published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leave 250-287). Also available in print.

Viruses may acquire mutations that result in a tropism shift. RNA viruses, such as Coronaviruses (CoVs), are susceptible to tropism shifts. A tropism shift occurs when a virus alters the tissue or cell type it infects, which can have important implications for disease pathogenesis, virulence, transmission, and treatment control. Tropism shifts can occur after cross-species jumps, as well as result from within-host evolution. Beyond the human host, CoVs can be highly pathogenic to a wide variety of wildlife and companion animals. A spillover event from animals to humans, resulting in a tropism shift, has occurred at some point in the evolutionary history of all three highly pathogenic human CoVs: severe acute respiratory syndrome coronavirus (SARS-CoV), middle eastern respiratory syndrome coronavirus (MERS), and severe acute respiratory syndrome 2 (SARS-CoV-2). Therefore, studying the evolution of CoVs in non-human animals may be of critical importance for pandemic prevention. This was the focus of my dissertation, to apply state-of-the-art codon models of evolution to a variety of CoV viral sequences to identify how natural selection may alter viral proteins priming them for tropism shifts. Statistical codon models can infer both which codon sites and genes have been subject to positive or negative selection, effectively differentiating signal between random mutations and those that may impact fitness. These models may also compare selection at homologous sites between different phenotypes (i.e., Spike protein sequences isolated from the gastrointestinal tract and those from macrophages) to identify where selection is acting differently between the phenotypes. In chapter 2 I examined a CoV sequence isolated from hospitalized humans in Malaysia that resembled a Canine Coronavirus (CCoV) to investigate how natural selection had shaped the Spike protein sequence in related animal CoV sequences priming it to jump into humans. In chapter 3 I compared the natural selection signals at specific codon positions in the Spike protein from sequences isolated from two separate feline tropisms (gastrointestinal and macrophage) to identity which adaptive mutations may be associated with the tropism shift and subsequent shift in virulence. This was performed on Feline Coronavirus (FCoVs), where almost 90% of all wild and domestic cats are gastrointestinally infected with FCoVs, and infection becomes highly pathogenic as a result of the shift in tropism to the macrophages. Since intra-host evolution can impact tropism shifts, in Chapter 4 I performed a detailed high-throughout analysis of intra-host evolution of RNAseq data of Equine Coronavirus (ECoV), as well as natural selection analyses of related embecoviruses that have colonized the human host. Taken together, I report on novel signals of natural selection across viral proteins, with an emphasis on Spike, on a diverse set of CoV clades that shed light on the complexities of coronavirus evolution as it relates to tropism shifts. / Biology

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