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HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains. / Ph. D.

Bluetongue (BT) is a non-contagious disease of mainly sheep but other ruminants like cattle, goats, and wild ruminants like alpacas, African antelopes and deer can also be affected. It is transmitted by Culicoides midges and its occurrence is seasonal, especially after good rains. The disease is subsiding when temperatures drop. The virus is distributed throughout the world in the tropical, subtropical and temperate areas where there are culicoides vectors which can transmit it (Tabachnick et al., 2011). This includes most countries in Africa, the Middle East, India, China, Australia, the United States of America, Canada and Mexico. Until 2008 24 BTV serotypes were known, but from 2008, data on the 25th serotype was published and recently, the 26th serotype has been identified (Hofmann et al., 2008; Maan et al., 2012a). In Africa 21 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze–dried live attenuated polyvalent BTV vaccine. Currently the vaccine used in the Southern African Development Community (SADC) region is produced by Onderstepoort Biological Products (OBP). The vaccine is constituted of fifteen serotypes of the bluetongue virus (BTV) divided into three separate bottles. Each bottle contains five serotypes. The inoculation procedures are that bottle B is given three weeks after bottle A and bottle C three weeks after bottle B. The full immunity is established three weeks after the last bottle. The vaccine is effective and it induces both humoral and cellular immune response (Dungu et al., 2004). However, the challenge with the vaccine is that during outbreaks, sheep might not have nine full weeks to develop protection against the disease; and the farmer loses money on treatment and death of animals. Hence the purpose of the study is to determine whether the number of serotypes in the vaccine can be reduced without affecting efficacy; thus shorten the time taken for the full development of immunity after vaccination of animals. This study is based on previously reported cross-neutralization of specific BTV serotypes in in vitro studies by Howell et al. (1970) and Dungu et al. (2004). Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control), 1, 8 (unrelated serotypes) 9, 10 and 11 in sheep using the serum neutralization test (SNT). The unvaccinated animals in all groups reacted to the challenge material. The animals vaccinated with and challenged with BTV-4, showed good immune response. Those animals that were vaccinated with BTV-4 and challenged with BTV-1 which is not directly related to BTV-4 (Howell et al., 1970), only 20% of the group was completely protected and did not show clinical signs other than a temperature reaction. The rest showed clinical signs, however the reaction was not as severe as the unvaccinated animal. The animals challenged with BTV-9 and 11 had good protection while those challenged with BTV-10, some showed good protection, some got very sick while others had mild clinical signs. The results showed that BTV serotype 4 do not only develop a specific immune response but can also protect against other serotypes. Future studies should be done looking at more serotypes but also look at the specific titres used per serotype in the vaccine. The development of cellular immunity should also be taken in consideration. With further studies it should be possible to develop a vaccine with fewer serotypes without compromising the immunity against the disease. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. Soybean yield losses of 10-40% have been reported as a consequence of BPMV infection. The complete nucleotide sequences of two strains, K-Ha1 and K-Ho1, were determined. Field isolates of BPMV were classified into two distinct subgroups (I and II) based on slot blot hybridization and sequence analyses. Full-length cDNA clones from which infectious transcripts can be produced were constructed for strains K-G7, K-Ho1 and K-Ha1. Whereas strains K-Ha1 and K-G7 induced mild or moderate symptoms in infected soybean plants, strain K-Ho1 produced very severe symptoms. Symptom severity was mapped to RNA1. Chimeric RNA1 constructs were generated by exchanging full or partial coding regions of the five RNA1-encoded mature proteins between the full-length cDNA clones of the three RNA1s and the resultant transcripts were inoculated onto soybean. The results showed that the coding regions of the protease co-factor (Co-pro) and the putative helicase (Hel) are determinants of symptom severity. Although symptom severity correlated well with accumulation of viral RNA, neither the Co-pro nor Hel protein could be demonstrated as a suppressor of RNA silencing. Furthermore, separate expression of the Co-pro or Hel proteins from a PVX vector induced necrosis on the inoculated leaves of Nicotiana benthamiana. Characterization of BPMV K-Ho1 indicated that it is a diploid reassortant, containing two distinct types of RNA1s and one type of RNA2. Examination of field isolates from various locations in the United States and Canada revealed that diploid reassortants are