Vaccination of influenza a virus decreases transmission rates in pigs

Limited information is available on the transmission and spread of influenza virus in pig populations with differing immune statuses. In this study we assessed differences in transmission patterns and quantified the spread of a triple reassortant H1N1 influenza virus in naïve and vaccinated pig populations by estimating the reproduction ratio (R) of infection (i.e. the number of secondary infections caused by an infectious individual) using a deterministic Susceptible-Infectious-Recovered (SIR) model, fitted on experimental data. One hundred and ten pigs were distributed in ten isolated rooms as follows: (i) non-vaccinated (NV), (ii) vaccinated with a heterologous vaccine (HE), and (iii) vaccinated with a homologous inactivated vaccine (HO). The study was run with multiple replicates and for each replicate, an infected non-vaccinated pig was placed with 10 contact pigs for two weeks and transmission of influenza evaluated daily by analyzing individual nasal swabs by RT-PCR. A statistically significant difference between R estimates was observed between vaccinated and non-vaccinated pigs (p < 0.05). A statistically significant reduction in transmission was observed in the vaccinated groups where R (95%CI) was 1 (0.39-2.09) and 0 for the HE and the HO groups respectively, compared to an Ro value of 10.66 (6.57-16.46) in NV pigs (p < 0.05). Transmission in the HE group was delayed and variable when compared to the NV group and transmission could not be detected in the HO group. Results from this study indicate that influenza vaccines can be used to decrease susceptibility to influenza infection and decrease influenza transmission

Contribution of newly discovered and emerging viruses to human disease

According to the World Health Organization, over 200 infectious diseases in humans originate from animals (zoonoses), posing significant threats to human health. Zoonotic agents account for the majority of emerging and re-emerging pathogens. The human-animal interface has been recognised as an important risk factor that facilitates viruses to cross the species barrier and establish infection in humans. This indicates a need to perform surveillance of human populations who are at high risk of zoonotic infection due to their frequent contact with animals, together with the animals to which humans are exposed. The VIZIONS (Vietnam Initiative on Zoonotic Infections) has been conducted to directly respond to that need. The large virus family Picornaviridae include known emerging pathogens that have major impacts on the economies and human and animal health (e.g. foot-and-mouth disease virus, hand foot and mouth disease virus). Some enteroviruses (EVs) and parechoviruses in this family have been shown to be able to infect both humans and animals while a number of new picornaviruses (new EV variants, cosaviruses, cardioviruses, hunniviruses) with unknown pathogenicity and zoonotic potential have been discovered. This thesis, as part of VIZIONS, hopes to address the following gaps in our knowledge of such viruses in six genera (Enterovirus, Parechovirus, Cosavirus, Cardiovirus, Kobuvirus and Hunnivirus) of the family Picornaviridae: 1) The prevalence and genetic diversity of picornaviruses in studied samples 2) The epidemiology and disease association of the identified viruses 3) The overlaps (if any) of picornaviruses circulating in animals and humans 4) Possible animal sources of picornavirus infections in humans In order to do that, over 2,000 faecal samples collected from a wide range of hosts (pigs, rats, bamboo rats, shrews, bats, chickens, ducks, boars, civets, porcupines, monkeys and humans) were screened for picornaviruses by nested PCR and real-time PCR assays. Detection frequencies varied between viruses and sample origins with kobuvirus as the most commonly detected virus, followed by EV, cardiovirus and hunnivirus. Parechovirus and cosavirus were not detected. Comparison of detection frequencies of viruses infecting pigs revealed a disease (diarrhoea) association with porcine kobuvirus (PKV) but not EV infections. However, differences in PKV viral loads between diarrhoeic and non-diarrhoeic pigs were not statistically significant (p = 0.22). In addition, the PKV VP1 sequences from the two pig categories were not phylogenetically distinct. EV VP1 sequences obtained from pigs and boars showed high genetic diversity with four previously known types and nine new types (EV-G8 to -G16). Analyses of complete genome sequences of two new EV types provided evidence for inter-type recombination with a putative breakpoint in the 2A coding region. Similarly, study on samples from monkeys showed endemic infection of EV but no overlap with EV variants in humans was observed. The majority of EV detected in monkeys were novel with evidence for chimeric genomes and putative recombination breakpoints in the 2A region. New criteria for the classification of EV were additionally proposed. Characterization by sequencing of VP4/VP2 and VP1 regions or complete genomes of picornaviruses in rats and bamboo rats also showed relatively high genetic diversity. While these viruses can infect different species of rats, they were again genetically different from viruses detected in the studied human populations. In summary, studies in this thesis provide substantial new information on the prevalence, genetic diversity and disease association of picornaviruses in the studied populations. However, picornaviruses detected from animals were consistently separate from those found in humans, consistent with a relatively limited zoonotic potential of members of the virus family

