Human coronaviruses were first discovered in 1960s and believed to be the causative agents of only mild respiratory tract infections. In 2003, the worldwide outbreak of Severe Acute Respiratory Syndrome caused by SARS-CoV had greatly boosted the research interest on coronaviruses. Two more human coronaviruses – HCoV-NL63 and HCoV-HKU1 were then discovered in 2004 and 2005 respectively. Events of recombination between different genotypes had also been detected in HCoV-OC43 and HCoV-HKU1 in natural circulating strains that are causing infections. Creation of novel genotypes is resulted, which may possibly associate with more severe disease. In this study, twenty seven HCoV-NL63 strains in Hong Kong from 2004 to 2012 were included in the investigation. RNA-Dependent RNA Polymerase gene, Spike gene and the Nucleocapsid gene of these strains were sequenced, followed by phylogenetic analysis and bootscan analysis. Antigenic drift from genotype A (in 2004-2006) to B (in 2003-2010) and C (in 2009-2012) were observed. Two local strains clustered with an American strain in all three genes, which the American strain had been formed by recombination between genotype A and C. Five recent strains from 2009 to 2012, along with two Beijing strains, may belong to a potential novel genotype. Two more strains were discovered with atypical genomic profile. Complete genome sequencing would be the further work for clear investigation on their sites of recombination. No clear association between the genotypes and clinical features had been observed. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Human coronaviruses, namely HCoV-229E and HCoV-OC43, have been identified as causal agent of upper respiratory tract infections for decades. The significance of human coronaviruses studies re-emerged after the SARS pandemic. Two novel human coronaviruses, HCoV-NL63 and HCoV-HKU1, were identified after the SARS pandemic. Up till now there are a total of five human coronaviruses being identified. After the discovery of HCoV-HKU1 in 2005, molecular epidemiology and genome studies identified the first natural recombination in human coronaviruses, which has led to the generation of different genotypes. A similar phenomenon was also observed in HCoV-OC43 in a subsequent study, resulting in the emergence of a novel genotype associated with pneumonia. Although HCoV-229E has been discovered for over forty years, information regarding its evolution and epidemiology is little and scattered. In this study, 32 HCoV-229E strains were collected from nasopharyngeal aspirates over a period of 8 years (from April 2004 to January 2012). Three genes, including RdRp (RNA-dependent-RNA polyermase), S (spike) and N (nucleocapsid), were sequenced and analyzed. Phylogenetic studies showed the existence of genetic drift among six chronological groups, including group 1 from 1979 to 1982, group 2 from 1982 to 1984, group 3 from 1990 to 1992, group 4 from 2001 to 2005, group 5 from 2005 to 2008 and group 6 from 2011 to 2012. One particular strain in 2006, HCoV-229E-HK06-24 displayed an incongruent position between the S and N gene, suggesting a possible recombination between group 4 and group 5. Additionally, five strains from 2011 to 2012 showed incongruent positions in RdRp comparing to other strains, which are suspected to be novel group 6. This study revealed the first evidence for a possible natural recombination event in HCoV-229E. The predominate group 6, which is genetically different from previous strains, may have been arisen by genetic drift. Further surveillance is required to monitor the genetic changes in HCoV-229E. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Nine years after the Severe Acute Respiratory Syndrome (SARS) pandemic in 2003, a novel human coronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV), emerged in the Middle East in 2012 and was believed to be of animal origin. Molecular study on the genome of MERS-CoV showed that it belongs to lineage C betacoronavirus and is closely related to the previously described Tylonycteris bat coronavirus HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat coronavirus HKU5 (Pi-BatCoV HKU5) identified in Tylonycteris pachypus and Pipistrellus abramus respectively. In this study, 597 respiratory and 934 alimentary specimens from 1174 bats of 12 different species were collected in 20 sampling sites of Hong Kong over seven years from November 2005 to November 2012. Degenerate primers designed from alignment of RdRp genes of lineage C betacoronaviruses were able to amplify partial RdRp sequences in two bat species only in this study, including 