Pox viruses have been demonstrated in over 60 types of wild and exotic birds as well as domestic birds. Avipox viruses have been isolated and characterised from fowls, quails, canaries, parrots and lovebirds. This work describes the first isolation of a poxvirus from Jackass penguins (Spheniscus dermersus) and the characterisation of the virus as a separate species of penguinpox virus.

Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.

Pouco se conhece a respeito da incidência ou prevalência da infecção pelo vírus da raiva em morcegos, ou ainda sobre a distribuição do vírus em tecidos e órgãos não nervosos. Os objetivos deste trabalho foram: i) verificar as espécies de morcegos mais freqüentemente envolvidas com a raiva no Estado de São Paulo, Sudeste do Brasil; ii) estudar a distribuição do vírus da raiva em tecidos e órgãos não nervosos de morcegos; iii) estudar os períodos de mortalidade das amostras de vírus da raiva encontradas nos cérebros e glândulas salivares de morcegos, após inoculação intracerebral em camundongos, e iv) comparação do isolamento do vírus da raiva no sistema camundongo e cultura de células de neuroblastoma (N2A). Entre abril de 2002 a novembro de 2003, 4.393 morcegos capturados de diferentes municípios do Estado de São Paulo foram enviados à Seção de Diagnóstico da Raiva do Instituto Pasteur de São Paulo. Destes, 82 (1,87%) foram positivos para raiva pela técnica de imunofluorescência aplicada aos materiais do cérebro e 33 morcegos pertenciam ao gênero Artibeus sp; 15 Myotis sp; 10 Epitesicus sp; 5 Lasiurus sp; 4 Nyctinomops sp; 4 Tadarida sp; 3 Histiotus sp; 1 Molossus sp; 1 Eumops sp e 6 vampiros Desmodus rotundus. A distribuição do vírus em diferentes órgãos foi examinada pela inoculação de camundongos e células N2A com suspensões a 20% preparadas a partir de fragmentos do cérebro, glândula salivar submandibular, pulmão, língua, coração, bexiga urinária, rins, gordura interescapular, músculo peitoral, trato genital (testículos ou ovários e útero) e estômago. O vírus foi prontamente recuperado de tecidos e órgãos não nervosos com diferentes graus de sensibilidade, tanto em camundongos como em células N2A, e os órgãos mais apropriados para o isolamento viral foram os cérebros e glândulas salivares. Os períodos máximos de mortalidade observados para os vírus presentes nos cérebros usualmente foram mais curtos que os das glândulas salivares, a média do período máximo &plusmn; desvio padrão calculado para os cérebros de morcegos hematófagos foi de 15,33 &plusmn; 2,08 dias e para as glândulas salivares, 11,33 &plusmn; 2,30 dias; para os morcegos insetívoros, 16,45 &plusmn; 4,48 dias para os cérebros e para as glândulas salivares, 18,91 &plusmn; 6,12 dias; e para os morcegos frugívoros, as suspensões cerebrais apresentaram período máximo médio 12,60 &plusmn; 2,13 dias e para as glândulas salivares, 15,67 &plusmn; 4,82 dias. O teste de ANOVA indicou existir diferenças significantes entre os períodos de mortalidade correspondentes às suspensões preparadas a partir dos cérebros de morcegos insetívoros (período mínimo) e glândulas salivares de morcegos insetívoros (período máximo) e entre glândulas salivares de morcegos insetívoros (período máximo) e cérebros de morcegos frugívoros (período mínimo), com p<0.001. O uso de células N2A para o primo-isolamento do vírus da raiva a partir de tecidos e órgãos não nervosos de morcegos, diferente de cérebros, não mostraram resultados consistentes, especialmente devido à contaminação bacteriana e fator toxicidade / Little is known about the incidence of infection or the prevalence rate of rabies in bats, or the distribution of virus in non-nervous tissues and organs. The aim of this work was to study: i) the most frequent species of bats involved with rabies virus infection in the State of São Paulo, Southeast Brazil; ii) the distribution of rabies virus in tissues and non-nervous organs of bats; iii) the mortality periods of virus found in brains and salivary glands of bats after intracerebral inoculation of mice, and iv) comparison of virus isolation in mice and N2A neuroblastoma cell culture. From April 2002 to November 2003, 4,393 bats captured from different municipalities of the State of São Paulo were sent to Rabies Diagnostic Section of the Instituto Pasteur de São Paulo - SP. Among these, 82 (1.87%) were found positive by the immunofluorescence technique applied to brain specimens and 33 bats were of the genus Artibeus sp; 15 Myotis sp; 10 Epitesicus sp, 5 Lasiurus sp, 4 Nyctinomops sp, 4 Tadarida sp, 3 Histiotus sp; 1 Molossus sp, 1 Eumops sp, and 6 vampires Desmodus rotundus. The distribution of virus in the organs was examined by inoculating mice and N2A cells with the 20% suspensions prepared from brain, submaxillary salivary gland, lungs, tongue, heart, urinary bladder, kidneys, brown fat, pectoral muscle, genital tract (testicles or ovaries and uterus), and stomach. The virus was promptly recovered from tissues and non-nervous organs at different degrees of sensitivity in both mice and N2A cells, and the most appropriate organs for the virus isolation were the brains and salivary glands. The maximum mortality periods found for the brain specimens usually were shorter than the salivary glands, the maximum mean perio &plusmn; standard deviation calculated for the brains taken from the vampire bats was 15.33 &plusmn; 2.08 days and for salivary glands, 11.33 &plusmn; 2.30 days; for the insectivorous bats the maximum for the brain suspensions was 16.45 &plusmn; 4.48 days and for the salivary glands, 18.91 &plusmn; 6.12; and for the frugivorous bats, the brain suspensions showed the maximum of 12.60 &plusmn; 2.13 days and the salivary glands, the mean maximum period of 15.67 &plusmn; 4.82 days. The ANOVA test indicated that the most significant differences in the mortality periods were between the suspensions prepared by the brains of insectivorous bats (minimum period) and salivary glands of insectivorous bats (maximum period); salivary glands of insectivorous bats (minimum period) and brains of frugivorous bats (minimum period) with p<0.001. The use of N2A cells for the prime isolation of rabies virus from tissues and non-nervous organs other than brains of bats did not show consistent results, especially due to bacterial contamination and toxicity factor

O presente trabalho descreve o desenvolvimento de uma técnica de nested-PCR para a detecção de ADN do vírus da laringotraqueíte infecciosa (VLTI) utilizando primers que amplificam uma região do gene que codifica a glicoproteína E viral. A técnica padronizada amplificou amostras isoladas e de campo, mostrando alta sensibilidade e especificidade tomando o isolamento em ovos embrionados SPF como técnica de referência. O seqüenciamento de uma amostra positiva por nested-PCR confirmou a identidade do produto amplificado. Foram submetidas a nested-PCR padronizada amostras de traquéia, pulmão e conjuntiva de 51 granjas de poedeiras comerciais procedentes da região de Bastos, Estado de São Paulo, que apresentou um surto caracterizado por sinais respiratórios, queda na produção de ovos e aumento da mortalidade. Vinte e três granjas foram positivas a nested-PCR e vinte e duas amostras foram isoladas. Considerando os resultados das duas técnicas, vinte e quatro granjas resultaram positivas. Não foram detectados os vírus das doenças de Newcastle, pneumovirose aviaria, nem Mycoplasma gallisepticum. O vírus da bronquite infecciosa das galinhas foi detectado em uma granja, e Mycoplasma synoviae em oito. A alta freqüência de ocorrência do VLTI, a alta concordância entre o quadro clínico observado e detecção do VLTI e o resultados do diagnóstico diferencial demonstram que o VLTI foi o agente etiológico causador de um surto de doença respiratória observado na região de Bastos, Estado de São Paulo. / The present work describes the development of a nested-PCR technique for the detection of Infectious laryngotracheitis virus (ILTV) DNA using primers to amplify a region that encodes the glycoprotein E. The standardized technique amplified isolated and from clinical strains, offering high sensitivity and specificity, using the isolation in SPF chicken embryos such as reference test. The identity of the amplified product of the nested-PCR was confirmed by DNA sequencing. Trachea, lung and conjunctiva samples from fifty-one commercial layer farms from Bastos region, São Paulo State, which showed an outbreak characterized by respiratory signs, decreased egg production and increased mortality, were tested by the standardized nested-PCR. Twenty-three farms were positive by nested-PCR and samples of twenty-two farms were isolated. Twenty-four farms were positives when the results of both techniques were considered. Newcastle disease virus, Avian Pneumovirus and Mycoplasma gallisepticum were not detected. Infectious bronchitis virus was detected in one farm and Mycoplasma synoviae was detected in eight farms. The high frequency of occurrence of ILTV, the high agreement between clinical signs observed and ILTV detection and the results of differential diagnosis demonstrate that ILTV was the etiological agent of an outbreak respiratory disease observed in Bastos region, São Paulo State.

