A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012)

Cases of vesicular and exanthematic disease by Vaccinia virus (VACV) have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goiás state, Midwestern Brazil (2010-2012), involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old) were affected in all cases and calves (2 to 9 months old) were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm), painful and coalescent papules (3-8 mm), which resulted in ulcers (5-25mm) and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to vgf gene of Orthopoxvirus was positive in samples collected from all outbreaks, and in some cases, genomic VACV sequences were identified by nucleotide sequencing of the PCR amplicons. Infectious virus was isolated in cell culture from scabs from one outbreak. Antibodies to Orthopoxvirus were detected in at least 3 or 4 animals in most outbreaks, by ELISA (outbreaks 1, 2, 3, 4, 5 and 7) or virus-neutralization (outbreak 6). Neutralizing titers ranging from 8 to 64 in outbreak 6. In all outbreaks, VACV infection was suspected based on the clinical and pathological findings and it was confirmed by laboratory tests. Upon the etiological confirmation, other agents associated with vesicular disease were discarded. In all outbreaks, at least one milker who handled the affected cows developed malaise, headache, fever, painful vesico-pustular lesions mainly in the hands, but also in the neck and nose. These results confirm the circulation of VACV in the region and call attention for a correct diagnosis and the adoption of prophylactic and control measures.

Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.

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.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Recombinant vaccines and the development of new vaccine strategies

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 4 - The reflection of parasite stock in the responsiveness of different vector species to chronic infection with different Trypanosoma cruzi stocks

Previous studies (1982,1987) have emphasized the superiority of sylvatic vector species over domestic species as xenodiagnostic agents in testing hosts with acute or chronic infections by T. cruzi "Y" stock. The present study, which is unique in that it contains data on both infectivity rates produced by the same stock in 11 different vector species and also the reaction of the same vector species to seven different parasite stocks, establishes the general validity of linking efficiency of xenodiagnosis to the biotope of its agent. For example, infectivity rates produced by "São Felipe" stock varied from 82.5% to 98.3% in sylvatic vectors but decreased to 42.5% to 71.3% in domestic species. "Colombiana" stock produced in the same sylvatic vectors infectivity rates ranging from 12.5% to 45%. These shrank to 5%-22.5% in domestic bugs. The functional role of the biotope in the vector-parasite interaction has not been eluddated. But since this phenomenon has been observed to be stable and easy to reproduce, it leads us to believe that the results obtained are valid. Data presented also provide increasing evidence that the infectivity rates exhibited by bugs from xenodiagnosis in chronic hosts, are parasite stock specific. For example, infectivity rates produced by "Berenice", "Y", "FL" and "CL" varied in R. neglectus from 26.3% to 75%; in P. megistus from 56.3% to 83.8%; in T. sordida from 28.8% to 58.8% in T. pseudomaculata from 41.3% to 66.3% and in T. rubrovaria from 48.8% to 85%. Data from xenodiagnosis in the same hosts, carrying acute infections by the same parasite stocks, gave the five sylvatic vectors a positive rating of approximately 100%, thus suggesting that the heavy loads of parasites circulating in the acute hosts obscured the characteristic interspecific differences for the parasite stock. Nonetheless these latter were revealed in the same hosts with chronic infections stimulated by very low numbers of the same parasite stocks. Certain observations here described lead us to speculate as to the possibility of further results from other parasite stocks, allowing the association of the infectivity rates produced in bugs by different parasite stocks with the isoenzymic patterns revealed by these stocks.

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.

Human case of gastric infection by a fourth larval stage of Pseudoterranova decipiens (Nematoda, Anisakidae)

Only three cases of human infection by anisakid nematodes have been reported in Chile since 1976. In the present case, an anisakid worm, identified as a fourth-stage Pseudoterranova decipiens larva, was removed with a gastroendoscopic biopsy clipper from the stomach of a 45 year-old man from southern Chile. The patient, who presented acute epigastric pain and a continuous sensation of having an empty stomach, reported having eaten smoked fish. The worm was fixed in 70% ethanol and cleaned in lactophenol for morphological study. The morphometric characteristics of the worm are described and drawn. Anisakid larvae in fish flesh can be killed by freezing or cooking.

HIV/tuberculosis co-infection: a request for a better surveillance

The increasing endemicity of tuberculosis resulting from causes such as immigration, poverty, a declining public health infrastructure and co-infection by HIV/Mycobacterium tuberculosis, is leading to a change in tuberculosis control programmes. One of the main reasons for the resurgence of tuberculosis is HIV infection - the risk of tuberculosis is greater in HIV patients than in the majority of the population as can be seen from numerous research projects. The need for systematic testing for HIV infection in all tuberculosis patients by undertaking confidential HIV tests on admission to a tuberculosis programme is brought out. This measure would increase the number of cases diagnosed and provide data for better surveillance of the co-infection.

Susceptibility of asthmatic children to respiratory infection

OBJECTIVE: A case-control study of patients with pneumonia was conducted to investigate whether wheezing diseases could be a risk factor. METHODS: A random sample was taken from a general university hospital in S. Paulo City between March and August 1994 comprising 51 cases of pneumonia paired by age and sex to 51 non-respiratory controls and 51 healthy controls. Data collection was carried out by two senior paediatricians. Diagnoses of pneumonia and presence of wheezing disease were independently established by each paediatrician for both cases and controls. Pneumonia was radiologically confirmed and repeatability of information on wheezing diseases was measured. Logistic regression analysis was used to identify risk factors. RESULTS: Wheezing diseases, interpreted as proxies of asthma, were found to be an important risk factor for pneumonia with an odds ratio of 7.07 (95%CI= 2.34-21.36), when the effects of bedroom crowding (odds ratio = 1.49 per person, 95%CI= 0.95-2.32) and of low family income (odds ratio = 5.59 against high family income, 95%CI= 1.38-22.63) were controlled. The risk of pneumonia attributable to wheezing diseases is tentatively calculated at 51.42%. CONCLUSION: It is concluded that at practice level asthmatics should deserve proper surveillance for infection and that at public health level pneumonia incidence could be reduced if current World Health Organisation's guidelines were reviewed as to include comprehensive care for this illness.