Evaluation of different transfection methodologies to achieve efficient expression of the NS1 dengue protein in HepG2 cells

HepG2, a human hepatocarcinoma cell line, has been used as a model to study infection by several pathogens including dengue virus. However, this cell line is notoriously difficult to be transfected with plasmid DNAs by traditional methods, which is a limitation for some studies involving heterologous gene expression. In the present work, we analyzed different protocols for transfection of HepG2 with the plasmid pcENS1, which encodes the dengue NS1 protein, in order to evaluate the best methodology for achieving high cell viability and transfection efficiency. We analyzed two transfection approaches using lipid-based methods (Lipofectamine and FuGENE 6) or electroporation by nucleofection. Expression of the recombinant NS1 protein was evaluated by immunofluorescence and flow cytometry. Transfection with either of the two lipid-based methods led to very low number of HepG2 cells expressing NS1 (3.9% and 6.8% with Lipofectamine and FuGene, respectively) and high cell death rates. On the other hand, the efficiency of cell transfection was remarkable higher with nucleofection when compared to these other methods, achieving 63% of cells expressing NS1 protein and more than 60% of viability in the optimized condition.

Serological Survey of Rabies Virus Infection among Bats in Brazil

Rabies is a disease caused by a virus belonging to the family Rhabdoviridae, genus Lyssavirus (RABV), and is characterized as a progressive and acute encephalitis that presents high lethality. Between 2002 and 2012, 2149 wild animals were found positive for RABV in Brazil where 79% were associated with the aerial cycle of the disease. As part of the active surveillance of rabies in Brazil, the objective of this study was to investigate the presence of RABV infection in bats captured in two different regions of Brazil. Bat specimens were collected in (i) the metropolitan region of Rio de Janeiro city, (ii) Igrapiúna Municipality, Bahia State, and (iii) Serra do Tabuleiro State Park (PEST) in Santa Catarina State. Forty-four bats from the two first areas were tested for RABV at Institute Jorge Vaitsman (IJV), reference laboratory for rabies in Rio de Janeiro State. Of the individuals captured and sent for the diagnosis of rabies, a total of 15 were identified as being hematophagous, all of them belonging to the species Desmodus rotundus. Twenty-four individuals were considered as frugivorous, four as insectivores and one individual (Lonchophylla peracchii) identified as having a pollen/nectar feeding habit. All specimen sent to RABV diagnosis presented negative results. Considering the active surveillance for RABV in Brazil, studies of fauna survey with serological characterization are important in the strengthening of epidemiological surveillance and protection of human life.

Human Rhinovirus Association with Influenza-Like Illness and Symptomatic Treatment for Acute Respiratory Infection in a Brazilian Southern City

Acute respiratory infections (ARI) are the world’s leading cause of morbidity and mortality. ARI impairs children’s education and have a huge impact on the economy. Human rhinovirus (HRV) is the most prevalent agent of ARI. In this study, a clinical and epidemiological surveillance in outpatients was carried to investiga-te the involvement of HRV in ARI cases in the city of Guarapuava, a Brazilian southern city. Attention was also given to the most common medications used for treating ARI symptoms. Samples from 135 patients were col-lected from Apr to Dec from 2014, HRV was identified in nearly 20% of samples, with symptoms ranging from common cold to Influenza-like Illness (ILI) and was more frequent in individuals with 10 or less years-old. Ne-arly two thirds of patients reported use of at least one class of drug during the ARI episodes, such as analgesi-cs, cough and cold preparations, and NSAIDs. In some cases and with no justifiable reason, patients also repor-ted the use of antibiotics, possibly contributing to the development of bacterial resistance. These results show a significant detection rate of HRV in ARI cases, and highlight the impact of this virus in the local population.

Assessment of Hepatitis B and Hepatitis C Knowledge and Attitudes in a Sample of Health Care Workers from Southeast Brazil

This study aims to assess the knowledge and attitudes about hepatitis B and C viruses (HBV and HCV) in health care workers (HCW). A total of 266 HCW from Rio de Janeiro State (Southeast Brazil) answered an instrument containing 17 questions to study viral hepatitis knowledge and 12 statements regarding attitude and behavior about HBV and HCV. Mean HCWs’ knowledge level was considered satisfactory (mean knowledge score was 12.21 ± 3.36), but almost half of HCW presented low knowledge level (score lower than 12.21). High knowledge level was observed in individuals with previous HBV vaccination. Mean attitude score was 39.06±10.47 showing high level of positive attitudes, particularly among dentists and professionals who had previous needle stick injury. In conclusion, almost half of HCW had low level of knowledge about HBV and HCV and some gaps were identified showing the importance of continuous viral hepatitis education to HCW in this setting.

