Most emerging infectious diseases in humans are zoonotic agents. Since the emergence of severe acute respiratory syndrome (SARS), swine-origin influenza and avian influenza epidemics, the study of novel and emerging viruses with zoonotic potential has been considered more and more important. Paramyxoviruses have been known for their potential to cross species barrier and infect new hosts. In the last decade, a number of novel and emerging paramyxoviruses have been reported in various animals. Our research group recently identified three novel bat paramyxoviruses, Tuhoko viruses 1, 2 and 3 (ThkPV-1, 2, and 3) from fruit bats in mainland China, an unclassified paramyxovirus, named Tailam virus (TlmPV) from Sikkim rats and a novel feline paramyxovirus, called Feline morbillivirus(FmoPV) from domestic cats in Hong Kong, suggesting that there is still a diversity of undescribed paramyxoviruses in animals. In this study, a novel porcine paramyxovirus, Swine parainfluenza virus 1 (SpiPV-1), was discovered and characterized from deceased pigs in Hong Kong. A total of 951 samples from 386 deceased pigs were collected, including 386 nasopharyngeal swab, 303 rectal swab, 153 blood, 56 lung and 53 liver samples. And SpiPV-1 was detected in 12 (3.1%) of 386 nasopharyngeal swab and 2 (0.7%) of 303 rectal swab samples by RT-PCR. All the blood, lung and liver samples showed negative results. The complete genome sequences of three strains (SpiPV-1 S033N, SpiPV-1 S119N and SpiPV-1 S206N) from three pigs were amplified and determined. The genome organization of SpiPV-1 is similar to that of viruses under genus Respirovirus, subfamily Paramyxovirinae. The genome contains six genes (3’-N-P/V/C-M-F-HN-L-5’) and putatively codes for the nucleocapsid (N), phosphoprotein (P/V/C), matrix (M), fusion (F), attachment (HN) and large (L) proteins.Like other respiroviruses, the P gene of SpiPV-1 can produce more than one protein, including P, V and W proteins by mRNA editing and C protein by alternative translation initiation. And phylogenetic analysis showed that in all six phylogenetic trees constructed byusing the N, P, M, F, HN and L genes, the three strains SpiPV-1 S033N, S119N and S206N formed a distinct cluster among the known respiroviruses and were most closely related to Sendai virus (SenPV) and Human parainfluenza virus 1 (HpiPV-1). The genome organization, P gene analysis and phylogenetic analysis all suggested that SpiPV-1 is a novel paramyxovirus under genus Respirovirus, subfamily Paramyxovirinae. Seven porcine samples positive for SpiPV-1 were cultured in five different cell lines for viral isolation. However, no cytopathic effect was observed and no viral replication was detected in any of the cell lines. The pathogenicity and emergent potential of SpiPV-1 remain to be determined. Further studies on serology and development of cell cultures for viral isolation may provide better insight into this novel paramyxovirus. / published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Interferon (IFN) is activated in response to virus infections and upregulates interferon-stimulated genes (ISGs) resulting in the expression of hundreds of proteins, many of which have direct or indirect antiviral activity. Parainfluenza virus 5 (PIV5) of the Paramyxoviridae family is a non-segmented negative sense single-stranded RNA virus with seven genes encoding eight proteins. Here we present that IFN induces alterations in the pattern of both virus transcription and translation and that ISG56 is primarily responsible for these effects. We report that when cells were treated with IFN post-infection, virus protein synthesis was inhibited while virus transcription levels were increased. These results suggest that ISG56 selectively inhibits the translation of viral mRNAs. In addition, the relationship of various PIV5 isolates was analysed by next generation sequencing. Four areas with a high degree of single nucleotide polymorphisms (SNPs) were identified and mapped to the intergenic regions of NP-V/P, M-F and HN-L, as well as the entire SH gene. Three of the isolates, the porcine strain SER and the canine strains CPI+ and CPI-, did not express an SH protein due to the lack of a start codon. A low degree of variation was found in the amino acid sequence of the HN glycoprotein suggesting that PIV5 may be less pressured to evolve in order to evade immune responses, such as neutralising antibodies.

