Hepatitis A virus subgenotyping based on RT-qPCR assays

Coralie Coudray-Meunier 0 3 Audrey Fraisse 0 3 Camlia Mokhtari 2 Sandra Martin-Latil 0 3 Anne-Marie Roque-Afonso 1 2 4 Sylvie Perelle 0 3 0 Universite Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses unit , 23 Avenue du General de Gaulle, 94706 Maisons-Alfort, cedex , France 1 Univ Paris-Sud, UMR-S 785 , Villejuif 94804 , France 2 AP-HP, Hopital Paul Brousse , Virologie, Villejuif 94804 , France 3 Universite Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses unit , 23 Avenue du General de Gaulle, 94706 Maisons-Alfort, cedex , France 4 INSERM U785 , Villejuif 94804 , France Background: The hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. HAV genotypes and subtypes differ in their geographic distribution and the incidence of HAV infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. Phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of HAV strains. In food microbiology, this approach is complicated by the low contamination levels of food samples. To date, real-time reverse-transcription PCR has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide HAV subtyping information. Results: Six subtype-specific RT-qPCR assays were developed for human HAV. The limit of detection of HAV was 50 genome copies/assay for subtype IIB, 500 genome copies assay for IA, IB, IIA and IIIB and 5000 genome copies/assay for IIIA. The specificity of the assays was evaluated by testing reference isolates and in vitro HAV RNA transcripts. No significant cross reactivity was observed. Subtyping results concordant with sequencing analysis were obtained from 34/35 clinical samples. Co-infection with a minor strain of a different subtype was suggested in 5 cases and a recombinant event in one case. Conclusions: These RT-qPCR assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples. - Background Hepatitis A virus (HAV) is a small, non-enveloped hepatotropic virus classified into the Hepatovirus genus within the Picornaviridae family. Its genome consists of an approximately 7.5 kilobase positive single-strand RNA comprising a 5 untranslated region (5UTR), a single open reading frame (ORF) that encodes both structural and non-structural proteins, and a 3 UTR with a short poly (A) tail. There is only one serotype of HAV. Genomic characterization of HAV has been carried out mainly by sequencing of strains from different geographic regions of the world. Firstly, using a short fragment of the VP1/2A junction region, strains were classified in to seven genotypes on the basis of >15% nucleotide variation between isolates, and the subgenotypes with >7.5% to <15% nucleotide variation [1]. Then, the complete genomic data indicated that genotypes II and VII should be considered a single genotype, based upon the complete VP1 sequence [2; 3]. So, by sequencing of the VP1/2A junction and the VP1 gene, three genotypes (I, II, III) divided in two subtypes (A and B) have been described for humans and three others (IV, V, VI) for primates [1-3]. HAV infection is the leading worldwide cause of acute viral hepatitis [4,5]. There are an annual estimated of 1.5 million cases of hepatitis A worldwide [6]. Optimal use of vaccination can significantly reduce the hepatitis A disease burden and the World Health Organization position on hepatitis A vaccines depend on the level of endemicity in countries. In highly endemic countries, large-scale vaccination programmes are not recommended. In countries of intermediate endemicity, large-scale childhood vaccination may be considered as a supplement to health education and improved sanitation. And in regions of low endemicity, vaccination against hepatitis A is indicated for individuals with increased risk of contracting the infection such as travelers to areas of intermediate or high endemicity [7]. HAVs geographical distribution is dependent on socioeconomic development and sanitation levels. In areas with high and very high endemicity (Africa, Middle East, India, Central and South America), where infections are mostly asymptomatic and epidemics are rare, 50% seroprevalence is reached between the ages of 5 and 14 [8]. In areas with moderate endemicity (Eastern Europe and south-eastern Asia), 50% seroprevalence is reached between the ages of 14 and 34 and epidemics can occur within the general population. In areas with low endemicity (North America, Western Europe and Australia), most of the population is still susceptible to HAV, particularly people over 50 years old, and the risk of fulminant hepatitis is higher. HAV is transmitted mainly by the fecal-oral route, either by person-to-person contact or by ingestion of contaminated water and food, particularly shellfish, soft fruits and raw vegetables [9-16]. HAV is stable in the environment and is particularly resistant to disinfectants, heating, pressure and low pH [4,17]. Contamination may occur during growth in the field as well as during processing, storage, distribution or final preparation. In developed countries, low incidence and low vaccine coverage have led to a high proportion of susceptible individuals, which creates a potential for expanded hepatitis A outbreaks when contaminated products are widely distributed [8]. The development of sensitive, reliable techniques for the detection of HAV in food and water samples contributes to the safety of these products [18]. However, detection of HAV on the basis of its infectivity is complicated by the absence of a reliable cell culture method and the low contamination levels of food samples. HAV detection is currently based on nucleic acid testing methods. The International Organization for Standardization/Technical specification (ISO/TS) 15216 standard was published in the first half of 2013 and will be published as ISO standard methods after validation. These protocols target the 5UTR which shows the lowest diversity across HAV genotypes [19-22]. Currently, HAV genotyping relies on amplification, sequencing and phylogenetic analysis of a portion of the viral genome. However, these techniques are time-consuming and may lack sensitivity, particularly with food samples, where the level of contamination by enteric viruses is often very low. Alternative approaches for HAV genotyping in complex samples (food, environmental) may help to better manage the risk. Indeed, although genotypes I and III are the most frequently reported worldwide, HAV genotypes and HAV strains differ in their geographic distribution [23,24]; strain genotyping can thus give clues to understanding food contamination routes. Currently, very few stu (...truncated)