O presente trabalho descreve o desenvolvimento de uma técnica de nested-PCR para a detecção de ADN do vírus da laringotraqueíte infecciosa (VLTI) utilizando primers que amplificam uma região do gene que codifica a glicoproteína E viral. A técnica padronizada amplificou amostras isoladas e de campo, mostrando alta sensibilidade e especificidade tomando o isolamento em ovos embrionados SPF como técnica de referência. O seqüenciamento de uma amostra positiva por nested-PCR confirmou a identidade do produto amplificado. Foram submetidas a nested-PCR padronizada amostras de traquéia, pulmão e conjuntiva de 51 granjas de poedeiras comerciais procedentes da região de Bastos, Estado de São Paulo, que apresentou um surto caracterizado por sinais respiratórios, queda na produção de ovos e aumento da mortalidade. Vinte e três granjas foram positivas a nested-PCR e vinte e duas amostras foram isoladas. Considerando os resultados das duas técnicas, vinte e quatro granjas resultaram positivas. Não foram detectados os vírus das doenças de Newcastle, pneumovirose aviaria, nem Mycoplasma gallisepticum. O vírus da bronquite infecciosa das galinhas foi detectado em uma granja, e Mycoplasma synoviae em oito. A alta freqüência de ocorrência do VLTI, a alta concordância entre o quadro clínico observado e detecção do VLTI e o resultados do diagnóstico diferencial demonstram que o VLTI foi o agente etiológico causador de um surto de doença respiratória observado na região de Bastos, Estado de São Paulo. / The present work describes the development of a nested-PCR technique for the detection of Infectious laryngotracheitis virus (ILTV) DNA using primers to amplify a region that encodes the glycoprotein E. The standardized technique amplified isolated and from clinical strains, offering high sensitivity and specificity, using the isolation in SPF chicken embryos such as reference test. The identity of the amplified product of the nested-PCR was confirmed by DNA sequencing. Trachea, lung and conjunctiva samples from fifty-one commercial layer farms from Bastos region, São Paulo State, which showed an outbreak characterized by respiratory signs, decreased egg production and increased mortality, were tested by the standardized nested-PCR. Twenty-three farms were positive by nested-PCR and samples of twenty-two farms were isolated. Twenty-four farms were positives when the results of both techniques were considered. Newcastle disease virus, Avian Pneumovirus and Mycoplasma gallisepticum were not detected. Infectious bronchitis virus was detected in one farm and Mycoplasma synoviae was detected in eight farms. The high frequency of occurrence of ILTV, the high agreement between clinical signs observed and ILTV detection and the results of differential diagnosis demonstrate that ILTV was the etiological agent of an outbreak respiratory disease observed in Bastos region, São Paulo State.