Existem diversos trabalhos publicados sobre a utilização de diferentes métodos imunológicos para diagnosticar infecções do trato respiratório causadas por vírus parainfluenza bovino tipo 3 (bPIV-3). Entretanto, é escassa a literatura sobre a utilização da técnica de isolamento viral. Até o presente momento não havia sido relatada a utilização da Transcrição Reversa - Reação em Cadeia da Polimerase (RT-PCR), na detecção de bPIV-3. O objetivo deste estudo foi contribuir para uma melhor caracterização dos bPIV-3 através do desenvolvimento de técnicas de RTPCR para a sua detecção. Utilizando-se uma amostra referência de bPIV-3 (SF-4) e uma amostra de bPIV-3 isolada no Rio Grande do Sul (amostra DIO) foram desenvolvidas técnicas de RT-PCR para a amplificação de diferentes regiões do gene da hemaglutinina-neuraminidase (HN). Após seqüenciamento parcial do gene HN e alinhamento das seqüências da amostra DIO, os resultados revelaram homologia de 99% em relação à amostra referência, 98% a 91% quando comparada com outras amostras de bPIV-3 previamente publicadas na rede e 79% a 80% quando comparada com as amostras de hPIV-3. Foi desenvolvida, também, uma técnica de RT-PCR para amplificação de parte do gene da HN do bPIV-3 e do hPIV-3. Nos experimentos de otimização, a técnica de RT-PCR, comparada com o isolamento viral, apresentou sensibilidade de 140 DICC50, boa especificidade e reprodutibilidade. Os resultados, após seqüenciamento da amostra de vírus bPIV-3 isolada no RS, apresentaram, grande homologia com os das amostras de bPIV-3 comparadas, especialmente a amostra referência. Os dados obtidos neste estudo mostraram que a RT-PCR desenvolvida para a detecção de PIV-3 foi capaz de amplificar um fragmento de 1009 bp do gene da HN de amostras de PIV-3 isoladas de bovinos e humanos, possibilitando a sua utilização em diagnóstico e em estudos epidemiológicos. / There are several published studies on the application of different immunological methods for diagnosis of respiratory infections caused by bovine type 3 Parainfluenza virus (bPIV-3). However, the literature on viral isolation procedure is very scarce. At present there is no report about the utilization of reverse transcription-polymerase chain reaction (RT-PCR) for bPIV-3 detection. The aim of this study was to contribute to a better caracterization of bovine type 3 Parainfluenza virus by developing RT-PCR techniques to its detection. A reference sample (SF-4) and a sample of bPIV-3 isolated in Rio Grande do Sul (DIO sample) were used to develop RT-PCR techniques by amplification of different regions of hemagglutinin-neuraminidase gene (HN). After HN gene partial sequencing of DIO sample and sequence alignment, the results revealed 99% homology when compared to reference sample, 98% the 91% homology in relation to bPIV-3 samples previously published and 79% the 80% if compared to hPIV-3 samples. It was also developed a RT-PCR for amplification of a part of bPIV-3 and hPIV-3 NH gene. In optimization experiments, compared to viral isolation procedure, the RTPCR displayed a sensitivity of 140 DICC50, good specificity and reproductibility. Results after sequencing of bPIV-3 sample isolated in RS displayed strong homology with those of bPIV-3 tested samples, specially the reference sample. Data obtained in this study showed that the RT-PCR technique developed for PIV- 3 detection was able to amplify an 1009 bp HN gene fragment of bovine and human PIV-3 samples which enables its utilization in diagnostic and epidemiological studies.