of frequent occurrence in natural populations of BPMV. The vary severe symptoms induced by BPMV K-Ho1 can be mimicked by inoculation of plants with a mixture of RNA1 transcripts from two distinct strain subgroups and RNA2 transcript from either subgroup. Plants inoculated with a mixture of transcripts containing two types of RNA1 from the same strain subgroup did not produce very severe symptoms. These are due to interactions between two distinct types of RNA1s. At present, no soybean cultivars with resistance to BPMV are commercially available. Therefore, the feasibility of cross protection as an alternative disease management strategy was studied. Two mild strains of BPMV (K-Da1 and K-Ha1), belonging to subgroup II, were tested for their ability to protect infected plants against a severe strain (K-Ho1). Inoculation of the soybean cultivar Essex on the primary leaves with either of the two mild strains conferred complete protection against challenge inoculation with the severe strain K-Ho1, regardless of the timing of challenge inoculation. Cross-protection was evident regardless of whether virions or BPMV-RNA were used as inocula. Cross protection was independent of the soybean cultivar used and method of virus inoculation, sap-inoculation or by the bean leaf beetle, vector of BPMV. Protection was complete and durable.

Doenças causadas por virus do gênero Begomovirus (família Geminiviridae) são incomuns em espécies de passifloras. Nos últimos dez anos, entretanto, foram encontrados no Brasil begomovirus infectando passifloras em pomares nos municipios de São Fidelis (RJ), Paragominas (PA), Patos de Minas (MG) e Araguari (MG). Estes isolados foram transmitidos mecanicamente para plantas de Nicotiana benthamiana, que apresentaram sintomas de mosaico e deformação foliar. Também foi possível a transmissão para plantas de Sida rhombifolia através de inoculação por biobalística com o DNA amplificado do isolado de São Fidelis. Estas plantas demonstraram sintomas de mosaico amarelo e deformação foliar. O DNA total extraído de plantas infectadas foi amplificado por RCA, sendo que o componente A (DNA-A) dos isolados de Paragominas e Patos de Minas foram sequenciados diretamente por \"primer walking\". O DNA-A dos isolados de São Fidelis e Araguari foram clonados e sequenciados. As sequências de nucleotideos dos isolados de Paragominas e de São Fidelis apresentaram 90% de similaridade ao Sida mottle virus (SiMoV), enquanto a sequência de nucleotídeos do isolado de Araguari apresentou 96% de similaridade ao Sida micrantha mosaic virus (SimMV). Assim esses isolados encontrados em maracujazeiro podem ser considerados estirpes do SiMoV e SimMV, respectivamente. Não foi possivel a transmissão desses isolados através de Bemisia tabaci biótipo B, no entanto, os insetos foram capazes de adquirir o vírus. O isolado de São Fidelis foi detectado separadamente na região onde se encontra a glandula salivar (cabeça e protórax) e na região posterior do inseto, indicando que o vírus transpos a barreira do mesenteron e circulou pela hemolinfa do inseto. Alves (2008) obteve uma forma atenuada do isolado de São Fidelis através de inoculações mecanicas em plantas de N. benthamiana. O DNA-A desta forma atenuada foi sequênciado e apresentou 90% de identidade ao isolado do qual se originou. A forma atenuada do begomovirus foi capaz de proteger plantas de maracujazeiro contra a estirpe severa do vírus. / Diseases caused by begomoviruses (family Geminiviridae) are hardly found in Passiflora species. In the last years, however, begomovirus infected passionflowers were found in orchards in the counties of São Fidelis (state of Rio de Janeiro), Paragominas (Pará), Araguari and Patos de Minas (Minas Gerais). These isolates were mechanically transmitted to Nicotiana benthamiana plants, which showed variable symptoms of mosaic and leaf distortion. Another susceptible host is Sida rhombifolia, which was biolistic inoculated with amplified DNA of São Fidelis isolate, and showed symptoms of yellow mosaic and leaf distortion. Total DNA extracted from field infected passiflora was amplified by RCA, and the DNA-A of Paragominas and Patos de Minas isolates were directly sequenced by primer walking. The A component of São Fidelis and Araguari isolates were cloned and also completely sequenced. The complete nucleotide sequence of DNA-A of Araguari isolate shared 96% identity with that of Sida micrantha mosaic virus (SimMV), whereas the DNA-A of Paragominas and São Fidelis isolates shared 90% identity with that of Sida mottle virus (SiMoV). These viruses may be consider as strains of SiMoV and SimMV, respectively. It was not possible to transmit these isolates by Bemisia tabaci biótipo B, although the insects were able to acquire the virus. São Fidelis isolate could be detected separately at salivary gland region and posterior region of the insect, indicating that the virus could cross the digestive tract and circulate in the hemolymph. Alves (2008) obtained a mild strain of São Fidelis isolate by mechanical inoculation in N. benthamiana plants. The mild isolate was able to protect passionflower against the severe isoalte of this begomovirus.

