A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus.

Cytomegalovirus infection in children with Down syndrome in a day-care center in Brazil

This study evaluates the transmission of CMV infection in 120 children aged 1 to 15 years with Down syndrome who attended a day-care center for handicapped children in São Paulo, Brazil. A blood sample was obtained from each children at the beginning of the study for detection of IgG and IgM cytomegalovirus (CMV) antibodies by an immunofluorescence assay. Samples of saliva and urine were obtained every 3 months from the children with CMV antibodies to detect shedding of the virus by culture in human foreskin fibroblasts, by detection of pp65 CMV-antigen and by a nested PCR assay. The prevalence of anti CMV-IgG antibodies was 76.6% (92/120), and IgM anti-CMV antibodies were detected in 13% (12/92) of the seropositive children. During the first viral evaluation, CMV was detected in the urine and/or saliva in 39/90 (43.3%) of the seropositive children. In the second and third evaluations, CMV was detected in 41/89 (46%) and in 35/89 (39.3%) children, respectively. Detection of CMV was shown both in urine and saliva in 28/39 (71.8%), 19/41(46.3%) and 20/35 (57.1%) of the children excreting the virus, respectively. Additionally, in 33/49 (67.4%) of the excreters CMV could be demonstrated in urine or saliva in at least two out of the three virological evaluations carried out sequentially in a six month period. Of the 28 initially seronegative children, 26 were re-examined for anti-CMV IgG antibodies about 18 months after the negative sample; seroconversion was found in 10/26 (38.5%). Taking all 536 samples of urine or saliva examined by virus culture and pp65 antigen detection during the study into account, 159 (29.6%) were positive by virus culture and 59 (11%) gave a positive result with the pp65 assay. These data demonstrate the high prevalence of CMV shedding and the high risk of CMV infection in children with Down syndrome attending a day-care center for mentally handicapped patients. The virus culture was more sensitive than the pp65 CMV antigen assay for CMV detection in both urine and saliva samples.

Detection of human herpesvirus 6 and 7 DNA in saliva from healthy adults from Rio de Janeiro, Brazil

In this study, we aimed to evaluate virus shedding in the saliva of healthy adults from the metropolitan region of the city of Rio de Janeiro, Brazil, in order to verify the prevalence of both human herpesviruses 6 and 7 (HHV-6, HHV-7). The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Saliva specimens were subjected to a multiplex polymerase chain reaction (PCR) to detect the presence of HHV-6A, HHV-6B and HHV-7. The total Roseolovirus DNA prevalence was 22.4%. The PCR detected a HHV-6 prevalence of 9.8%, with HHV-6A detected in 7.1% of the samples and HHV-6B in 2.7%. HHV-7 DNA was revealed in 12.6% of the studied cases. Multiple infections caused by HHV-6A and 7 were found in 2.1% of the samples. No statistical differences were observed regarding age, but for HHV-7 infection, an upward trend was observed in female patients. Compared to studies from other countries, low prevalence rates of herpesvirus DNA were detected in saliva from the healthy individuals in our sample. PCR methodology thus proved to be a useful tool for Roseolovirus detection and it is important to consider possible geographic and populations differences that could explain the comparatively low prevalence rates described here.

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a)

The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ) and both proteins (BoHV-5gEΔTKΔ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8) or BoHV-5gEΔTKΔ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Herança da resistência a Watermelon mosaic virus em melancia

Entre as doenças que ocorrem na cultura da melancia (Citrullus lanatus), a virose ocasionada por Watermelon mosaic virus (WMV) se destaca entre as principais, sendo a resistência genética a forma mais indicada de controle. Dessa forma, é importante o conhecimento do controle genético da resistência que se pretende trabalhar. Objetivando estudar a herança da resistência ao WMV em melancia, foram realizados cruzamentos entre o cultivar Crimson Sweet (CS) suscetível e a linha L26 resistente. Populações segregantes e não segregantes obtidas dos cruzamentos foram inoculadas com um isolado de WMV e avaliadas quanto ao aparecimento de sintomas e à presença do vírus por testes de ELISA indireto contra antissoro específico para WMV. A hipótese de herança monogênica foi avaliada em diferentes graus médios de dominância e pelo método da máxima verossimilhança. Foram obtidas variâncias genética (σ²G), ambiental (σ²E), fenotípica (σ²F2), aditiva (σ²A) e de dominância (σ²D), herdabilidades nos sentidos amplo (h²a) e restrito (h²r). A herança monogênica foi rejeitada. O grau médio de dominância indicou efeito de dominância completa. As herdabilidades no sentido amplo foram baixas; portanto, constatou-se que o controle da resistência a WMV nas populações de melancia estudadas é do tipo oligogênica, com presença de efeitos aditivos e não aditivos e presença de genes maiores e poligenes.