Existem diversos trabalhos publicados sobre a utilização de diferentes métodos imunológicos para diagnosticar infecções do trato respiratório causadas por vírus parainfluenza bovino tipo 3 (bPIV-3). Entretanto, é escassa a literatura sobre a utilização da técnica de isolamento viral. Até o presente momento não havia sido relatada a utilização da Transcrição Reversa - Reação em Cadeia da Polimerase (RT-PCR), na detecção de bPIV-3. O objetivo deste estudo foi contribuir para uma melhor caracterização dos bPIV-3 através do desenvolvimento de técnicas de RTPCR para a sua detecção. Utilizando-se uma amostra referência de bPIV-3 (SF-4) e uma amostra de bPIV-3 isolada no Rio Grande do Sul (amostra DIO) foram desenvolvidas técnicas de RT-PCR para a amplificação de diferentes regiões do gene da hemaglutinina-neuraminidase (HN). Após seqüenciamento parcial do gene HN e alinhamento das seqüências da amostra DIO, os resultados revelaram homologia de 99% em relação à amostra referência, 98% a 91% quando comparada com outras amostras de bPIV-3 previamente publicadas na rede e 79% a 80% quando comparada com as amostras de hPIV-3. Foi desenvolvida, também, uma técnica de RT-PCR para amplificação de parte do gene da HN do bPIV-3 e do hPIV-3. Nos experimentos de otimização, a técnica de RT-PCR, comparada com o isolamento viral, apresentou sensibilidade de 140 DICC50, boa especificidade e reprodutibilidade. Os resultados, após seqüenciamento da amostra de vírus bPIV-3 isolada no RS, apresentaram, grande homologia com os das amostras de bPIV-3 comparadas, especialmente a amostra referência. Os dados obtidos neste estudo mostraram que a RT-PCR desenvolvida para a detecção de PIV-3 foi capaz de amplificar um fragmento de 1009 bp do gene da HN de amostras de PIV-3 isoladas de bovinos e humanos, possibilitando a sua utilização em diagnóstico e em estudos epidemiológicos. / There are several published studies on the application of different immunological methods for diagnosis of respiratory infections caused by bovine type 3 Parainfluenza virus (bPIV-3). However, the literature on viral isolation procedure is very scarce. At present there is no report about the utilization of reverse transcription-polymerase chain reaction (RT-PCR) for bPIV-3 detection. The aim of this study was to contribute to a better caracterization of bovine type 3 Parainfluenza virus by developing RT-PCR techniques to its detection. A reference sample (SF-4) and a sample of bPIV-3 isolated in Rio Grande do Sul (DIO sample) were used to develop RT-PCR techniques by amplification of different regions of hemagglutinin-neuraminidase gene (HN). After HN gene partial sequencing of DIO sample and sequence alignment, the results revealed 99% homology when compared to reference sample, 98% the 91% homology in relation to bPIV-3 samples previously published and 79% the 80% if compared to hPIV-3 samples. It was also developed a RT-PCR for amplification of a part of bPIV-3 and hPIV-3 NH gene. In optimization experiments, compared to viral isolation procedure, the RTPCR displayed a sensitivity of 140 DICC50, good specificity and reproductibility. Results after sequencing of bPIV-3 sample isolated in RS displayed strong homology with those of bPIV-3 tested samples, specially the reference sample. Data obtained in this study showed that the RT-PCR technique developed for PIV- 3 detection was able to amplify an 1009 bp HN gene fragment of bovine and human PIV-3 samples which enables its utilization in diagnostic and epidemiological studies.