Doenças causadas por virus do gênero Begomovirus (família Geminiviridae) são incomuns em espécies de passifloras. Nos últimos dez anos, entretanto, foram encontrados no Brasil begomovirus infectando passifloras em pomares nos municipios de São Fidelis (RJ), Paragominas (PA), Patos de Minas (MG) e Araguari (MG). Estes isolados foram transmitidos mecanicamente para plantas de Nicotiana benthamiana, que apresentaram sintomas de mosaico e deformação foliar. Também foi possível a transmissão para plantas de Sida rhombifolia através de inoculação por biobalística com o DNA amplificado do isolado de São Fidelis. Estas plantas demonstraram sintomas de mosaico amarelo e deformação foliar. O DNA total extraído de plantas infectadas foi amplificado por RCA, sendo que o componente A (DNA-A) dos isolados de Paragominas e Patos de Minas foram sequenciados diretamente por \"primer walking\". O DNA-A dos isolados de São Fidelis e Araguari foram clonados e sequenciados. As sequências de nucleotideos dos isolados de Paragominas e de São Fidelis apresentaram 90% de similaridade ao Sida mottle virus (SiMoV), enquanto a sequência de nucleotídeos do isolado de Araguari apresentou 96% de similaridade ao Sida micrantha mosaic virus (SimMV). Assim esses isolados encontrados em maracujazeiro podem ser considerados estirpes do SiMoV e SimMV, respectivamente. Não foi possivel a transmissão desses isolados através de Bemisia tabaci biótipo B, no entanto, os insetos foram capazes de adquirir o vírus. O isolado de São Fidelis foi detectado separadamente na região onde se encontra a glandula salivar (cabeça e protórax) e na região posterior do inseto, indicando que o vírus transpos a barreira do mesenteron e circulou pela hemolinfa do inseto. Alves (2008) obteve uma forma atenuada do isolado de São Fidelis através de inoculações mecanicas em plantas de N. benthamiana. O DNA-A desta forma atenuada foi sequênciado e apresentou 90% de identidade ao isolado do qual se originou. A forma atenuada do begomovirus foi capaz de proteger plantas de maracujazeiro contra a estirpe severa do vírus. / Diseases caused by begomoviruses (family Geminiviridae) are hardly found in Passiflora species. In the last years, however, begomovirus infected passionflowers were found in orchards in the counties of São Fidelis (state of Rio de Janeiro), Paragominas (Pará), Araguari and Patos de Minas (Minas Gerais). These isolates were mechanically transmitted to Nicotiana benthamiana plants, which showed variable symptoms of mosaic and leaf distortion. Another susceptible host is Sida rhombifolia, which was biolistic inoculated with amplified DNA of São Fidelis isolate, and showed symptoms of yellow mosaic and leaf distortion. Total DNA extracted from field infected passiflora was amplified by RCA, and the DNA-A of Paragominas and Patos de Minas isolates were directly sequenced by primer walking. The A component of São Fidelis and Araguari isolates were cloned and also completely sequenced. The complete nucleotide sequence of DNA-A of Araguari isolate shared 96% identity with that of Sida micrantha mosaic virus (SimMV), whereas the DNA-A of Paragominas and São Fidelis isolates shared 90% identity with that of Sida mottle virus (SiMoV). These viruses may be consider as strains of SiMoV and SimMV, respectively. It was not possible to transmit these isolates by Bemisia tabaci biótipo B, although the insects were able to acquire the virus. São Fidelis isolate could be detected separately at salivary gland region and posterior region of the insect, indicating that the virus could cross the digestive tract and circulate in the hemolymph. Alves (2008) obtained a mild strain of São Fidelis isolate by mechanical inoculation in N. benthamiana plants. The mild isolate was able to protect passionflower against the severe isoalte of this begomovirus.