Epidemiology and molecular biology of elephant endotheliotropic herpesvirus 1 in the Asian elephant Elephas maximus

Herpesviruses are ubiquitous and are found worldwide, most animal species can be infected with multiple herpesviruses. Some cause clinical disease and others remain symptomatic throughout life. Herpesviruses are found in both captive and wild animals including Asian elephants (Elephas maximus). Elephant Endothelioltropic Herpesvirus (EEHV) has been reported in both captive and wild Asian elephants, with a number of cases being reported in North America, Europe and Asia. It has been suggested that EEHV is associated with haemorrhagic disease, which has been attributed to a number of Asian elephant deaths, affecting mostly juveniles and calves. Clinical signs can vary from weight loss, lethargy, depression, cyanosis of the tongue and sudden death. Molecular testing using qPCR has enabled the detection of individual variants of EEHV, this thesis investigates the EEHV1 variant. EEHV1 has been highlighted as the variant that is more frequently associated with deaths. This thesis includes five studies investigating different aspects of EEHV. Including, the relationship between pregnancy and EEHV viral shedding, the use of an amended human protocol for culturing endothelial cells, EEHV tissue tropism, a potential genetic or familial link between EEHV associated deaths and the detection of potential co-pathogens. The main findings from this thesis include: 1) the use of a longitudinal study investigating a potential link between the physiological stress of pregnancy and EEHV viral shedding. This study suggested there was no link between pregnancy and EEHV viral shedding however other stressors may be involved. 2) Using an amended human umbilical vein endothelial cell protocol, the culture of Asian elephant endothelial cells was successful. The cells from this study may be used in subsequent drug testing and vaccine development. 3) Quantitative PCR was used to determine EEHV1 tropism in tissues from two deaths associated with the virus. Tropism appeared to be for the heart and liver. 4) This thesis provides results from a preliminary study into a potential link between EEHV associated deaths. The data from an Asian elephant genogram shows there is the possibility of a genetic or familial link, which requires further investigation. 5) A number of tissues from deaths associated with EEHV and or death from other causes were investigated for the presence of potential co-pathogens, including the presence of encephalomyocarditis virus (EMCV), using microarray technology. The results indicated there were no co-pathogens present in the tissues. This thesis adds to the current published data, and includes the first known preliminary study investigating a potential genetic link between elephant deaths due to EEHV

Epidemiology and molecular biology of elephant endotheliotropic herpesvirus 1 in the Asian elephant Elephas maximus

Herpesviruses are ubiquitous and are found worldwide, most animal species can be infected with multiple herpesviruses. Some cause clinical disease and others remain symptomatic throughout life. Herpesviruses are found in both captive and wild animals including Asian elephants (Elephas maximus). Elephant Endothelioltropic Herpesvirus (EEHV) has been reported in both captive and wild Asian elephants, with a number of cases being reported in North America, Europe and Asia. It has been suggested that EEHV is associated with haemorrhagic disease, which has been attributed to a number of Asian elephant deaths, affecting mostly juveniles and calves. Clinical signs can vary from weight loss, lethargy, depression, cyanosis of the tongue and sudden death. Molecular testing using qPCR has enabled the detection of individual variants of EEHV, this thesis investigates the EEHV1 variant. EEHV1 has been highlighted as the variant that is more frequently associated with deaths. This thesis includes five studies investigating different aspects of EEHV. Including, the relationship between pregnancy and EEHV viral shedding, the use of an amended human protocol for culturing endothelial cells, EEHV tissue tropism, a potential genetic or familial link between EEHV associated deaths and the detection of potential co-pathogens. The main findings from this thesis include: 1) the use of a longitudinal study investigating a potential link between the physiological stress of pregnancy and EEHV viral shedding. This study suggested there was no link between pregnancy and EEHV viral shedding however other stressors may be involved. 2) Using an amended human umbilical vein endothelial cell protocol, the culture of Asian elephant endothelial cells was successful. The cells from this study may be used in subsequent drug testing and vaccine development. 3) Quantitative PCR was used to determine EEHV1 tropism in tissues from two deaths associated with the virus. Tropism appeared to be for the heart and liver. 4) This thesis provides results from a preliminary study into a potential link between EEHV associated deaths. The data from an Asian elephant genogram shows there is the possibility of a genetic or familial link, which requires further investigation. 5) A number of tissues from deaths associated with EEHV and or death from other causes were investigated for the presence of potential co-pathogens, including the presence of encephalomyocarditis virus (EMCV), using microarray technology. The results indicated there were no co-pathogens present in the tissues. This thesis adds to the current published data, and includes the first known preliminary study investigating a potential genetic link between elephant deaths due to EEHV

