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

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

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

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van't Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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.

Source: Masters Abstracts International, Volume: 48-02, page: . Adviser: David Hewett-Emmett. Includes bibliographical references.

Bei bhPIV3 handelt es sich um ein bovines Parainfluenzavirus Typ 3 (bPIV3), dessen Ober-flächenproteingene gegen jene des humanen Parainfluenzavirus Typ 3 (hPIV3) ausgetauscht wurden. Dieses ursprünglich als experimenteller Impfstoff gegen hPIV3 entwickelte Virus wurde darüber hinaus als Impfvektor zur Expression anderer viraler Antigene verwendet. Im Rahmen der hier vorgestellten Arbeit wurden die ersten bhPIV3-basierten Vektoren für nicht-virale Antigene hergestellt und in einem ersten Versuch evaluiert. Dazu wurden ein reverses Genetiksystem zur Herstellung rekombinanter bhPIV3 in einem neuen Labor aufgebaut und fünf neue rekombinante Viren erhalten, welche zusätzlich Antigene des Mycobacterium tuberculosis (M. tb.) exprimieren. Balb/c-Mäuse wurden intranasal mit den bhPIV3-Vektoren infiziert, so dass sowohl deren Replikation als auch der induzierte protektive Effekt gegenüber M. tb.-Neuinfektionen getestet werden konnte. In einem ersten Versuch zeigte sich, dass eine Immunisierung mit den rekombinanten Viren allein keine Schutzwirkung entfaltet. Als Boost-Impfung nach Gabe des Bacille Calmette Guérin (BCG) zeigten einige Vektoren jedoch einen signifikanten protektiven Effekt. In einem Folgeversuch konnten diese Beobachtungen jedoch bislang nicht bestätigt werden, so dass weitere Versuche durchzuführen sind, bevor eine endgültige Aussage bezüglich des hervorgerufenen Schutzeffektes getroffen werden kann. In einem weiteren Tierversuch wurde gezeigt, dass die Baumwollratte ein Tiermodell darstellt, in dem bhPIV3 erheblich schlechter repliziert als hPIV3. Trotz der eingeschränkten Replikation induzierte bhPIV3 neutralisierende Antikörpertiter gegen hPIV3, die mit durch hPIV3 induzierten Titern vergleichbar waren. Mit Hilfe eines neu generierten rekombinanten Virus, welches das grün fluoreszierende Protein EGFP exprimiert, konnte ein Weg aufgewiesen werden, die Bestimmung neutralisierender Antikörpertiter deutlich zu vereinfachen. / The initial objective of this project was to establish a reverse genetic system for generation of recombinant bovine/human parainfluenza virus type 3 (bhPIV3), a bovine PIV3 (bPIV3) in which the bhPIV3 glycoprotein genes are replaced by their counterparts of human PIV3 (hPIV3). In addition, methods needed to characterise virus infectivity, genetic integrity and relevant in vitro phenotypes were established. The reverse genetics system was used to add individual mycobacterium tuberculosis (M. tb.) open reading frames (ORFs) as supernumerary gene units to the bhPIV3 genome and to rescue bhPIV3 vectors that expressed M. tb. antigens. In addition, a similar vector expressing the enhanced green fluorescent protein (EGFP) was constructed. Following the in vitro characterization of the derived viral vectors, the M. tb. vectors were evaluated for their efficacy to protect against M. tb. aerosole challenge in the Balb/c mouse model for tuberculosis. Although, in a single experiment, vaccination with bhPIV3 vectors alone did not confer any protection against M. tb. challenge, a boost with selected bhPIV3 vectors after Bacille Calmette Guérin (BCG) priming was successful in conferring protective efficacy against M. tb. challenge. A repeat of this challenge study could not confirm the initial observation, and further experiments are needed to determine whether the observed protection can be reliably reproduced. Evaluation of the bhPIV3 vectors in the cotton rat model showed that this small animal model is suitable to evaluate the attenuation phenotype of bhPIV3 compared to human parainfluenza virus type 3 (hPIV3). Although replication of bhPIV3 was highly restricted compared to hPIV3, hPIV3 neutralizing antibody titers induced by bhPIV3 infection were similar to those induced by hPIV3 infection. Studies with bhPIV3 expressing EGFP led to a new fluorescence based assay to determine hPIV3 neutralizing antibody titers. This assay could save time and resources in hPIV3 serology.