West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic. / Master of Science / West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging flaviviruses transmitted by mosquito bites, primarily among perching birds. However, mosquitoes can also transmit these viruses to animals and humans, especially in regions where these viruses are prevalent. The immune system, which defends against pathogens and other diseases, usually combats these viruses effectively, preventing most people from developing symptoms. The immune system has two main branches: the innate immune system, which confers immediate defense, and the adaptive immune system that includes antibodies and certain long-lasting memory cells, that can fight off infections years after the initial exposure to the same or similar disease-causing agents. Occasionally, the immune system fails to fight these viruses, particularly in the elderly or those with chronic diseases, leading to fever or severe brain inflammation called encephalitis. Currently, WNV and SLEV are circulating in the Americas, while WNV and USUV are present in European countries. Due to similar transmission methods, infection patterns, and geographical overlap, individuals might be sequentially infected with WNV and USUV in Europe, and WNV and SLEV in the Americas in their lifetime. These viruses also share common antigens, which can induce similar immune responses. Therefore, the immune response to one virus might protect against another with similar antigens. It has been reported that the immune response induced by WNV can protect against encephalitis caused by USUV or SLEV. However, it remains unclear whether this cross-protection is mediated by antibodies or a certain type of immune cells called T cells. This study investigates whether antibodies induced by WNV infection can protect against SLEV or USUV in a mouse model. Plasma, the part of blood containing antibodies, is referred to as convalescent plasma when collected after an individual has recovered from an infection or disease. Human WNV convalescent plasma was tested against SLEV and USUV using a plaque reduction neutralization test to determine the antibodies’ ability to prevent viral infection in a laboratory setting. Human WNV convalescent plasma effectively prevented SLEV and USUV from infecting cells. We then developed a mouse model that could be infected with SLEV or USUV and mimic human disease. Groups of mice were systematically transferred with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum one day before the infection with WNV, SLEV, or USUV. Disease conditions, such as weight loss, reduced movement, hunchback, fur loss, and occasional paralysis, were monitored until the infected mice were humanely euthanized. After euthanasia, the brains of the mice were collected to measure viral load and examine signs of encephalitis. We observed asymptomatic disease outcomes reflecting natural human infection. Both human and mouse WNV convalescent samples reduced viral load in the blood for a period in both SLEV and USUV-challenged groups. Mice treated with human WNV convalescent plasma showed a trend of lower SLEV in their brains. Additionally, mice treated with mouse WNV convalescent serum had lower SLEV titers in their brains compared to those treated with mouse normal serum. Overall, these findings suggest that human WNV convalescent plasma provides some crossprotection against SLEV- and USUV-induced diseases. Understanding the mechanism of crossprotection is crucial for developing therapeutics against these viruses and predicting disease outcomes in areas where multiple viruses of the Japanese encephalitis virus serocomplex are prevalent.

