Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only 30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

The Australian paralysis may be the missing link tick in the transmission of Hendra virus from bats to horses to humans

Hendra virus is a new virus of the family Paramyxoviridae. This virus was first detected in Queensland, Australia, in 1994; although, it seems that the virus has infected fruit-eating bats (flying-foxes) for a very long time. At least 2 humans and 15 horses have been killed by this virus since it first emerged as a virus that may infect mammals other than flying-foxes. Hendra virus is thought to have moved from flying-foxes to horses, and then from horses to people. There is a reasonably strong hypothesis for horse-to-human transmission: transmission of virus via nasal discharge, saliva and/or urine. In contrast, there is no strong hypothesis for flying-fox-to-human transmission. I present evidence that the Australian paralysis tick, Ixodes holocyclus, which has apparently only recently become a parasite of flying-foxes, may transmit Hendra virus and perhaps related viruses from flying-foxes to horses and other mammals. (C) 2003 Elsevier Science Ltd. All rights reserved.

Detection of West Nile Virus Infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in-New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Epizootic activity of Murray Valley encephalitis and Kunjin viruses in an aboriginal community in the Southeast Kimberley region of Western Australia: Results of mosquito fauna and virus isolation studies

We undertook annual surveys of flavivirus virus activity in the community of Billiluna of Western Australia in the southeast Kimberley region between 1989 and 2001. Culex annulirostris was the dominant mosquito species, particularly in years of above average rains and flooding. Murray Valley encephalitis (MVE) virus was isolated in 8 of the 13 years of the study from seven mosquito species, but more than 90% of the isolates were from Cx. annulirostris. The results suggest that MVE virus is epizootic in the region, with activity only apparent in years with average or above average rainfall and increased numbers of Cx. annulirostris. High levels of MVE virus activity and associated human cases were detected only once (in 1993) during the survey period. Activity of MVE virus could only be partially correlated with wet season rainfall and flooding, suggesting that a number of other factors must also be considered to accurately predict MVE virus activity at such communities.

Distribution of Sugarcane mosaic virus in sugarcane plants

Variation in the concentration of virus in different parts of the plant has implications for virus-indexing programs. To allow more reliable detection of Sugarcane mosaic virus (SCMV), the distribution of the virus in sugarcane plants after artificial inoculation was studied using a reverse transcription polymerase chain reaction (RT-PCR) assay. Leaves of susceptible and moderately resistant sugarcane were mechanically inoculated with SCMV 6 weeks after planting. Weekly for 8 weeks after inoculation, plants were examined for mosaic symptoms and samples of leaves, roots and tillers were tested by RT-PCR to detect virus. SCMV moved from the point of inoculation to younger leaves, roots and tillers and eventually to leaves that emerged prior to inoculation. The pattern of SCMV distribution in moderately resistant and susceptible cultivars was not substantially different. However, the virus moved more slowly in the moderately resistant than in the susceptible cultivar. Young leaves proved to be the most suitable tissue for testing.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Análise transcriptômica da interação mamoeiro-Papaya Meleira Virus

O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas nas regiões tropicais e subtropicais do mundo. O Brasil faz parte do grupo dos países que mais produzem e exportam mamão no mundo. O Espírito Santo e a Bahia são responsáveis por mais de 70% da área brasileira produtora deste fruto. Porém, doenças causadas por microrganismos infecciosos afetam de modo considerável sua produção. Entre as principais doenças, destaca-se a meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV), que ainda não possui uma cultivar resistente. Interessantemente os sintomas somente são desencadeados após a frutificação. Os mecanismos moleculares envolvidos no desenvolvimento dos sintomas e na resposta de defesa da planta ao PMeV ainda não foram esclarecidos. Para entender os pontos chaves desta interação, que permitam o desenvolvimento de metodologias de melhoramento genético, um estudo transcriptômico foi abordado. A tecnologia RNA-seq foi usada para o sequenciamento do transcriptoma a partir de plantas com 3, 6 e 8 meses de idade após plantio, inoculadas e não inoculadas com o PMeV. Os genes diferencialmente expressos nos 3 tempos e nas duas condições foram preditos e analisados. Estas análises revelaram um padrão de expressão geral dos genes envolvidos nesta interação. Foram encontrados 21 genes com o perfil de expressão alterado nas plantas inoculadas exclusivamente nos seis meses de idade. Destes, 8 genes envolvidos em processos de respostas de defesa e morte celular, resposta ao estresse e resposta ao estímulo biótico e abiótico foram reprimidos; enquanto os demais (13 genes), envolvidos principalmente em processos metabólicos primários, biogêneses, diferenciação e ciclo celular, comunicação e crescimento celular, bem como processos envolvidos em reprodução, e desenvolvimento da floração, foram superexpressos. Estes resultados sugerem que, aos seis meses de idade, a planta é obrigada a alterar seu programa de expressão gênica, direcionando a resposta para os processos próprios do desenvolvimento, requeridos nesse estádio fisiológico, que primam sob a resposta ao estresse, fato que finalmente leva ao desenvolvimento dos sintomas.

