Mauro Pires Moraes

Genome of Bovine Herpesvirus 5

ABSTRACT Here we present the complete genomic sequence of bovine herpesvirus 5 (BHV-5), an alphaherpesvirus responsible for fatal meningoencephalitis in cattle. The 138,390-bp genome encodes 70 putative proteins and resembles the α2 subgroup of herpesviruses in genomic organization and gene content. BHV-5 is very similar to BHV-1, the etiological agent of infectious bovine rhinotracheitis, as reflected by the high level of amino acid identity in their protein repertoires (average, 82%). The highest similarity to BHV-1 products (≥95% amino acid identity) is found in proteins involved in viral DNA replication and processing (UL5, UL15, UL29, and UL39) and in virion proteins (UL14, UL19, UL48, and US6). Among the least conserved (≤75%) are the homologues of immediate-early (IE) proteins BICP0, BICP4, and BICP22, the three proteins being longer in BHV-5 than in BHV-1. The structure of the BHV-5 latency-related (LR) region departs markedly from that of BHV-1 in both coding and transcriptional regulatory regions. Given the potential significance of IE genes and the LR region in virus-neuron interactions, it is likely these differences contribute to BHV-5 neuropathogenicity.

Enhanced Antiviral Activity against Foot-and-Mouth Disease Virus by a Combination of Type I and II Porcine Interferons

ABSTRACT Previously, we showed that type I interferon (alpha/beta interferon [IFN-α/β]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 109 PFU of human adenovirus type 5 expressing porcine IFN-α (Ad5-pIFN-α) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-γ) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-α, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 108 PFU of Ad5-pIFN-α, low- or high-dose Ad5-pIFN-γ, or a combination of Ad5-pIFN-α and low- or high-dose Ad5-pIFN-γ and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-α or a low dose of Ad5-pIFN-γ developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-γ was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-γ or with only high-dose Ad5-pIFN-γ were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.

Interferon-Induced Protection against Foot-and-Mouth Disease Virus Infection Correlates with Enhanced Tissue-Specific Innate Immune Cell Infiltration and Interferon-Stimulated Gene Expression

ABSTRACT Previously, we demonstrated that type I interferon (IFN-α/β) or a combination of IFN-α/β and type II IFN (IFN-γ) delivered by a replication-defective human adenovirus 5 (Ad5) vector protected swine when challenged 1 day later with foot-and-mouth disease virus (FMDV). To gain a more comprehensive understanding of the mechanism of protection induced by IFNs, we inoculated groups of six swine with Ad5-vectors containing these genes, challenged 1 day later and euthanized 2 animals from each group prior to (1 day postinoculation [dpi]) and at 1 (2 dpi) and 6 days postchallenge (7 dpi). Blood, skin, and lymphoid tissues were examined for IFN-stimulated gene (ISG) induction and infiltration by innate immune cells. All IFN-inoculated animals had delayed and decreased clinical signs and viremia compared to the controls, and one animal in the IFN-α treated group did not develop disease. At 1 and 2 dpi the groups inoculated with the IFNs had increased numbers of dendritic cells and natural killer cells in the skin and lymph nodes, respectively, as well as increased levels of several ISGs compared to the controls. In particular, all tissues examined from IFN-treated groups had significant upregulation of the chemokine 10-kDa IFN-γ-inducible protein 10, and preferential upregulation of 2′,5′-oligoadenylate synthetase, Mx1, and indoleamine 2,3-dioxygenase. There was also upregulation of monocyte chemotactic protein 1 and macrophage inflammatory protein 3α in the skin. These data suggest that there is a complex interplay between IFN-induced immunomodulatory and antiviral activities in protection of swine against FMDV.

Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Current inactivated vaccines require approximately 7 days to induce protection, but before this time vaccinated animals remain susceptible to disease. Previously, we demonstrated that intramuscular (IM) inoculation of a replication-defective human adenovirus type 5 (Ad5) vector containing a porcine interferon α gene (pIFNα) can protect swine challenged 1 day later by intradermal (ID) injection with FMDV A24 Cruzeiro from both clinical disease and virus replication. To extend these studies to other FMDV serotypes, we demonstrated the effectiveness of Ad5-pIFNα against ID challenge with O1 Manisa and Asia-1 and against A24 Cruzeiro in a direct contact challenge model. We also showed that an Ad5 vector containing the pIFNβ gene can protect swine against ID challenge with A24 Cruzeiro. Further, IM inoculation of a 10-fold lower dose of Ad5-pIFNα at 4 sites in the neck compared with 1 site in the hind limb can protect swine against ID challenge. These studies demonstrate the ability of Ad5-delivered type I IFN to rapidly protect swine against several FMDV serotypes and suggest that various modifications of this approach may enable this strategy to be successfully used in other FMD susceptible species.