Vírus da raiva provenientes de 23 morcegos de espécies hematófagas, frugívoras e insetívoras foram caracterizados geneticamente pelo seqüenciamento completo da região que codifica a nucleoproteína N. A análise filogenética das seqüências, incluindo lyssavirus e isolados de morcegos do Chile e Estados Unidos, mostrou que os diferentes isolados do vírus da raiva foram de modo geral segregados em quatro grupos genéticos distintas: morcegos hematófagos, morcegos insetívoros 1, 2 e 3. Os morcegos insetívoros 1 constituiram-se por isolados de Eptesicus furinalis: BR-EF1, BR-EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; os morcegos insetívoros 2 consistiram de isolados de Molosssus spp: BR-MM1, BR-MM2 e BR- MA1 e os morcegos insetívoros 3 isolados de Nictinomops laticaudatus: BR-NL1 e BR-NL3. A homologia de nucleotídeos entre cada grupo de morcegos insetívoros 1, 2 e 3 foi maior que 99%, 97% e 99%, respectivamente. O grupo de morcegos hematófagos foi representado pelos isolados de: 3 morcegos hematófagos Desmodus rotundus (BR-DR1, BR-DR2 e BR-DR3); 5 morcegos frugívoros Artibeus lituratus BR-AL1, BR-AL2, BR-AL3, BR-AL4 e Artibeus planirostris BRAP1; 2 morcegos insetívoros (BR-MR1 e BR-EA2) e 2 de espécies não identificadas (BR-BAT1 e BR-BAT2). Entre as amostras seqüenciadas foram selecionadas cinco (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR-DR1) e um isolado de cão (BR-C) para os estudos de patogenicidade em camundongos albinos suíços inoculados pela vias intracerebral (IC) e intramuscular (IM). Todas as amostras quando inoculadas em camundongos pela via IC apresentaram-se patogênicas, provocando a morte dos mesmos num período de 4 a 14 dias pós-inoculação. No entanto, 500DLIC50 das mesmas amostras inoculadas pela via IM levaram a uma mortalidade de camundongos de: 60% (BR-DR1); 50% (BR-C, BR-NL); 40% (BR- AL3); 9,5% (BR-MM1); 5,2% (BR-EF10). As mesmas amostras foram utilizadas para a verificação de proteção cruzada, conferida por vacina comercial de uso animal, de camundongos que receberam uma ou duas doses de vacina pela via subcutânea (SC) e desafiados pelas vias IC e IM. Camundongos inoculados com duas doses de vacina foram protegidos quando desafiados pela via IC, com todas as amostras testadas. Quando os camundongos receberam uma dose da mesma vacina houve proteção parcial daqueles desafiados com as amostras de vírus PV e BR-C. Houve proteção de 100% dos camundongos desafiados pela via IM, com exceção daqueles vacinados com uma dose de vacina e desafiados com a amostra PV que apresentaram um índice de 66% de sobreviventes. Os resultados indicam a possibilidade de existir variantes do vírus da raiva espécies específicas circulando em morcegos. Sugerem ainda, que espécies de morcegos hematófagos, frugívoros e insetívoros compartilham o mesmo polimorfismo de vírus. A vacina comercial contra a raiva contendo vírus inativado e de uso veterinário protegeu os camundongos contra o desafio com as diferentes amostras testadas, sugerindo que as vacinas usualmente utilizadas são efetivas no tratamento profilático da raiva transmitida por morcegos, apesar da marcada diferença de neurovirulência dos diferentes isolados quando inoculados em camundongos pela via IM. / Twenty-three rabies viruses isolated from hematophagous, frugivorous and insectivorous bats were characterized genetically by complete sequencing of the region coding the nucleoprotein N. The phylogenetic analysis of the sequences, including the lyssavirus and the bat isolates from Chile and USA revealed that the isolates were segregated into four distinct genetic lineages: those related to the vampire bats and to the insectivorous bats 1, 2 and 3. The isolates related to the insectivorous bats 1 were from the Eptesicus furinalis: BR-EF1, BR- EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; those of the insectivorous bats2 included the isolates from Molosssus spp: BR-MM1, BR-MM2 and BR-MA1 and the group 3, by the isolates from the Nictinomops laticaudatus: BR-NL1 and BR-NL3. The homology among each group of the insectivorous bats 1, 2 and 3 were greater than 99%, 97% and 99%, respectively. The lineage related to vampire bats was represented by