Existem diversos trabalhos publicados sobre a utilização de diferentes métodos imunológicos para diagnosticar infecções do trato respiratório causadas por vírus parainfluenza bovino tipo 3 (bPIV-3). Entretanto, é escassa a literatura sobre a utilização da técnica de isolamento viral. Até o presente momento não havia sido relatada a utilização da Transcrição Reversa - Reação em Cadeia da Polimerase (RT-PCR), na detecção de bPIV-3. O objetivo deste estudo foi contribuir para uma melhor caracterização dos bPIV-3 através do desenvolvimento de técnicas de RTPCR para a sua detecção. Utilizando-se uma amostra referência de bPIV-3 (SF-4) e uma amostra de bPIV-3 isolada no Rio Grande do Sul (amostra DIO) foram desenvolvidas técnicas de RT-PCR para a amplificação de diferentes regiões do gene da hemaglutinina-neuraminidase (HN). Após seqüenciamento parcial do gene HN e alinhamento das seqüências da amostra DIO, os resultados revelaram homologia de 99% em relação à amostra referência, 98% a 91% quando comparada com outras amostras de bPIV-3 previamente publicadas na rede e 79% a 80% quando comparada com as amostras de hPIV-3. Foi desenvolvida, também, uma técnica de RT-PCR para amplificação de parte do gene da HN do bPIV-3 e do hPIV-3. Nos experimentos de otimização, a técnica de RT-PCR, comparada com o isolamento viral, apresentou sensibilidade de 140 DICC50, boa especificidade e reprodutibilidade. Os resultados, após seqüenciamento da amostra de vírus bPIV-3 isolada no RS, apresentaram, grande homologia com os das amostras de bPIV-3 comparadas, especialmente a amostra referência. Os dados obtidos neste estudo mostraram que a RT-PCR desenvolvida para a detecção de PIV-3 foi capaz de amplificar um fragmento de 1009 bp do gene da HN de amostras de PIV-3 isoladas de bovinos e humanos, possibilitando a sua utilização em diagnóstico e em estudos epidemiológicos. / There are several published studies on the application of different immunological methods for diagnosis of respiratory infections caused by bovine type 3 Parainfluenza virus (bPIV-3). However, the literature on viral isolation procedure is very scarce. At present there is no report about the utilization of reverse transcription-polymerase chain reaction (RT-PCR) for bPIV-3 detection. The aim of this study was to contribute to a better caracterization of bovine type 3 Parainfluenza virus by developing RT-PCR techniques to its detection. A reference sample (SF-4) and a sample of bPIV-3 isolated in Rio Grande do Sul (DIO sample) were used to develop RT-PCR techniques by amplification of different regions of hemagglutinin-neuraminidase gene (HN). After HN gene partial sequencing of DIO sample and sequence alignment, the results revealed 99% homology when compared to reference sample, 98% the 91% homology in relation to bPIV-3 samples previously published and 79% the 80% if compared to hPIV-3 samples. It was also developed a RT-PCR for amplification of a part of bPIV-3 and hPIV-3 NH gene. In optimization experiments, compared to viral isolation procedure, the RTPCR displayed a sensitivity of 140 DICC50, good specificity and reproductibility. Results after sequencing of bPIV-3 sample isolated in RS displayed strong homology with those of bPIV-3 tested samples, specially the reference sample. Data obtained in this study showed that the RT-PCR technique developed for PIV- 3 detection was able to amplify an 1009 bp HN gene fragment of bovine and human PIV-3 samples which enables its utilization in diagnostic and epidemiological studies.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Human parainfluenza 3 (HPIV3) is a member of the Paramyxoviridae, a single strain negative-sense non-segmented RNA virus in the order Mononegavirales. It is a respiratory pathogen with a broad spectrum of presentations for which there is currently neither a vaccine nor licensed treatment for HPIV3. To date most research on HPIV3 has been conducted using significantly culture adapted reference strains. Therefore, minimally adapted clinical strains were grown in two cell culture systems: immortalised and primary. Plaque phenotype, growth kinetics and inflammatory response triggered were evaluated and it was found that there is a range of phenotypes exhibited by clinical strains with potential implications in vivo. To examine the genetic diversity of circulating strains of HPIV3 in the UK, a new amplicon based sequencing pipeline for whole genome sequencing of HPIV3 was developed and validated. A short hypervariable region in the HPIV3 genome was identified and evaluated as a potential candidate for subsequent phylogenetic analysis compared to whole genome data. This method was then applied to tracking an HPIV3 outbreak that took place on a paediatric oncology ward. It was found to be a point-source outbreak and the clinical impact in this setting, as well as the infection control procedures involved were evaluated. Finally a robust in vitro model for the evaluation of potential therapeutic candidates for HPIV3, based on a panel of minimally passaged clinical strains as well as a culture-adapted reference strain, was set up. This model was applied to three potential inhibitors of HPIV3: ribavirin, favipiravir and zanamivir. The results showed that clinical strains were at least as susceptible to ribavirin and favipiravir as the laboratory reference strain and significantly more susceptible to zanamivir. This indicates that further work on minimally adapted clinical strains is essential to further the understanding of this important virus.