Molecular characterization of faecal RNA virome of healthy chickens using next-generation sequencing

Abstract

The incidence of emerging and re-emerging diseases has been on the rise, affecting both wild and domestic animals. Globally, it is noteworthy that major disease outbreaks that have caused significant morbidity and mortality in poultry systems, other animal species and human populations, have been attributed to viruses originating from animals including birds. Some of these viral outbreaks, especially those characterized by highly unstable RNA genomes have escalated into epidemics or even pandemics. Instances of RNA viral outbreaks, notably associated with animal origins, include severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), Ebola, Swine flu, and Middle East respiratory syndrome (MERS), Spanish flu, Asian flu, and Hong Kong flu. Globally, substantial losses in poultry, have been attributed to RNA viral-linked infections including Newcastle disease, avian influenza, avian leukosis, Gumboro disease, bronchitis, and acute enteritis. In South Africa, the poultry industry has remained the largest agricultural sector, with significant contribution to the nation’s gross domestic product from proceeds of poultry meat and eggs. Interestingly, chicken is the most farmed poultry bird in South Africa and a major source of protein consumed across all income classes. As a result of the increasing demand for chicken in South Africa, its consumption has outweighed its local production, leading to importations. Though the nation’s chicken production strives to remain competitive for its growing demand, however, this goal has been threatened by the rising production cost and infectious disease outbreaks including those of viral origin in flocks. Therefore, it has remained imperative to carry out an in-depth evaluation of these factors, particularly infectious diseases that are associated with suboptimal performance, lowered productivity, and chicken mortality in poultry production. The productive performance and feed utilization rate of chickens are significantly impacted and reliant upon the state of health and proper functioning of their gastrointestinal tract (GIT). Chicken’s GIT is the site of metabolism and may contain diverse microorganisms including fungi, bacteria and viruses whose composition and abundance vary remarkably across its growth stages. Among these organisms, viruses have been implicated in major infectious diseases leading to seasonal culling of poultry birds. These viral diseases cause low productivity in chickens due to immune suppression, subclinical growth impedance, and malabsorption. Chicken flocks are homogenous, often crowded and possess similar genetic features, leaving them vulnerable to viral infections. Thus, with infected birds being initially asymptomatic and the viruses unidentified, these viruses spread rapidly causing outbreaks leading to substantial colossal losses. Poor GIT health, even in the absence of a recognized disease state, can affect poultry performance and result in low productivity. Unfortunately, studies on ix the GIT of farm animals and birds are relatively scarce, from the African continent, though there are a few studies from other continents available with information on the prokaryotic microbiome of birds GIT, with chickens being more studied because of its economic importance. Nevertheless, virome studies on birds including chickens are relatively few, despite being implicated in major outbreaks. Viruses, unlike bacteria lack a universal gene marker for identification and the low amount of their nucleic acid in biological samples makes their identification difficult. Importantly, studies have characterized one or a few of these known viruses using non-NGS molecular methods. However, these approaches do not represent the occurrence of these viruses in natural settings and ignore certain factors such as virus-virus interactions, bird age, host taxonomy and community structure dynamics which have been shown to influence the emergence and abundance of viruses in birds. In addition, while non-NGS methods effectively study each viral species or fewer viruses under experimental settings, they are flawed by the limitation of characterizing only known viruses. Hence, characterizing the viruses present in the GIT of chickens using high-throughput technologies, has remained important to determine the key viral agents associated with poultry infectious outbreaks. The use of viral metagenomics through the NGS approach has allowed the investigation of viruses including novel viruses in animal samples and birds, regardless of the sample type. This approach offers a combined advantage of speed and high-throughput recovery of viruses. While the information on the virome composition of African birds is scarce, the data on their RNA virome including chickens are even scantier despite the continuous evolution of RNA viruses and their associated disease outbreaks. Therefore, it has become paramount to characterize RNA viruses in chicken’s GIT using metagenomic NGS (mNGS). South Africa being the highest poultry producing country in the African continent has been plagued by many seasonal outbreaks of RNA viral diseases in flocks. Hence, determining the complex RNA viral constituents