three isolates from the D. rotundus (BR-DR1, BR-DR2 e BR-DR3); five from the fruit bats Artibeus lituratus (BR-AL1, BR-AL2, BR-AL3, BR-AL4) and Artibeus planirostris (BRAP1); two from insectivorous bats (BR-MR1 and BR-EA2) and two from unidentified species (BR-BAT1 and BR-BAT2). Among the sequenced amples, five bat isolates (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR- DR1) and one dog isolate (BR-C) were selected for the study of their pathogenicity in Swiss mice, inoculating through intracerebral (IC) and intramuscular (IM) routes. All the isolates, when inoculated via IC, were pathogenic, provoking death in 4 - 14 post inoculation days. However, mice inoculated with 500ICLD50 of the same isolates through IM route were found with different death rates: 60.0% (BR-DR1); 50.0% (BR-C, BR-NL); 40.0% (BR-AL3); 9.5% (BR-MM1) and 5.2% (BR-EF10). The same isolates were used for the assessment of cross protection conferred by a commercial vaccine of veterinary use. The mice were vaccinated subcutaneously, receiving either one or two shots of vaccine, and challenged through IC and IM routes. Mice receiving two shots were protected against all the isolates, when challenged intracerebrally. Mice receiving one shot were found only partially protected against the challenge with the fixed PV strain and BR-C isolate. Mice challenged intramuscularly showed 100.0% of protection, with the exception of those vaccinated with one dose and challenged with PV strain, which were found with 66.0% of survivors. These results indicate the possibility of the existence of rabies virus variants circulating in different species of bat population. The data also suggest that the vampires, frugivorous and insectivorous bats share the same lineage of rabies viruses. The commercial vaccine has protected the mice against the challenge with different rabies virus isolates, suggesting that the vaccines usually employed in the field are effective, although some marked difference in neurovirulence by IM inoculation was found among the isolates tested.

Entre as cucurbitáceas cultivadas no Brasil, a abobrinha de moita (Cucurbita pepo L.) apresenta grande importância econômica, notadamente no Estado de São Paulo, a maior área de plantio. No Brasil, já foram encontrados nove vírus capazes de infectar esta espécie. Os mais freqüentes e responsáveis por prejuízos significativos à produção da abobrinha de moita são os do mosaico do mamoeiro – estirpe melancia (Papaya ringspot virus - type W – PRSV-W) e do mosaico amarelo da abobrinha (Zucchini yellow mosaic virus – ZYMV), devido principalmente à sua alta sensibilidade. A premunização das plantas com estirpes fracas vem sendo investigada há mais de 10 anos no Brasil e surge como uma alternativa interessante para controle destas viroses. A premunização de abobrinha de moita só foi estudada até o momento em plantas da variedade Caserta. O presente trabalho teve o objetivo de dar continuidade a estes estudos avaliando o efeito de estirpes premunizantes PRSV-W-1 e ZYMV-M na produção quantitativa e qualitativa de quatro variedades comerciais de C. pepo: Samira, Novita Plus, AF-2847 e Yasmin. Os resultados obtidos em experimentos conduzidos em estufa plástica mostraram que as plantas destas variedades infectadas com a estirpe fraca ZYMV-M, não exibiram sintomas foliares acentuados e não apresentaram alterações na produção quantitativa e qualitativa de frutos, quando comparada com a produção das plantas sadias. No entanto, as plantas dessas mesmas variedades infectadas com a estirpe PRSV-W-1, só ou em mistura com a estirpe ZYMV-M, exibiram sintomas acentuados de mosaico foliar e alteração significativa na qualidade dos frutos, caracterizada pelo escurecimento da casca. Não houve alteração significativa na quantidade de frutos produzidos por essas plantas e no peso médio dos frutos. Quando plantas dessas variedades foram infectadas somente com a estirpe fraca PRSV-W-1 e conduzidas paralelamente em condições de campo e estufa plástica, constatou-se intensificação dos sintomas nos frutos e nas folhas das plantas infectadas conduzidas principalmente em estufa, porém a produção quantitativa mais uma vez não foi alterada. A variedade Samira mostrou-se a mais sensível ao PRSV-W-1 em ambas as condições. Os resultados sugerem que fatores ambientais, além da interação da estirpe fraca com a variedade de abobrinha, parecem interferir na expressão de sintomas nos frutos das plantas infectadas com a estirpe PRSV-W-1. Também foi objeto de estudo desse trabalho a identificação da melhor hospedeira, entre algumas cucurbitáceas, para a multiplicação e manutenção das duas estirpes fracas, e a avaliação da eficiência dos afídeos Myzus nicotianae e M. persicae na transmissão da estirpe ZYMV-M. Os resultados mostraram que a estirpe ZYMV-M parece atingir as maiores concentrações em C. pepo cv. Caserta e em C. melo cv. Casca-de-Carvalho. Para a estirpe PRSV-W- 1, as melhores hospedeiras foram C. pepo cv. Caserta, seguida de C. lanatus cv. Crimson Sweet e C. sativus cv. Safira. M. nicotianae e M. persicae transmitiram a estirpe fraca ZYMV-M para 11,7% e 0% das plantas de abobrinha de moita cv. Caserta, respectivamente. Enquanto a estirpe severa foi transmitida para 47,0% e 80% das plantas teste, respectivamente. / Zucchini squash (Cucurbita pepo L.) is widely cultivated in Brazil, especially in the State of São Paulo, which is the major producer. Nine viruses capable to infect this species have already been described in Brazil. The most frequent and responsible for significant yield losses are the potyviruses Papaya ringspot virus - type W (PRSV-W) and Zucchini yellow mosaic virus (ZYMV), for which zucchini squash is highly susceptible and intolerant. Preimmunization with mild strains of both viruses have been investigated in Brazil for the last 10 years and appears as and interesting option for the control of these viruses. As preimmunization have been evaluated only for zucchini squash cv. Caserta, the purpose of the present work was to investigate the effect of the mild strains PRSV-W-1 and ZYMV-M on the quantitative and qualitative yield of four other commercial zucchini squash varieties: Samira, Novita Plus, AF-2847 e Yasmin. The results of an experiment carried out under plastic-house showed that mild strain ZYMV-M induced mild symptoms on the leaves of the plants of all varieties, but did not affect the yield of marketable fruits, as compared to those from the respective healthy controls. On the other hand, plants from all varieties exhibited accentuated mosaic and leaf malformation when infected with mild strain PRSV-W-1, alone or in mixture with ZYMV-M. The amount of fruits harvested (number and average weight) from these plants was similar to that from the respective healthy controls. However, the quality of the fruits was severely affected, since the mild strain PRSV-W-1 induced alteration on the texture and color of the skin. When plants infected only with mild strain PRSV-W-1 were grown simultaneously under field and plastic-house conditions, it was noticed that leaf and fruit symptoms were more intense on those maintained in the plastic-house. Once more the quantitative fruit yield was not affected by this mild strain. Zucchini squash cv. Samira was the most sensitive to PRSV-W-1 under both conditions. Together theses results suggest that environmental variables, besides the interaction between the variety and the mild strain, might influence the expression of symptoms shown by plants infected with PRSV-W-1. In addition to this, the present work also tried to identify the most appropriate host, among some cucurbit species/varieties, for multiplication of both mild strains, and the efficiency of two species of aphids (Myzus nicotianae and M. persicae) on the transmission of the mild strain ZYMV-M. The mild strain ZYMV-M attained the highest concentration in C. pepo cv. Caserta and Cucumis melo cv. Casca-de-Carvalho. The most appropriate hosts for multiplication of mild strain PRSV-W-1 were C. pepo cv. Caserta followed by Citrullus lanatus cv. Crimson Sweet and C. sativus cv. Safira. M. nicotianae and M. persicae transmitted the mild strain ZYMV-M to 11.7% and 0% of the test-plants of zucchini squash cv. Caserta. The transmission of the severe strain of ZYMV by both species of aphids occurred for 47% and 80% of the test-plants, respectively.

O Passion flower little leaf mosaic virus (PLLMV) foi encontrado causando danos severos em plantios de maracujazeiro (Passilora edulis f. flavicarpa) em dois municípios do Estado da Bahia no ano de 2001. Nesses locais foi constatada que a incidência deste begomovirus estava relacionada à colonização das plantas por Bemisia tabaci, cujo biótipo não foi identificado. Até o momento este vírus não parece constituir grave ameaça a cultura do maracujazeiro, o que aparentemente esta relacionado ao fato de P. edulis f. flavicarpa não ser preferida para a alimentação desse aleyrodídeo. Assim, os objetivos deste trabalho foram: a) selecionar espécies silvestres de Passiflora resistentes a este begomovirus que possam ser úteis em futuro programa de melhoramento genético; b) identificar possíveis hospedeiros alternativos do patógeno entre algumas espécies da vegetação espontânea e cultivadas e c) avaliar se adultos de B. tabaci biótipo B presentes no Estado de São Paulo são capazes de transmitir esse vírus. A reação de espécies silvestres de Passiflora foi avaliada por enxertia em maracujazeiro amarelo infectado que serviu como de fonte de inóculo. As avaliações foram feitas por meio da expressão de sintomas, análise de PCR e teste de recuperação do vírus para maracujazeiro amarelo. As espécies P. alata, P. quadrangularis, P. morifolia, P. serrato-digitata, P. suberosa e P. foetida foram suscetíveis ao PLLMV, enquanto P. caerulea, P. cincinnata, P nitida, P. mucronata e P. giberti se mostraram resistentes a este vírus. No estudo de hospedeiros alternativos, primeiramente o PLLMV foi inoculado mecanicamente nas seguintes espécies vegetais: Capsicum annuum, Chenopodium quinoa, Solanum lycopersicon, S. tuberosum, P. edulis f. flavicarpa, Phaseolus vulgaris, Nicotiana benthamiana e Sida sp.. Somente N. benthamiana foi infectada sistemicamente. Posteriormente foram feitas tentativas de transmissão desse begomovirus por meio de enxertia, usando-se como fonte de inóculo (porta-enxerto), plantas infectadas de N. benthamiana. Foram avaliadas as seguintes espécies vegetais: S. pimpinellifolium, S. lycopersicon, S. tuberosum, N. benthamiana, D. stramonium, C. annuum, N. glutinosa e Sida rhombifolia. O vírus foi transmitido somente para plantas de S. pimpinellifolium. As sucessivas transmissões mecânicas do PLLMV em N. benthamiana permitiram a seleção de uma estirpe fraca e protetora deste begomovirus. B. tabaci biótipo B não foi capaz de transmitir o PLLMV. / The Passion flower little leaf mosaic virus (PLLMV) was found causing severe damage in passion flower (Passilora edulis f. flavicarpa ) orchards in two counties of Bahia state, in 2001. The high incidence of this begomovirus was related to the colonization of plants by Bemisia tabaci, whose biotype was not indentified. To date this virus doesnt seem to be a serious threat to the passion flower cultivation, which apparently is related to the fact that P. edulis f. flavicarpa is not a preferred specie for whitefly feeding. The aim of this work were: a) to select wild species of Passifloraceae resistant to PLLMV, that may be useful in future breeding program; b) to indentify some possible alternative hosts of the pathogen among some weed and cultivaded species and c) to evaluat whether adults of B. tabaci biotype B are capable of transmitting this virus. The reaction of wild species of Passifloracea was evaluated by grafting on infected yellow passion fruit that served as a source of inoculum. The evaluation of these plants were done by means of symptoms, PCR test and biological recovery of the virus to yellow passion fruit. The sepecies P. alata, P. quadrangularis, P. morifolia, P. serrato-digitata, P. suberosa and P. foetida were susceptible to PLLMV, while P. caerulea, P. cincinnata, P nitida, P. mucronata and P. giberti were resistant to this virus. In the study of alternative hosts, at first, PLLMV was mechanically inoculated in the following species: Capsicum annuum, Chenopodium quinoa, Solanum lycopersicon, S. tuberosum, Passiflora edulis f. flavicarpa, Phaseolus vulgaris, Nicotiana benthamiana and Sida sp. Only N. benthamiana was systematically infected. Subsequently, graft transmission of PLLMV was carried out using infected N. benthamiana as source of inoculum (root stock). The following species were evaluated: Solanum pimpinellifolium, S. lycopersicon, S. tuberosum, N. benthamiana, D. stramonium, C. annuum, N. glutinosa and Sida rhombifolia.The virus was transmited only to S. pimpinellifolium. Successive mechanical transmissions of PLLMV in N. benthamiana led to the selection of a mild and protective strain of this begomovirus. B. tabaci biotype B was not able to transmit the PLLMV.