Date of Award

2008

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Helen O'Shea

Abstract

Rotavirus infections are considered to be the most common cause of severe gastroenteritis in children under the age of 5 years, and are responsible for up to 611,000 deaths annually, mainly in developing countries. From 1997 to 1999, 16 hospital laboratories in the Republic of Ireland reported to detection of 4,643 cases of rotaviral disease, while in 2004 and 2005 and increase of 1,600 and 2,251 rotavirus cases were reported. These rotavirus cases have a significant impact on the healthcare system with the minimum cost per case being €728.40. A large percentage of the infections were reported in neonates, suggesting inadequate maternal derived protection from existing strains, or the existence of novel strains circulating in Ireland to which there is little or no immunity in the population. The most efficient method of control of rotavirus infections is the implementation of suitable rotavirus vaccination programme. Prior to the implementation of any such vaccination programme information on the epidemiology of strains currently circulating in the Irish population needs to be gathered, along with the identification of any novel strains.

Between 2003 and 2006, 558 rotavirus positive stool samples were collected from two major hospitals in the southern regions of Ireland. Subsets of these samples were characterised using polyacrylamide gel electrophoresis (n=89) and polymerase chain reaction mediated typing (n=249 G typed, n=255 P typed). The predominant types found to be circulating over the 3 years of the study were G1P[8] (65.5%), G3P[8] (13.8%), and G1G3P[8] (9.2%). However, novel types which had not previously been reported in the Irish population were also identified (e.g. G2P[6], G1P[8]P[9], and G9P[8]P[9]). Significantly, protection to these types is not offered by any vaccines currently under evaluation, and as a result they could escape the immune protection offered by an outdate vaccine.

Rotavirus infections in young calves remain an important problem in bovine herds worldwide. Substantial economic loss occurs due to increased morbidity and mortality, treatment costs, and reduced growth rates of the calves as a result of rotavirus infection Little data is currently available on the epidemiology of rotavirus infections in bovines.

One hundred and three rotavirus positive bovine faecal samples were collected from the Cork Regional Veterinary Laboratory, between 2004 and 2005. A subset of these samples was selected for RNA extraction. Polyacrylamide Gel Electrophoresis analysis, RT-PCR and G and P typing. The common types found in the bovine population were G6P[5]P[11] (24.24%), G6P[5] (18.18%), G6P[11] (15.15%), G6G10P[11] (12.12%), and G10P[11] (10.6%). The ciinent vaccine available against bovine rotavirus infections is directed against strains of G6P[5]. According to the results of this study, this vaccine would not offer protection against all of the current circulating bovine rotavirus types, and consequently, without this protection, factors such as calf morbidity and mortality, treatment costs and reduced growth rates will not be fully minimised.

Norovirus gastroenteritis occurs in all age groups. It is the most common cause of gastroenteritis in the community and between 1 and 3% of people become infected with norovirus each year. Norovirus disease is a community based disease and is often found in hospital and residential institutions. It results in significant economic losses, along with the shut down of hospital wards and procedures. The transmission route is usually person to person although food and waterborne sources have been identified. To date confirmation of norovirus infections in medical laboratories is carried out by electron microscopy. For molecular detection strategies such as PCR there is a lack of sensitivity as a consensus set of primers capable of detecting all norovirus strains has yet to be developed. Many research institutions use an array of primer sets to detect norovirus.

Eighty Two faecal samples were collected from patient suffering from suspected norovirus infections between 2005 and 2006. All samples were tested for the presence of norovirus by enzyme immunoassay, RT-PCR using a panel of primer sets, and real time RT-PCR. Both GGI and GGII norovimses were identified by enzyme immunoassay while only GGII noroviruses were identified by the molecular based methods. Twelve of the 82 samples (14.63%) were found to be positive for both GGI and GGII noroviruses by enzyme immunoassay however, these samples are not thought to be true dual infections as it was thought that possible antibody cross reactivity was occurring in the GGI enzyme immunoassay.

Enteric adenoviruses have been shown to be a substantial cause of paediatric gastroenteritis in various parts of the world, and are considered to be the second most common cause of viral gastroenteritis, next to rotavirus in young children. Worldwide adenoviral gastroenteritis is mainly caused by type Ad40 and Ad41, with Ad41 being the predominant type. Little information on the causative type of adenoviral gastroenteritis is available although both Ad40 and Ad41 have been reported. No information on the possible genetic variability with Irish adenoviral samples was reported prior to this.

Ninety five adenovirus positive samples were collected from patients suffering from gastroenteritis between 2002 and 2007. These samples were subjected to typing by PCR and RFLP assays. The genetic variability within the hexon gene, to which neutralising antibodies are directed, was also assessed by DNA sequencing analysis. All adenovirus samples were found to be of Ad41 origin, and a high level of similarity was observed within the hexon gene suggesting that antigenic drift is not occurring within the Irish samples.

The transmission of gastrointestinal disease in humans due to the consumption of contaminated food and water is a significant public health concern. Human enteric viruses can exist in the environment for long periods of time due to their extreme resistance to unfavourable conditions. Transmission of gastroenteritis has often been reported to occur by consumption of contaminate food and water, in particular the transmission of norovirus. In contrast to bacterial food and water quality monitoring in Ireland the presence of enteric viruses is not routinely analysed. The absence of useful, easy, rapid, and reliable methods to concentrate and detect theses viruses may contribute to the lack of viral monitoring in Ireland. The aim of this investigation was to assess the drinking water quality in the southern regions of Ireland from both public and private sources. The quality of bottled water which waste bottled within Ireland was also assessed, along with shellfish samples taken over a three year period (2004- 2007) from a single mussel/oyster farm in order to determine if contaminated shellfish were acting as a reservoir for enteric viruses.

In total 14 water samples were collected and tested, along with 5 shellfish samples. Following concentration and extraction procedures all drinking water samples were found to be negative for the presence of enteric viruses, while one shellfish sample was found to be positive for rotavirus and norovirus by enzyme immunoassay and adenovirus by PCR.

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