Aids e estomatite aftóide recidivante

O estado de imunodeficiência em pacientes HIV positivos tem sido causa de episódios severos de Estomatite Aftóide Recidivante (EAR). OBJETIVO: Este estudo objetiva estabelecer evidências da relação entre o surgimento (ou agravamento) de EAR com o estado de imunossupressão causado pelo vírus HIV, por meio da contagem de células CD4+, CD8+ e da carga viral infectante. FORMA DE ESTUDO: estudo de série. MATERIAL E MÉTODO: Noventa e quatro pacientes HIV (1)-positivos (25 mulheres e 69 homens) com EAR foram acompanhados no ambulatório de Aids da Divisão de Clínica ORL do Hospital das Clínicas da FMUSP no período de janeiro de 1998 a dezembro de 2003. A idade dos pacientes variou de 19 a 63 anos (média = 35,3 anos). RESULTADO: Os pacientes com Aids e soropositivos apresentaram, respectivamente, oito aftas e duas aftas por surto. Da mesma maneira, os pacientes portadores de úlceras do tipo major apresentaram menor contagem de células CD8+, CD4+ e relação CD4+/CD8+ e maior valor médio da carga viral do que os pacientes portadores de aftas herpetiformes e minor. Entre os portadores de aftas minor e herpetiforme não houve diferença estatística. CONCLUSÕES O aparecimento das lesões, principalmente as do tipo major, está diretamente relacionado ao estado imunitário do paciente soropositivo, acarretando déficits nutricionais e piora na qualidade de vida. Desta forma, o diagnóstico e tratamento da EAR é um desafio que não deve ser desprezado.

O papel da imunofluorescência direta na fisiopatologia e no diagnóstico diferencial da estomatite aftóide recorrente

A estomatite aftóide recorrente é doença caracterizada por aparecimento periódico de aftas na mucosa oral, cuja etiologia e fisiopatologia não estão bem explicadas. Estudos recentes com imunofluorescência direta mostram resultados controversos. Alguns revelam que o distúrbio básico está relacionado à imunidade humoral, enquanto outros apontam alterações da imunidade celular. Formas atípicas de estomatite aftóide podem fazer diagnóstico diferencial com doenças vésico-bolhosas como pênfigo vulgar. OBJETIVO: Verificar a presença de imunecomplexos na mucosa de pacientes com estomatite aftóide e utilidade do método no diagnóstico diferencial com dermatopatias bolhosas. CASUÍSTICA E MÉTODO: 23 pacientes portadores de estomatite aftóide, de modo prospectivo, foram incluídos no estudo. Todos foram submetidos à biópsia de mucosa sob anestesia local para retirada de dois fragmentos. Um deles foi enviado para exame histológico e, outro, para ser realizada a imunofluorescência direta. RESULTADOS: As 23 amostras no exame histológico revelaram processo inflamatório inespecífico ulcerado. As amostras enviadas para imunofluorescência resultaram negativas e apenas uma revelou presença de complemento em membrana basal. CONCLUSÃO: Baseado em nossos resultados, concluímos que pacientes portadores de EAR não apresentam depósitos de imunecomplexos na mucosa da cavidade bucal e a imunofluorescência é útil no diagnóstico diferencial entre a doença e dermatopatias bolhosas.

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.

Surface plasmon resonance as a tool in the functional analisys of an immunodominant site in foot-and-mouth disease virus

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments. Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide. Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide ? mAb 4C4 complex showed paratope ? epitope interactions identical to all FMDV peptide ? mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope ? paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).

Virus-Resistance Genes: The Mouse Model

Os Homens e os animais diferem grandemente nas suas respostas às infecções virais. Os vírus podem induzir, em alguns, sintomas ligeiros, enquanto que em outros podem provocar patologias graves mesmo mortais. Acumulam-se evidências que o património genético é um dos factores primordiais a condicionar e contribuir para a complexidade das interações vírus-hospedeiro. A identificação de genes com papel na resposta à infecção viral tornouse pois o tema de investigação de muitos laboratórios, com o objectivo de elucidar os processos fisiopatológicos que regem e determinam esse tipo de resposta. Neste artigo de revisão pretende-se ilustrar como o modelo murino têm sido utilizado para a identificação de genes de resistência viral, e como estes podem funcionar como base para a descoberta de genes homólogos em outras espécies. na elucidação dos mecanismos de resistência, e em novos componentes da reunir todos os genes de resistência viral descobertos em murganhos.