Investigation of Ehrlichia spp., Anaplasma spp. and Rickettsia spp. in ectoparasites collected from domestic animals, Rio de Janeiro State, Brazil

The aim of this study was to determine the occurrence of emerging arthropod-borne pathogens Anaplasma, Ehrlichia and Rickettsia infection in ticks (Acari: Ixodidae) and fleas (Insecta: Siphonaptera) collected from dogs and horses within municipality of Itaboraí, Rio de Janeiro State, Southern Brazil. Samples from 280 ticks and two fleas were subjected to family or/and genus specific PCR for Anaplasmataceae, Ehrlichia and Rickettsia, followed by DNA sequencing to ensure pathogen identity. In ticks Rhipicephalus sanguineus collected from dogs the DNA of Anaplasma platys and Ehrlichia canis was detected in 6.8% and 2.2% samples respectively. In two R. sanguineus confection with two pathogens was observed. In Dermacentor nitens ticks, collected from horses Francisella-like endosymbiont was found in 42.8% samples. DNA of Rickettsia felis and Wolbachia pi-petens was detected in fleas Ctenocephalides canis fleas. No DNA of Rickettsia was found in tested ticks. The findings contribute to our knowledge of tick-borne bacteria, ticks and endosymbionts distribution in Brazil.

Parameters for MDBK cell growth on microcarriers and BoHV-1 virus production

Bovine herpes virus 1 (BoHV-1) is an important veterinary agent , which causes infectious bovine rhinotra-cheitis. This disease affects the respiratory tract or genitals, causing weight loss, reduced milk production and abor-tion. Several vaccines against BoHV-1 have been developed. In this paper, we study the parameters for MDBK growth on microcarriers (Cytodex 1) and for BoHV-1 virus production. The cell culture attached to microcarriers is an effi-cient method to enlarge the surface of cell growth and for large-scale cell production. Our studies reveal that MDBK adhered to MCs in 30 minutes and that initial agitation of culture did not influence on the efficiency of adhesion or cell growth. In our experiments, we detected no relevant influence of agitation on initial cell adhesion of MDBK to MCs. The maximum cell yield was similar to all initial conditions of agitation studied. The maximum yield obtained in culture started with 15, 20 and 30 cells / MC, was respectively.8.7 x105, 9.3 x105 and 9.8 x105 cells / ml . The cellular distribution on the MCs at the beginning of the culture was more heterogeneous in higher initial densities. After three medium exchanges during MDBK cell culture, the increase in the final yield was 100% higher than that from culture performed without medium change (0.93 x 106 cells / mL). Replacing 50% of the culture medium with fresh medium after 24 hours of growth, the concentration of glucose (5 mM) and glutamine (1.8 mM) were almost completely res-tored. In these studies, BoHV-1 infections of MDBK were performed after 48, 72 and 86 hours with daily exchanges of 50% of the medium. The increase in viral titer was proportional to the number of viable cells present at the time of infection. The best result of BoHV-1 production was achieved when the infection was performed from 86 hours of cell culture, reaching about 3.7 x108 (TCID50/ml) after 24-48 hours of infection, being on average four times higher when compared to the culture in which the infection was performed with 48 hours of culture and approximately 2 times greater than the crop whose infection occurred after 72 hours of culture. The best yields are obtained when viral infections were performed on cultures with higher cell densities. The best result for the production of BoHV-1 occur-red with 10 MOI, 48 hours after infection, which yield was 24 and 41% superior to the MOI 0.1 and 1, respectively.

Tissular Distribution of Argentinean Strains of Bovine Herpesvirus Type 4 (BoHV-4) in Experimentally-Infected Calves

Although bovine herpesvirus type 4 (BoHV-4) is primarily associated with reproductive disorders of cattle, it can produce a variety of clinical signs. To determine the distribution, the presence and type of microscopic lesions caused by BoHV-4 strains of different genotypes an in vivo model, calves were infected with three phylogenetically different Argentinean BoHV-4 strains. Samples from nasal and ocular secretions, peripheral blood leukocytes, tissues and serum were analyzed. BoHV-4 was isolated from nasal and ocular secretions at 7 and 14 days post-inoculation (dpi). Viral DNA was detected by nested PCR in peripheral blood leukocytes at 14 and 21 dpi for two out of three strains and in tissues, such as nervous system, trachea, pulmonary and retropharyngeal lymph nodes, spleen and kidney, at 21 dpi. Antibody levels detected by viral seroneutralization test were mostly low and varied widely for the different strains. The tissue distribution of the BoHV-4 strains and the variations observed in the levels of neutralizing anti-bodies indicate that certain differences can be established among the patterns of biological behavior of each strain. This is an initial step to get insight into the biological characteristics of Argentinean BoHV-4 isolates. However, further evaluation involving a higher number of inoculated animals will be required to be conclusive on this aspect.

Seroprevalence of Human Herpesvirus Type 2 in a Reference Center for Pregnant Women in Rio de Janeiro, Brazil

Introduction: Pregnant women stand as an relevant group for research about Human Herpesvirus (HHV-2) infection owing to the risk of mother-to-child transmission. Methods: Women attending in a prenatal care center were tested for HHV-2 IgM and IgG by ELISA. Quantitative PCR test was the chosen method to ascertain viremia. Results: The seroprevalence of IgG and IgM anti-HHV-2 was 20.6% and 2.2% respectively. HHV-2 viremia was found in one pregnant woman with HHV-2 IgM, leading to the assumption of primary infection. Conclusion: The significantly high prevalence of HHV-2 found and the ascertainment of primary infection in a pregnant woman underline the need for constant HHV-2 follow-up and diagnosis in order to avoid sexual transmission.