Rita Maria Zucatelli Mendonça

Purification and Characterization of an Anti-Apoptotic Protein Isolated from Lonomia obliqua Hemolymph

Previously it was reported that supplementation of insect cell culture with Lonomia obliquahemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58–63). In this work the anti‐apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf‐9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti‐apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf‐9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliquahemolymph is due to the presence of potent anti‐apoptotic factors.

Co-infection between influenza virus and flagellated bacteria

Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibiotics, the bacteria remained resistant in several of the co-infected samples (48.39%). These bacteria, considered opportunistic invaders from environmental sources, are associated with viral infections in upper respiratory tract of hosts. The protease (elastase), secreted by Stenotrophomonas maltophilia plays a role in the potentiation of influenza virus infection. Proteolytic activity was detected by casein agar test. Positive samples from animals and humans had either a potentiated influenza infectivity or cytopathic effect (CPE) in MDCK and NCI H292 cells, Stenotrophomonas maltophilia were always present. Virus and bacteria were observed ultrastructurally. These in vitro findings show that microbial proteases could contribute to respiratory complications by host protease activity increasing inflammation or destroying endogenous cell protease inhibitors.

Antiviral activity of the hemolymph of Lonomia obliqua (Lepidoptera: Saturniidae)

Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3+/-1.25 x 10(7) to 2.1+/-1.5 x 10(5) TCID(50)mL(-1)) in measles virus production and a 61-fold reduction (from 2.8+/-1.08 x 10(9) to 4.58+/-1.42 x 10(7)mL(-1)) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response.

Investigation of influenza in migrating birds, the primordial reservoir and transmitters of influenza in Brazil

Birds are the most important reservoirs of the influenza virus. Its maintenance in its natural hosts, including man, allows the influenza virus to reassorts its strains. The recent report of an avian influenza A (H5N1) virus in humans, was in a child with fatal respiratory illness in China, 1997. The current study was conducted to elucidate the transportation of the influenza by birds that migrate, annually, through the both Northern and Southern hemispheres, with special attention paid to the Vireo olivaceus [Juruviara(BR) or Red-eyed vireo(USA)] species, which travels from the USA to Brazil, and vice versa, and the Elaenia mesoleuca [Tuque(BR) or (USA)] species that flies over the entire Southern Hemisphere. There are two species of birds, which breed and migrate in Sao Paulo State, Brazil, and which were demonstrated to carry Influenza virus, were selected. The viral particles isolated were observed by electron microscopy. The influenza virus was detected by the House Duplex/PCR and Gloria molecular biology tests. The results demonstrated that the Elaenia mesoleuca and Vireo olivaceus bird species are carrying the Influenza virus whilst crossing both the Northern and Southern hemispheres. To understand the role that these migrating birds may play in epidemic influenza, in Brazil, characterization of avian influenza subtypes will be done.

Influenza em animais heterotérmicos

The objective was to study Orthomyxovirus in heterothermic animals. Blood samples from snakes (genus Bothrops and Crotalus) and from toads and frogs (genus Bufo and Rana) were collected to evaluate the red cell receptors and antibodies specific to influenza virus by the hemagglutination and hemagglutination inhibition tests, respectively. Both snakes and toads kept in captivity presented receptors in their red cells and antibodies specific to either influenza virus type A (human and equine origin) or influenza type B. The same was observed with recently captured snakes. Concerning the influenza hemagglutination inhibition antibodies protective levels were observed in the reptiles’ serum, against influenza type A and type B. Unlike the toads, 83.3% of the frogs presented mean levels of Ab 40HIU for some influenza strains. It was concluded that heterothermic animals could offer host conditions to the influenza virus and also susceptibility to the infection. Key-words: Orthomyxovirus. Heterothermic animals. Receptors. Specific antibodies. Influenza virus. 1. Laboratorio de Virologia do Instituto Butantan, Sao Paulo, SP. 2. Laboratorio de Genetica do Instituto Butantan, Sao Paulo, SP. 3. Laboratorio de Herpetologia do Instituto Butantan, Sao Paulo, SP. Endereco para correspondencia: Dra. Dalva Assuncao Portari Mancini. Divisao de Desenvolvimento Cientifico/Laboratorio de Virologia/IB. Av. Vital Brasil 1500, 05503-900 Sao Paulo, SP. Tel: 55 11 3726-7222 ramal 2152 e-mail: labvirol@bol.com.br dapmancini@butantan.gov.br. Recebido para publicacao em 8/2/2003 Aceito em 27/2/2004 Para melhor entendimento sobre a funcao do receptor celular no reconhecimento do virus influenza, tem sido desenvolvidas pesquisas relacionadas a especificidade de ligacao desse virus isolado de varias especies animais 10 . Bossart e cols verificaram atraves da microscopia eletronica, os fenomenos conhecidos de adsorcao (atraves da hemaglutinina) e subsequente eluicao (pela neuraminidase) do virus influenza sobre a membrana de eritrocitos de ave e de humano. Essa interacao do virus a celula vermelha e comparado ao que ocorre as celulas hospedeiras. Os virus influenza tipo A mostram habilidade, entre seus subtipos, de ligacao aos receptores atraves dos terminais dos acidos sialicos, contendo oligossacarideos, mas, sao distintos quanto a especificidade de receptor, o que provavelmente os diferencia quanto a virulencia aos hospedeiros . Quanto ao tropismo desse virus, verifica-se que as celulas receptoras virus ARTIGO/ARTICLEO objetivo foi pesquisar Ortomyxovirus em animais heterotermicos. Coletou-se sangue de serpentes dos generos Bothrops e Crotalus e de sapo e ras dos generos Bufo e Rana, para a deteccao dos receptores de hemacias e anticorpos especificos, ao virus influenza, pelos testes de hemaglutinacao e inibicao da hemaglutinacao, respectivamente. Pelo teste de hemaglutinacao, verificou-se que serpentes e sapos em cativeiro apresentaram receptores em suas hemacias para o virus influenza, humano e equino do tipo A e tipo B. O mesmo ocorreu com serpentes recem chegadas. Quanto ao teste de inibicao da hemaglutinacao dos soros dos repteis observou-se titulos protetores de anticorpos aos virus influenza tipo A (origens humana e equina) e tipo B. Com soro de sapo nao se observou reacao de inibicao da hemaglutinacao porem, 83,3% das ras obtiveram medias de 40UIH para algumas cepas. Conclui-se que animais heterotermicos podem oferecer condicoes de hospedeiros aos virus influenza, assim como susceptibilidade a infeccao.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09%) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64%) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13%) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51%) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 masculineC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

Termoestabilidade da vacina contra a raiva, tipo Fuenzalida & Palacios, uso humano

Ten lots of Fuenzalida & Palacios type antirabies vaccine for human use, produced at the Instituto Butantan (Sao Paulo, Brazil) were stored at temperatures of 45, 37, 28 and 2-8oC. The potency of each lot was determined in samples taken at varied time intervals using the NIH method and lots presenting antigenic values > 0,3 were considered satisfactory for use. After 2 hours at 45oC the antigenic value of one out of 10 lots tested was found to be less than the minimum required value. At 37oC all lots maintained satisfactory antigenic values until the third day of storage, whilst at 28 and 2-8oC the potency was fully maintained, respectivelly for 10 and 360 days. At the ideal temperature of 2-8oC, 100% of the tested vaccines maintained the minimum required antigenicity for a longer period (16 months) than the expiry time of 6-12 months usually recommended for this type of biological produced in Latin American and Caribbean countries. Thus, the obtained data suggested that in countries still producing Fuenzalida & Palacios type vaccine, the expiry time could be extended to 16 months, what could prevent the unnecessary discarding of products still in useful condition.

Influenza viruses in adult dogs raised in rural and urban areas in the state of São Paulo, Brazil

In 1970, searching for the interspecies transmission of influenza viruses led to the first study on influenza viruses in domestic animals. Birds and mammals, including human beings, are their natural hosts; however, other animals may also play a role in the virus epidemiology. The objective was to investigate the incidence of influenza viruses in adult dogs raised in rural (9, 19.56%) and urban (37, 80.43%) areas in the state of São Paulo, Brazil. Dog serum samples were examined for antibodies to influenza viruses by the hemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Dogs from rural areas presented antibodies to influenza A H3N2, and influenza A H7N7 and H3N8. In rural areas, dog sera displayed mean titers as 94.37, 227.88, 168.14, 189.62 HIU/25 µL for subtypes H1N1, H3N2, H7N7, H3N8, respectively. About 84% and 92% of dogs from urban areas exhibited antibodies to human influenza A H1N1 and H3N2, respectively, with statistical difference at p < 0.05 between the mean titers of antibodies to H1N1 and H3N2. About 92% and 100% were positive for H7N7 and H3N8, respectively. In dogs from urban areas, the mean titers of antibodies against influenza A H1N1, H3N2, H7N7 and H3N8, were 213.96, 179.42, 231.76, 231.35 HIU/25 µL respectively. The difference among them was not statistically significant at p > 0.05. In conclusion, these dogs were positive for both human and equine influenza viruses. The present study suggests the first evidence that influenza viruses circulate among dogs in Brazil.

Anti-human influenza protector antibody detected in horses as a zoonotic viruses

O objetivo deste estudo foi investigar em cavalos, a incidencia do virus influenza e seu ciclo de transmissao interespecies. Portanto, levantamento sorologico foi realizado em soro de cavalos, confrontados com ambas cepas, as especificas (equino) e nao especificas (humana) deste virus. Sangrias de cavalos realizadas nos anos de 1999 e de 2000, forneceram soros que, apos tratamento com Caolim (20%) e hemacias de galo(50%) para remocao dos anticorpos inespecificos, foram titulados contra ambas referidas cepas, atraves do teste de Inibicao da Hemaglutinacao (recomendado pela OMS). Os resultados, demonstraram que as respostas sorologicas dos cavalos apresentaram reacao cruzada entre as cepas especificas e as nao especificas. As porcentagens de titulos IH obtidos foram de 62,75% e de 60,65% para as cepas especificas A/Eq1 (H7N7) e A/Eq2 (H3N8), respectivamente. E as cepas nao especificas essas porcentagens foram de: 79,05% para A (H1N1), de 94,45% para A (H3N2) e de 77,75% ao tipo B. O mais relevante nestes dados comparativos com virus influenza, foi a alta porcentagem de resposta protetora a cepa nao especifica comparada aquela especifica, detectada nos soros equinos. Considerando o fato de que o tipo B, deste virus, ser restrito a especie humana, portanto a resposta de protecao nos cavalos sugere uma direta transmissao interspecies, como em viroses zoonoticas. Os autores relatam pela primeira vez este tipo de evento no Brasil.

PRESENCE OF RESPIRATORY VIRUSES IN EQUINES IN BRAZIL

Equines are susceptible to respiratory viruses such as influenza and parainfluenza. Respiratory diseases have adversely impacted economies all over the world. This study was intended to determine the presence of influenza and parainfluenza viruses in unvaccinated horses from some regions of the state of São Paulo, Brazil. Blood serum collected from 72 equines of different towns in this state was tested by hemagglutination inhibition test to detect antibodies for both viruses using the corresponding antigens. About 98.6% (71) and 97.2% (70) of the equines responded with antibody protective titers (≥ 80 HIU/25µL) H7N7 and H3N8 subtypes of influenza A viruses, respectively. All horses (72) also responded with protective titers (≥ 80) HIU/25µL against the parainfluenza virus. The difference between mean antibody titers to H7N7 and H3N8 subtypes of influenza A viruses was not statistically significant (p > 0.05). The mean titers for influenza and parainfluenza viruses, on the other hand, showed a statistically significant difference (p < 0.001). These results indicate a better antibody response from equines to parainfluenza 3 virus than to the equine influenza viruses. No statistically significant differences in the responses against H7N7 and H3N8 subtypes of influenza A and parainfluenza 3 viruses were observed according to the gender (female, male) or the age (≤ 2 to 20 years-old) groups. This study provides evidence of the concomitant presence of two subtypes of the equine influenza A (H7N7 and H3N8) viruses and the parainfluenza 3 virus in equines in Brazil. Thus, it is advisable to vaccinate equines against these respiratory viruses.