present in the GIT of South African chickens is imperative. In this study, the diversity and abundance of the total RNA viruses found in healthy South African chickens was studied using the mNGS technique.This was achieved through optimized enrichment strategies for better virus recovery using the Illumina Miseq sequencing. The use of Novel Enrichment Technique of VIRomes (NetoVIR) standardized sample preparation protocol, whole transcriptome amplification (WTA) and QIAseq FX library preparation method while using the non-invasive faecal sampling method. The effect of age (2, 4 and 7 weeks) and seasons (winter and summer) were studied as factors that may modulate the abundance and/or diversity of viruses in the GIT of chickens. This was achieved using established ecological metrics of alpha and beta x diversities and their result was statistically evaluated. In addition, the evolutionary relatedness of some of the identified viruses were explored using phylogenetic analysis. The results obtained from the RNA virome investigation of 10 asymptomatic, commercially bred South African chickens revealed a total of 48 RNA viral species. The identified viruses spanned across 11 orders, 15 families and 21 genera. The viral families such as Coronaviridae, Picornaviridae, Reoviridae, Astroviridae, Caliciviridae, Picorbirnaviridae and Retroviridae were the most abundant. Among these families, picornaviruses, reoviruses, astroviruses, picobirnaviruses and coronaviruses were most prevalent at 100%, 88.9%, 81.5%, 81.5% and 74% occurrence across the 27 samples, respectively. Specifically, virus genera such as Rotavirus, Orthoreovirus, Gammacoronavirus, Sicinivirus and Megrivirus relatively prevailed in the 2 weeks faecal samples regardless of season. Significantly, Rotavirus G and Avian Orthoreovirus with high abundance observed at 2 weeks, drastically decreased by the 7th week of development and this may be attributed to their stable, fully developed immune system compared to their juvenile stages. Furthermore, the complete genome of novel chicken astroviruses (CAstV) and genomes of many previously known viruses, including pathogenic avian viruses, mammalian, fungal and plant viruses were identified in this study. Additionally, results from the investigated factors (age and season), showed that there was no effect on viral shedding within samples in a group (alpha diversity) for age (P = 0.146) and season (P = 0.242), which was contrasting to beta diversity (between groups) metrics that indicated that viral diversity and abundance was significantly influenced by age (P = 0.01099) and season (P = 0.00099). More viruses were abundant in the 2 weeks and 4 weeks samples, while for the two seasons, the winter samples had more viruses. Interestingly, for age, this outcome could be attributed to the higher viral susceptibility of chickens at juvenile and intermediate ages as a result of their weaker, still developing immune system while for season, it could be deduced that due to temperature differences of the two seasons, more viruses thrive at winter compared to summer season. Furthermore, the outcomes of the viral evolutionary relatedness demonstrated global distribution and distinctiveness in terms of some specific genotypes or virus lineages for identified viruses. Taken together, the results obtained from this study show that viral structure in the GIT of South African chickens are diverse. It was noted that chickens might carry pathogenic viruses even in the absence of an observable disease condition where pathogenesis may be triggered under certain conditions. Furthermore, the relative abundance profiles of specific avian viruses may be dependent on the age of the bird investigated. Based on the samples analysed, the overall GIT viral abundance in chickens within the same group may be homogenous. However, the viral diversity and abundance xi of chickens GIT may vary between different chicken groups characterised by distinct features, for instance, age and season, provided other underlining nutritional and environmental factors are considered. Undoubtedly, based on the chicken faecal samples studied and the diverse viruses recovered/characterized, mNGS has proven to be a valuable tool for effectively studying the virome in the GIT of avian chickens. Overall, this viral metagenomic study offers some insights into the diversity and composition of RNA viruses circulating in commercially bred chickens in South Africa and this information would be helpful towards understanding the key RNA viruses present in chicken’s GIT at early, intermediate and mature stages of growth. In addition, this study has provided baseline data that will be handy for research endeavours aiming to compare RNA virome structure between healthy and diseased chickens. The identification of some pathogenic viruses in apparently healthy/asymptomatic chickens provides information that may be beneficial for further epidemiological studies looking to decipher the transition dynamics of gut viruses in chicken host from being asymptomatic carriers to diseased condition, aimed at averting illnesses and improving chicken gut health. This is a significant stride towards better preparedness for emerging or reoccurring viral infections from chickens in South Africa and beyond.