Epidemiology of emerging human-infective RNA viruses: discovery, geographical extent, and disappearance

Previous investigations into human infectious diseases have revealed RNA viruses as major etiological agents. Given the recent rate of newly detected human-infective RNA viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome coronavirus, and Bundibugyo ebolavirus, targeting virus discovery in high-risk regions, characterizing viruses with the greatest likelihood of spreading and establishing sustained infection in humans would benefit better preparedness for future outbreaks. There is a lack of evidence on determinants of spatio-temporal patterns in the discovery of human-infective RNA viruses, though previous studies have attempted to identify hotspots of emerging infectious diseases caused by various pathogens. There are also no quantitative studies exploring predictors of geographical extent and the disappearance for all currently known human-infective RNA viruses. This thesis aimed to address the following gaps. 1. Identifying predictors discriminating between areas with and without discovery of human-infective RNA viruses and predicting discovery hotspots, at both global and regional scales. Predictors identified include socio-economic, climatic, land use, and biodiversity variables. 2. Prediction of the geographical extent and the disappearance of human-infective RNA viruses, using features such as taxonomy, virus structure, transmission mode, host range, origin, and clinical presentation. 3. Taking SARS-CoV-2 as an example, investigating how predictors related to demographics, socioeconomics, travel, healthcare, co-morbidities, readiness, geography, COVID-19 testing, and interventions have affected the epidemic of the disease it caused—coronavirus disease 2019 (COVID-19)—between countries in the WHO African Region. In order to address the gaps outlined above, I firstly geocoded the first reports of 223 human-infective RNA viruses at the global scale. Using a Poisson boosted regression tree (BRT) model, I identified GDP growth, GDP, and urbanization as top predictors of virus discovery, and predicted discovery hotspots including both historical hotspots—eastern North America, Europe, central Africa, eastern Australia, and north-eastern South America, and new hotspots—East and Southeast Asia, India, and Central America. Stratified analyses suggested discovery of vector-borne viruses and strictly zoonotic viruses was more correlated with climatic variables and biodiversity, whereas the discovery of non-vector-borne viruses and human-transmissible viruses was more strongly correlated with GDP and urbanization. Next, I focused on comparisons of the specific predictors of RNA virus discovery in three different regions with different GDP—United States, China, and Africa. A similar methodology as the global analysis was used on each region separately, the results showed that predictors such as GDP and land use continued to be top predictors in three regions, but climate and biodiversity variables were consistently less important predictors than at a global scale. To identify predictors of the geographical extent and the disappearance (no record of infection in the literature for the past ten years or more), I collated information for 223 human-infective RNA viruses on their geographical extents and persistence in causing human infections from peer-reviewed literature. By fitting Bernoulli BRT models, I observed that viral features that predicted wide geographic extent included transmissibility between humans, a +ssRNA genome, narrow host range [i.e. infecting humans only or humans and other non-human primates (NHP) only], and having a reservoir host in a NHP. Viruses were more likely to disappear if they were incapable of transmission between humans, have had a localised geographic extent, a dsRNA genome, were non-pathogenic and non-fatal, were firstly discovered through active discovery programmes rather than passive investigation of the aetiology, and were transmitted by vectors and direct contact. Results for both geographical extent and virus disappearance did not change after factoring out reporting effort. I concluded that multiple characteristics determined the geographical extent and disappearance of human-infective RNA viruses; however, transmission mode and structure were consistently the most important predictors of the geographical extent and disappearance of human-infective RNA viruses. Host range was an important predictor of geographical extent, though less important for disappearance. Geographical extent, clinical presentation and discovery process all contributed to the probability of a virus disappearing. To understand the differences between epidemics of COVID-19 between countries of the WHO African Region, I selected the timing of the first case and the mortality rate in the first and second waves as the three outcomes. By applying a series of statistical models including Cox proportional hazards regression models, generalized linear mixed models and multinomial logistic regression models, I found that COVID-19 in Africa arrived earlier and caused greater mortality in countries with more pre-pandemic international connectivity and a more urban population. Mortality was exacerbated by high HIV prevalence. The stringency and timing of government restrictions on behaviour were not associated with a lower per capita mortality rate. A more urban population and a higher infectious disease resilience score were associated with more stringent restrictions and/or a higher per capita mortality rate. The predictor set for the first and second waves were similar, and first wave per capita mortality was a significant predictor of second wave per capita mortality. In summary, studies in this thesis showed that there were variations in predictors of discovery both between virus types and geographical regions, and identified high-risk regions for virus discovery beyond their historical extent. The studies also provided proof-of-principle for the prediction of attributes such as mortality, geographical extent, and disappearance for new human-infective RNA viruses. These results help identify priority regions for investment in surveillance systems for new human-infective viruses, and to make risk assessments once they have emerged