The measurement by serological means of the impact of the Hong Kong/68 influenza virus on a population

In order to obtain evidence on the size of the impact of the Hong Kong/68 variant of influenza A2 virus on the population of São Paulo, Brazil, serum samples taken in 1967 before this variant appeared and during successive years after it appeared were examined for their antibody content. Haemagglutination-inhibition tests performed on a total of 2726 serum samples from adults showed a sharp decrease in 1969 of the proportion of sera without antibody to the Hong Kong/68 variant and a corresponding mercase in the proportion with high titres. It was concluded that about three-quarters of the adult population became infected at some time after the variant appeared, the majority in the first year of prevalence.

Dados preliminares sobre partículas semelhantes a virus em células de triatomíneos

Nos tubos de Malpighi de Triatoma infestans Klug e de Panstrongylus megistus Burmeister foram encontradas estruturas víricas distribuídas no citoplasma, no interior de citolissomos ou outros tipos de glóbulos, formando às vezes arranjos para cristalinos envolvidos por membrana, e entre plasmalemas de células contíguas. Outros tecidos dos mesmos insetos apresentam arranjos paracristalinos desses virus. A análise citoquímica ao nível da microscopia eletrônica indica serem estes virus compostos de RNP. Não são encontradas alterações morfológicas drásticas nos tecidos infectados. Admite-se que tais virus tenham sido ingeridos durante uma alimentação de sangue infectado de ave.

Soro-epidemiologia do virus de Epstein-Barr (VEB)

É feita revisão dos estudos soro-epidemiológicos do vírus de Epstein-Barr (vírus EB), mostrando a ubicuidade deste vírus, bem como sua relação com doenças malígnas e não malígnas do homem. O desenvolvimento de novas técnicas laboratoriais e o acúmulo, nos últimos 10 anos, de grande número de estudos soro-epidemiológicos, levaram à conclusão de que, além da associação etiológica com a mononucleose infecciosa, torna-se cada vez mais evidente a ligação do vírus EB com o linfoma de Burkitt e com o carcinoma do nasofaringe. Estas observações, além de sua extraordinária importância intrínseca, tem sido de grande utilidade para a melhor compreensão dos possíveis mecanismos e efeitos das infecções virais no homem.

Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.

Peroral susceptibility of Aedes albifasciatus and Culex pipiens complex mosquitoes (Diptera: Culicidae) from Argentina to western equine encephalitis virus

The transmission cycle of western equine encephalitis (WEE) virus in South America is unknown. A WEE virus strain was isolated from Aedes albifasciatus in Argentina during the WEE epizootic of 1982-83. Also, Culex pipiens from Argentina was reported to be able to transmit WEE virus experimentally, but other results indicate that Cx. pipiens from the USA is refractory to this virus. We determined the susceptibility of Argentina strains of Ae. albifasciatus and Culex pipiens complex mosquites to infection by WEE virus by the oral route. Adult females were fed on chicks infected with a WEE virus strain isolated in Cordoba Province, Argentina, or were fed on a blood/virus suspension. Each mosquito ingested between 10(1.6) to 10(6.4) vero cell plaque-forming units of virus. Each of 28 Ae. albifasciatus was positive for virus from the fourth day postfeeding, and there was evidence for virus replication. In contrast, 0/44 Cx. p. quinquefasciatus and only 1/15 Cx. p. pipiens was positive. Aedes albifasciatus is susceptible to infection by WEE virus and should be considered a potential vector of this virus in Argentina. Both subspecies of Cx. pipiens are refractory to peroral infection by WEE virus and probably do not play a role in the WEE virus cycle in Argentina.

A case-control study on the association of hepatitis B virus infection and hepatocellular carcinoma in Northeast Brazil

Hepatitis B virus (HBV) serological markers were investigated in 40 incident cases of hepatocellular carcinoma (HCC) and in two age and sex matched control groups, comprising 40 patients with other cancers and 80 healthy individuals, resident in Bahia, Brazil. Serologic tests were done by radioimmunoassay. The study observed high proportion of seropositivity to HBsAg (42.5%) and of those presenting HBsAg or antiHBc (65.0%) among HCC cases, higher in men than women and in those aged 17 to 30 years old. HBsAg seropositivity among HCC patients was greater than in the control group with other cancers (7.5%) and in healthy controls (2.5%), corresponding to odds ratio estimates of 15.0 (95% CI 3.29, 68.30) and 33.0 (95% CI 9.13, 119.28), both statistically significant. HBeAg was not observed and antiHBe was present in 41.2% of cases, suggesting the absence of viral replication, possibly with viral DNA intergration into the hepatocyte genome. The presence of cirrhosis was associated with HBsAg seropositivity among HCC cases. A history of chronic alcoholism is shown to be more frequently related to those cases with cirrhosis. This study highlights the relevant association between HCC and HBV in Northeast Brazil, particularly for young individuals, and the high risk of development of HCC for HBsAg carriers.

RC-IAL cell line: sensitivity of rubella virus grow

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL² with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.