Pouco se conhece a respeito da incidência ou prevalência da infecção pelo vírus da raiva em morcegos, ou ainda sobre a distribuição do vírus em tecidos e órgãos não nervosos. Os objetivos deste trabalho foram: i) verificar as espécies de morcegos mais freqüentemente envolvidas com a raiva no Estado de São Paulo, Sudeste do Brasil; ii) estudar a distribuição do vírus da raiva em tecidos e órgãos não nervosos de morcegos; iii) estudar os períodos de mortalidade das amostras de vírus da raiva encontradas nos cérebros e glândulas salivares de morcegos, após inoculação intracerebral em camundongos, e iv) comparação do isolamento do vírus da raiva no sistema camundongo e cultura de células de neuroblastoma (N2A). Entre abril de 2002 a novembro de 2003, 4.393 morcegos capturados de diferentes municípios do Estado de São Paulo foram enviados à Seção de Diagnóstico da Raiva do Instituto Pasteur de São Paulo. Destes, 82 (1,87%) foram positivos para raiva pela técnica de imunofluorescência aplicada aos materiais do cérebro e 33 morcegos pertenciam ao gênero Artibeus sp; 15 Myotis sp; 10 Epitesicus sp; 5 Lasiurus sp; 4 Nyctinomops sp; 4 Tadarida sp; 3 Histiotus sp; 1 Molossus sp; 1 Eumops sp e 6 vampiros Desmodus rotundus. A distribuição do vírus em diferentes órgãos foi examinada pela inoculação de camundongos e células N2A com suspensões a 20% preparadas a partir de fragmentos do cérebro, glândula salivar submandibular, pulmão, língua, coração, bexiga urinária, rins, gordura interescapular, músculo peitoral, trato genital (testículos ou ovários e útero) e estômago. O vírus foi prontamente recuperado de tecidos e órgãos não nervosos com diferentes graus de sensibilidade, tanto em camundongos como em células N2A, e os órgãos mais apropriados para o isolamento viral foram os cérebros e glândulas salivares. Os períodos máximos de mortalidade observados para os vírus presentes nos cérebros usualmente foram mais curtos que os das glândulas salivares, a média do período máximo &plusmn; desvio padrão calculado para os cérebros de morcegos hematófagos foi de 15,33 &plusmn; 2,08 dias e para as glândulas salivares, 11,33 &plusmn; 2,30 dias; para os morcegos insetívoros, 16,45 &plusmn; 4,48 dias para os cérebros e para as glândulas salivares, 18,91 &plusmn; 6,12 dias; e para os morcegos frugívoros, as suspensões cerebrais apresentaram período máximo médio 12,60 &plusmn; 2,13 dias e para as glândulas salivares, 15,67 &plusmn; 4,82 dias. O teste de ANOVA indicou existir diferenças significantes entre os períodos de mortalidade correspondentes às suspensões preparadas a partir dos cérebros de morcegos insetívoros (período mínimo) e glândulas salivares de morcegos insetívoros (período máximo) e entre glândulas salivares de morcegos insetívoros (período máximo) e cérebros de morcegos frugívoros (período mínimo), com p<0.001. O uso de células N2A para o primo-isolamento do vírus da raiva a partir de tecidos e órgãos não nervosos de morcegos, diferente de cérebros, não mostraram resultados consistentes, especialmente devido à contaminação bacteriana e fator toxicidade / Little is known about the incidence of infection or the prevalence rate of rabies in bats, or the distribution of virus in non-nervous tissues and organs. The aim of this work was to study: i) the most frequent species of bats involved with rabies virus infection in the State of São Paulo, Southeast Brazil; ii) the distribution of rabies virus in tissues and non-nervous organs of bats; iii) the mortality periods of virus found in brains and salivary glands of bats after intracerebral inoculation of mice, and iv) comparison of virus isolation in mice and N2A neuroblastoma cell culture. From April 2002 to November 2003, 4,393 bats captured from different municipalities of the State of São Paulo were sent to Rabies Diagnostic Section of the Instituto Pasteur de São Paulo - SP. Among these, 82 (1.87%) were found positive by the immunofluorescence technique applied to brain specimens and 33 bats were of the genus Artibeus sp; 15 Myotis sp; 10 Epitesicus sp, 5 Lasiurus sp, 4 Nyctinomops sp, 4 Tadarida sp, 3 Histiotus sp; 1 Molossus sp, 1 Eumops sp, and 6 vampires Desmodus rotundus. The distribution of virus in the organs was examined by inoculating mice and N2A cells with the 20% suspensions prepared from brain, submaxillary salivary gland, lungs, tongue, heart, urinary bladder, kidneys, brown fat, pectoral muscle, genital tract (testicles or ovaries and uterus), and stomach. The virus was promptly recovered from tissues and non-nervous organs at different degrees of sensitivity in both mice and N2A cells, and the most appropriate organs for the virus isolation were the brains and salivary glands. The maximum mortality periods found for the brain specimens usually were shorter than the salivary glands, the maximum mean perio &plusmn; standard deviation calculated for the brains taken from the vampire bats was 15.33 &plusmn; 2.08 days and for salivary glands, 11.33 &plusmn; 2.30 days; for the insectivorous bats the maximum for the brain suspensions was 16.45 &plusmn; 4.48 days and for the salivary glands, 18.91 &plusmn; 6.12; and for the frugivorous bats, the brain suspensions showed the maximum of 12.60 &plusmn; 2.13 days and the salivary glands, the mean maximum period of 15.67 &plusmn; 4.82 days. The ANOVA test indicated that the most significant differences in the mortality periods were between the suspensions prepared by the brains of insectivorous bats (minimum period) and salivary glands of insectivorous bats (maximum period); salivary glands of insectivorous bats (minimum period) and brains of frugivorous bats (minimum period) with p<0.001. The use of N2A cells for the prime isolation of rabies virus from tissues and non-nervous organs other than brains of bats did not show consistent results, especially due to bacterial contamination and toxicity factor

O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

A rubéola é uma doença infecciosa aguda, normalmente com um curso clínico suave. Porém quando adquirida nas primeiras 12 semanas de gestação, pode causar severos defeitos de nascimento, conhecida como Síndrome da Rubéola Congênita (SRC). A caracterização genética do vírus da rubéola é feita analisando a seqüência da região hipervariável do gene da glicoproteína E1. Este estudo, apresenta a primeira caracterização molecular de do vírus da rubéola isolados no estado de São Paulo, Brasil. As amostras (sangue, urina, swab de orofaringe, explante de fígado, produto de aborto e fluido cerebrospinal) foram coletados entre 1997 e 2004 de pacientes com sintomas clínicos de rubéola. O gene E1 vírus da rubéola foi amplificado pela reação em cadeia da polimerase em cadeia diretamente de espécimes clínicos e isolados, e os fragmentos de DNA obtidos foram seqüenciados. As seqüências foram alinhadas para a analise filogenética, com seqüências representativas dos diferentes genótipos do vírus da rubéola. Vinte e nove isolados foram obtidos, incluindo isolados relacionados com insuficiência hepática aguda, encefalite e infecções congênitas. A análise filogenética mostrou que 19 dos 29 virus da rubéola isolados pertencem ao genótipo 1a, e 10 pertencem ao genótipo 1G. Este trabalho demonstrou dois genótipos do vírus da rubéola circularam simultaneamente entre os anos de 1997-2004. / Rubella is an acute infectious disease with normally a mild clinical course. However, infections during pregnancy, especially before week 12 of gestation (WG), can cause severe birth defects known as congenital rubella syndrome (CRS). Genetic characterization of wild-type rubella virus is based on sequence analysis of a hypervariable region of the glycoprotein E1 gene. This study presents the first molecular characterization of isolates from São Paulo, Brazil. Samples (blood, urine, oropharyngeal swab, explanted liver, product of conception and cerebrospinal fluid) were collected between 1997 and 2004 from patients with clinical symptoms of rubella. The rubella virus E1 gene coding region was amplified by reverse transcriptase polymerase chain reaction directly from clinical specimens and isolates, and the resulting DNA fragments were sequenced. Sequences were assigned to genotypes by phylogenetic analysis with rubella virus reference sequences. Twenty-nine isolates were obtained, including isolates from acute liver failure, encephalitis and congenital infections. Phylogenetic analysis showed that 19 out of 29 isolated in the São Paulo strains of rubella virus belonged to genotype 1a, and 10 strains to genotype 1G. This work demonstrated two genotypes of RV circulated simultaneously between years 1997 and 2004 in the state of São Paulo. The information reported in this paper may be useful for contributes to understand better the molecular epidemiology of RV in São Paulo, Brazil.

O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.