Novel Antiviral Therapeutics to Control Foot-and-Mouth Disease

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8u2009mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8u2009mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24u2009h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Evolução da imunidade passiva em fêmeas bovinas da raça holandesa

The zinc sulfate turbidity test (ZST) was used to determine the immunoglobulin concentration in 1.530 serum samples collected from 81 cows (28 days before parturition to 28 days after parturition - n=579). Heifers born from these cows were also tested untd 210 days of live (n=951) with the same test Levels of Igs in cows decreased during the two weeks prepartum, indicating a transfer of circulating Igs to the mammary gland. The average serum Igs at 24 hours after birth in 76 female calves was 11.66 mg/ml with a standart deviation (SD) of 7 4. Failure of passive transfer (FPT) was considered to occur m animals with levels of lgs lower than one SD below average (4.2 mg/ml). FPT animals had lower levels of serum Igs than the other group unlil 56 days of age. High correlation (r=O.91) between ZST and serum electrophoresis was observed Sodium sulfite precipitation and glutaraldehyde coagulation tests were less efficient than ZST.

A retrospective search for bovine respiratory syncytial virus (BRSV) antigens in histological specimens by immunofluorescence and immunohistochemistry

Bovine respiratory syncytial virus (BRSV) has been only sporadically identified as a causative agent of respiratory disease in Brazil. This contrasts with frequent reports of clinical and histopathological findings suggestive of BRSV-associated disease. In order to examine a possible involvement of BRSV in cases of calf pneumonia, a retrospective search was performed for BRSV antigens in histological specimens submitted to veterinary diagnostic services from the states of Rio Grande do Sul and Minas Gerais. Ten out of 41 cases examined (24.4%) were positive for BRSV antigens by immunohistochemistry (IPX). Eight of these cases (19.5%) were also positive by indirect immunofluorescence (IFA), and 31 cases (75.6%) were negative in both assays. In the lungs, BRSV antigens were predominantly observed in epithelial cells of bronchioles and less frequently found in alveoli. In one case, antigens were detected only in the epithelium of the alveolar septae. The presence of antigen-positive cells was largely restricted to epithelial cells of these airways. In two cases, positive staining was also observed in cells and cellular debris in the exudate within the pulmonary airways. The clinical cases positive for BRSV antigens were observed mainly in young animals (2 to 12 month-old) from dairy herds. The main microscopic changes included bronchointerstitial pneumonia characterized by thickening of alveolar septae adjacent to airways by mononuclear cell infiltrates, and the presence of alveolar syncytial giant cells. In summary, the results demonstrate the suitability of the immunodetection of viral antigens in routinely fixed tissue specimens as a diagnostic tool for BRSV infection. Moreover, the findings provide further evidence of the importance of BRSV as a respiratory pathogen of young cattle in southeastern and southern Brazil.

Fator estimulante de colônias de granu-lócitos e macrófagos (GM-CSF) não aumenta a eficácia ou potência da vacina de subunidades da febre aftosa em suínos

Foot-and-mouth disease (FMD) is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5) vector containing the FMDV capsid (P1-2A) and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24). An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C), however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF). However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.

Relationship between passive immunity and morbidity and weight gain in dairy cattle

Serum samples from 87 calves from a dairy herd in Southern Brazil were collected to determine the levels of passive transfer and its relationship to morbidity and mean daily weight gain (MDG). Serum immunoglobulin (Ig) levels were measured by the zinc sulfate turbidity test at 24 hours of age in the calves. The average serum Ig level was 11.40g/l. Fourteen out of 87 calves (16.1%) showed serum Ig levels one standard deviation below the mean (4.59g/l) and were considered as having failure of passive immunity transfer (FPT). The occurrence of diarrhea from birth to weaning was higher in the FPT group (100%) than the normal group (90.7%) but the difference was not significant. The occurrence of signs of respiratory disease was similar in both groups (35.7% for FPT and 36.9% for the normal group). The mean daily gain from birth to 13-16 months of age in the FPT group was significantly (P>0.05) lower than in the group with normal serum Ig levels. The difference in MDG from birth to weaning between the groups was not significant. These results demonstrate the importance of passive immunity in cattle, and also provide regional parameters for the evaluation of FPT in cattle.

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein

Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.