Metagenomic surveillance of viruses at the human-animal interface

Zoonotic viruses are a major contributor to emerging infectious diseases, and continuously burden public health systems. Early detection and effective response to viral emergence require an overview of what viruses are circulating in animal hosts, which of these can and do infect at-risk human populations, and which pose the greatest risk of further spread. However, knowledge of such epidemiological patterns is generally biased towards known pathogens of humans and of economically important livestock species. With metagenomic sequencing, one can begin to address these biases by generating a more representative picture of what viruses are present in different host species living in a shared environment. Vietnam is considered a high-risk setting for the emergence of zoonoses, due to its high population and livestock densities and the prevalence of socio-cultural practices involving frequent close contacts between humans, livestock and wildlife. The Vietnam Initiative on Zoonotic Infections (VIZIONS) was established to improve our understanding of zoonotic emergence in this context. Over 2000 faecal samples and rectal swabs were collected from humans and a variety of farmed animals, and subjected to metagenomic sequencing. In this thesis, I use viral taxonomic classification methods to identify and characterise the viruses present in these samples. I investigate any signals for (putative) zoonotic viruses, and assess whether they could represent emerging public health threats. I also evaluate the roles and challenges of metagenomic surveillance for emerging viruses at the human-animal interface. The first part of this thesis focuses on the development and testing of a viral taxonomic classification pipeline. I describe the basic steps of this pipeline, and the rationale behind the chosen methods. Next, I test the pipeline on a subset of samples and viruses for which diagnostic quantitative PCR (qPCR) data were available for comparison. Receiver operating characteristic (ROC) curve analysis showed that the pipeline accurately distinguishes qPCR positive from qPCR-negative samples, and read pair counts correlate well with qPCR cycle threshold values. Investigation of samples with discordant qPCR and metagenomic results indicated that taxonomic misclassification by the pipeline plays a minor role in these discrepancies. Additionally, I found that, for each of the tested viruses, negative samples have variable read pair counts (“background noise”) that correlate with the total number of read pairs assigned to the virus across all samples of the same sequencing run. I hypothesise that this is due to “index switching”, a form of cross-contamination, and model the association. The findings of these investigations allow me to incorporate additional steps into the pipeline to counteract misclassification, and to use signal thresholds that take into account the effect of index switching cross-contamination. In the second part of this thesis, I focus on the characterisation of viruses identified with the taxonomic classification pipeline. I present an overview of the mammalian viruses found in samples from humans, swine and rats from Dong Thap province. After removing likely contaminants, I categorize the remaining viruses according to their zoonotic potential. Seven of these viruses are known or generally presumed to be zoonotic; three are only found in the animal study populations, but four – Rotavirus A, Picobirnavirus, Human associated cyclovirus 8, and Mammalian orthoreovirus – are shared between human and animal populations. Comparison of signals suggests that viral chatter (Rotavirus A) and cross-species transmission within a more generalist ecology (Picobirnavirus, Human associated cyclovirus 8) are plausible in this setting. Additionally, three putative novel zoonoses are identified, but knowledge gaps hinder extensive interpretation. I evaluate the relevance of these 10 zoonotic and putative novel zoonotic viruses as potential emerging public health threats, and highlight the knowledge gaps that need to be addressed before the risks of these viruses can be properly assessed. Finally, I interpret my findings in the general context of disease emergence, and evaluate the roles and challenges of viral metagenomics as a tool in the surveillance for emerging infectious disease

Aptamers to the hepatitis C virus polymerase

Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ‘best binding’ aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and Φ6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals