Perfil da infecção pelo vírus da diarreia viral bovina (BVDV) em um rebanho bovino leiteiro de alta produção e com programa de vacinação contra o BVDV

A infecção pelo vírus da diarreia viral bovina (BVDV) foi avaliada em um rebanho bovino leiteiro de alta produção com histórico de problemas reprodutivos e de vacinação regular contra o BVDV. A identificação do vírus foi realizada por RT-PCR em soro sanguíneo e o perfil sorológico por vírus-neutralização. Inicialmente, 100% (n=692) dos animais do rebanho foram avaliados com relação à presença de infecção ativa pelo BVDV por meio da RT-PCR. Quatro meses após, todos os animais positivos (n=29) na primeira avaliação foram avaliados novamente pela RT-PCR, assim como todos os animais que nasceram (n=72) e os que apresentaram problemas reprodutivos (n=36) no intervalo entre a primeira e a segunda colheita de sangue. Os resultados finais do estudo possibilitaram identificar 27 animais transitoriamente infectados e três animais persistentemente infectados (PI). A sorologia, realizada apenas nos animais positivos na primeira avaliação pela RT-PCR e nas vacas que apresentaram problemas reprodutivos entre a primeira e a segunda RT-PCR, demonstrou grande flutuação nos títulos de anticorpos neutralizantes, além de soroconversão na maioria dos animais. Foram identificados aumentos nos títulos de anticorpos neutralizantes que variaram entre 3 e 8 log2, indicando infecção ativa no rebanho. A circulação viral no rebanho avaliado foi responsável pela expressão de sinais clínicos da esfera reprodutiva em animais com baixo título de anticorpos e consequente falha na proteção fetal. Os resultados demonstram que o controle da infecção pelo BVDV apenas por meio da vacinação regular em rebanhos com animais PI pode não ser eficaz na profilaxia dessa virose.

Caracterização morfológica e imuno-histoquímica de lesões em casos de aborto bovino bacteriano e viral no sul de Minas Gerais

O presente estudo avaliou a participação de agentes bacterianos e virais em abortos em bovinos de propriedades rurais do sul de Minas Gerais. Foi realizada análise histopatológica e imuno-histoquímica dos casos de aborto recebidos pelo Setor de Patologia Veterinária da Universidade Federal de Lavras no período de 1999 a 2013. De 60 fetos analisados, em 30 (50%) foram observadas lesões microscópicas. Destes, oito apresentavam lesões compatíveis com infecção por agentes bacterianos e três apresentaram lesões sugestivas de agentes virais. Dos abortos bacterianos, um feto tinha lesões compatíveis com leptospirose, caracterizadas por icterícia e colestase, nefrite intersticial linfoplasmocítica e nefrose tubular. Sete fetos apresentaram pneumonia ou broncopneumonia purulenta; num deles havia também pleurite e peritonite fibrinosas; e em dois desses fetos houve imunomarcação para Brucella abortus. Dos três fetos com lesões sugestivas de aborto viral ocorreu imunomarcação anti-Herpesvírus bovino em um. Os resultados demonstram a ocorrência de abortos de origem bacteriana e viral na Região do estudo e que medidas profiláticas devem ser adotadas nas propriedades. O trabalho demonstra também que a imuno-histoquímica (IHQ); associada à histopatologia; é uma ferramenta útil e viável para o diagnóstico, especialmente quando provas microbiológicas e/ou sorológicas não estão disponíveis.

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs

Abstract: In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals.

The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

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 values 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.

DNA vaccines for viral diseases

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Recombinant viruses as vaccines against viral diseases

Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

HEV, TTV and GBV-C/HGV markers in patients with acute viral hepatitis

The aim of the present study was to evaluate the prevalence of HEV, TTV and GBV-C/GBV-C/HGV in patients with acute viral hepatitis A, B and non-A-C. We evaluated sera of 94 patients from a sentinel program who had acute hepatitis A (N = 40), B (N = 42) and non-A-C (N = 12); 71 blood donors served as controls. IgM and anti-HEV IgG antibodies were detected by enzyme immunoassay using commercial kits. TTV and GBV-C/HGV were detected by nested PCR; genotyping was done by sequencing and phylogenetic analysis. Anti-HEV IgG was present in 38, 10 and 17% of patients with hepatitis A, B and non-A-C. Four patients with hepatitis A and 1 with non-A-C hepatitis also had anti-HEV IgM detected in serum. TTV was detected in 21% of patients with acute hepatitis and in 31% of donors. GBV-C/HGV was detected in 9% of patients with hepatitis, and in 10% of donors. We found TTV isolates of genotypes 1, 2, 3, and 4 and GBV-C/HGV isolates of genotypes 1 and 2. Mean aminotransferase levels were lower in patients who were TTV or GBV-C/HGV positive. In conclusion, the detection of anti-HEV IgM in some acute hepatitis A cases suggests co-infection with HEV and hepatitis E could be the etiology of a few cases of sporadic non-A-C hepatitis in Salvador, Brazil. TTV genotype 1, 2, 3 and 4 isolates and GBV-C/HGV genotype 1 and 2 strains are frequent in the studied population. TTV and GBV-C/HGV infection does not appear to have a role in the etiology of acute hepatitis.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Human T-cell lymphotropic virus type I (HTLV-I) proviral DNA viral load among asymptomatic patients and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.

Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.