) are completely protected when challenged 1 day later with virulent foot-and-mouth disease virus (FMDV). In the current study, we examined the duration of protection afforded swine by Ad5-pIFNalpha and the ability of a combination of Ad5-pIFNalpha and a FMDV subunit vaccine delivered by Ad5-A24 (an Ad5 vector containing the capsid coding region of FMDV serotype A24 Cruzeiro and the 3C proteinase coding region of FMDV serotype A12) to induce immediate as well as long-lasting protection against homologous FMDV challenge. Groups of swine were inoculated with Ad5-pIFNalpha and challenged with virulent FMDV A24 1, 3, 5, and 7 days postinoculation (dpi) or 1 day preinoculation. All animals challenged 1 and 3dpi were completely protected from disease. The animals in the remaining groups had either no clinical signs of disease or clinical signs were delayed and less severe compared to the control group. Swine inoculated with a combination of Ad5-pIFNalpha and Ad5-A24 and challenged 5dpi were all completely protected from disease and developed a significant FMDV-specific neutralizing antibody response.

Adenovirus-Mediated Type I Interferon Expression Delays and Reduces Disease Signs in Cattle Challenged with Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is an economically important disease of livestock. Eliminating FMD outbreaks in previously disease-free countries often relies on restriction of animal movement and massive slaughter of infected and in-contact susceptible animals. To develop a more effective and humane FMD control strategy, we explored the possibility of using type I interferon (IFN-alpha/beta) as a novel anti-FMD agent. We have demonstrated previously that swine inoculated with replication-defective human adenovirus type 5 (Ad5) vector expressing porcine IFN-alpha (Ad5-PoIFN-alpha) were completely protected from FMD virus (FMDV) challenge. To extend this approach to bovines, we constructed Ad5 vectors that express bovine IFN-alpha or IFN-beta (Ad5-BoIFN-alpha and Ad5-BoIFN-beta). Cells infected with these viruses produced high levels of biologically active BoIFN-alpha/beta, but despite expression in vitro, no detectable IFN-induced biologic activity was found in cattle inoculated with Ad5-BoIFN-alpha. Because PoIFN-alpha inhibits FMDV replication in bovine cells, we evaluated the potential use of PoIFN-alpha against FMD in cattle. In cattle inoculated with Ad5-PoIFN-alpha, the appearance of vesicles was delayed after challenge with FMDV and disease was less severe than in control animals. One Ad5-PoIFN-alpha-inoculated animal never developed clinical disease. Similarly, although all the Ad5-PoIFN-alpha-inoculated animals developed viremia, it was delayed for 1 day as compared with the control group. These results suggest that in vivo expression of PoIFN-alpha partially protected cattle from FMD.

Constitutive Expression of Alpha Interferon by Skin Dendritic Cells Confers Resistance to Infection by Foot-and-Mouth Disease Virus

ABSTRACT The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-β) mRNA but not IFN-α mRNA. However, these DC secreted both IFN-α and IFN-β proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-α/β, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-α protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-α in uninfected animals. This is the first demonstration of the constitutive expression of IFN-α in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs--one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48--recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R value suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Assessing the variability of Brazilian Vaccinia virus isolates from a horse exanthematic lesion: coinfection with distinct viruses

During the last bovine vaccinia (BV) outbreaks, several Vaccinia virus (VACV) strains were isolated and characterised, revealing significant polymorphisms between strains, even within conserved genes. Although the epidemiology of VACV has been studied in BV outbreaks, there is little data about the circulation of the Brazilian VACV isolates. This study describes the genetic and biological characterisation of two VACV isolates, Pelotas 1 virus (P1V) and Pelotas 2 virus (P2V), which were obtained concomitantly from a horse affected by severe cutaneous disease. Despite being isolated from the same exanthematic clinical sample, P1V and P2V showed differences in their plaque phenotype and in one-step growth curves. Moreover, P1V and P2V presented distinct virulence profiles in a BALB/c mouse model, as observed with other Brazilian VACV isolates. Sequencing and phylogenetic analysis of four different genes demonstrated that the isolates are segregated in different VACV clusters. Our results raise interesting questions about the diversity of VACV isolates in Brazil.

Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006)

Bovine herpesviruses types 1 and 5 (BoHV-1; BoHV-5) are genetically and antigenically closely related such they can not be distinguished by routine diagnostic tests. As BoHV-1 has been historically associated with respiratory and genital disease, herpesviruses isolated from these clinical syndromes have been tentatively - and sometimes definitively - diagnosed as BoHV-1. Likewise, cases of herpetic neurological infection in cattle have been generally attributed to BoHV-5. This study reports the identification of 40 herpesvirus isolates from different clinical specimens and syndromes in central-southern Brazil, Argentina and Uruguay (1987-2006) by the use of a PCR able to differentiate between BoHV-1 and BoHV-5. BoHV-1 isolates (n=16) were identified in cases of respiratory disease (n=3), vulvovaginitis and/or balanoposthitis (n=3), in semen of healthy bulls (n=5) and in cases of neurological disease (n=5). Viruses identified as BoHV-5 (n=24) were isolated predominantly from cases of neurological disease (n=21), but also from semen of healthy bulls (n=2) and from a spleen of a calf with systemic disease (n=1). These results show that both BoHV-1 and BoHV-5 are not strictly associated with their respective diseases; yet are frequently involved in clinical conditions otherwise attributed to the other virus. These findings also reinforce the need of correctly identifying the herpesvirus isolates as to better understand their pathogenesis and epidemiology.

Molecular Identification of Bovine Papillomaviruses Associated with Cutaneous Warts in Southern Brazil

Bovine papillomaviruses (BPVs) are widespread pathogens mainly associated with benign, self-limiting, cutaneous lesions (warts). At least 8 viral types, defined by serology or nucleotide sequences of the L1 gene, have been identified to date. Different serotypes are associated with the specific type and morphology of the lesion and with particular geographical regions. This article describes the molecular identification of papillomaviruses from Brazilian cattle (n = 48) and horses (n = 1) through partial amplification and sequencing of the L1 gene. Bovine papillomavirus–1 (BPV-1) was identified in warts from 29 cattle (59%), BPV-6 from 9 cattle (18%), and BPV-2 in 8 lesions (16%). Warts of 2 cattle harbored L1 sequences of a new BPV type (BAA5), otherwise identified almost exclusively in healthy skin. The newly proposed BPV type “BR-UEL-4” was identified in a sarcoid tumor of a horse. Thus, the present report provides information on the main types of BPV involved in bovine papillomatosis in Brazil and reveals a new viral type associated with equine sarcoid, which to date has been attributed exclusively to BPV-1 and BPV-2.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric beta-galactosidase gene. Subsequently, using the BoHV-5 gE virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE) and TK + gE (BoHV-5 gE/TK) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE, BoHV-5 TK and BoHV-5 gE/TK) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is immunogenic for calves and confers protection upon homologous challenge and BoHV-1 challenge.

A recombinant bovine herpesvirus 5 lacking thymidine kinase and glycoprotein E genes (BoHV-5gEΔTKΔ) was evaluated as a live experimental vaccine. In a first experiment, ten-months-old calves were vaccinated intramuscularly (n=9) or remained as controls (n=8) and 42 days later were challenged with BoHV-5 or BoHV-1 intranasally. The four control calves challenged with BoHV-5 developed severe depression and neurological signs and were euthanized in extremis at days 13 and 14 pos-infection (pi); the five vaccinated animals challenged with BoHV-5 remained healthy. The titers of virus shedding were reduced (p<0.01) from days 3 to 7 post-infection (pi) in vaccinated animals. Control and vaccinated calves challenged with BoHV-1 presented mild transient respiratory signs; yet the magnitude of virus shedding was reduced (p<0.05) in vaccinated animals (days 5, 9 and 11pi). In a second experiment, young calves (100-120 days-old) were vaccinated (n=15) or kept as controls (n=5) and subsequently challenged with a BoHV-1 isolate. Control calves developed moderate to severe rhinitis and respiratory distress; two were euthanized in extremis at days 5 and 9 pi, respectively. In contrast, vaccinated animals were protected from challenge and only a few developed mild and transient nasal signs. The duration and titers of virus shedding after challenge were reduced (p<0.05) in vaccinated animals comparing to controls. In both experiments, vaccinated animals developed antibodies to gE only after challenge. These results demonstrate homologous and heterologous protection and are promising towards the use of the recombinant BoHV-5gEΔTKΔ in vaccine formulations to control BoHV-5 and BoHV-1 infections.