8 lesser bamboo bats and 34 Japanese pipistrelle, with 200-bp sequences shared 99-100% and 96-100% nucleotide identities to Ty-BatCoV HKU4 and Pi-BatCoV HKU5 respectively. Phylogenetic analysis showed that Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were closely related to but still distinct from MERS-CoV, implying that the two bat coronaviruses are probably not the direct ancestor virus of MERS-CoV. The infection of Pi-BatCoV HKU5 is independent of sex (P = 0.494) but juvenile Japanese pipistrelles were more susceptible to the infection (P = 0.000101) while the infection of Ty-BatCoV HKU4 is independent of both sex (P = 0.464) and age (P = 0.193). Additionally, the mean body weight of the adult lesser bamboo bats positive for Ty-BatCoV HKU4 was significantly lower than that of the negative ones (P = 0.03). On the other hand, there was no significant difference in the mean body weights between the positive and negative bats for Pi-BatCoV HKU5 among the juvenile Japanese pipistrelles (P = 0.06). Continuous surveillance study of coronaviruses on different bat species as well as other potential intermediate animal hosts should be carried on the evolution study of the lineage C betacoronaviruses. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Bats and birds are known to be the reservoirs of a number of zoonotic diseases. The capacity of flight and the diversity of these animals may make them special in maintaining and disseminating diverse viruses leading to instances of emerging zoonoses. In particular bats are increasingly recognized to be reservoirs of a wide range of viruses, including Nipah, Ebola and severe acute respiratory syndrome (SARS) coronaviruses. In most instances these viruses appear to establish long-term persistence in bats. In this thesis I report the identification of novel astroviruses from different insectivorous species of apparently healthy bats sampled in Hong Kong and in 11 provinces of Mainland China with high positive rates. Astroviruses are important causes of diarrhea in many animal species, including humans. This study revealed a remarkably high genetic diversity of bat astroviruses, which form novel distinct phylogenetic groups in the genus Mamastrovirus. Evidence for varying degrees of host restriction for bats astroviruses has been found. The finding of diverse astroviruses in Miniopterus bats captured within a single cave habitat in Hong Kong illustrates a very unusual virus host relationship between astroviruses and these bats. Surveillance of astroviruses in rodents, the only mammal with species numbers surpassing that of bats, has revealed a novel astrovirus in only 1.6 % of the faecal samples of urban brown rat (Rattus norvegicus) in Hong Kong in marked contrast with the prevalence and diversity of astroviruses in bats. Rat astrovirus was phylogenetically related to human astroviruses MLB1 which was detected from clinical samples from diarrhoeal patients in Hong Kong in this study. The unusually high positive rates of astroviruses in bats have been again highlighted. Avastroviruses were detected in 7.1% of the aquatic wild bird samples. Avastrovirus have also been detected in doves in Hong Kong, pond herons and a less whistling duck in Cambodia. A phylogenetic analysis of these novel astroviruses together with other previously known astroviruses revealed that avastrovirus can be divided into 3 monophyletic groups. On the other hand, avian coronaviruses was detected in 12.5% of the aquatic wild bird samples. Phylogenetic analysis of these avian coronaviruses has led us to suggest taxonomic separation of these viruses into two groups as gammacoronaviruses and deltacoronaviruses. Frequent interspecies transmissions of gammacoronaviruses between duck species were demonstrated. Analysis of the avian viral sequences and host mitochondrial DNA sequences suggested that some coronaviruses may have coevolved with birds from the same order. With the discoveries of coronaviruses and astroviruses in mammalian and birds, we now have a better understanding on the diversity and ecology of these two virus families in wildlife. These findings provide new insights into the ecology and evolution of these viruses in nature and have revealed possible inter-species transmissions of these viruses. The role of bats as a reservoir of viruses with potential to pose zoonotic threats to human health was also reinforced. Studies of the virus ecology in wildlife as demonstrated in this thesis will help formulating better strategies for controlling emerging diseases in the future. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy