PCR detection and analysis of potentially zoonotic Hepatitis E virus in French rats

Abstract

Background: Hepatitis E virus has been detected in a wide range of animals. While Genotypes 1-2 of this virus infect only humans, 3-4 can spread from animals to humans and cause sporadic cases of human disease. Pig, and possibly also rats, may act as a reservoir for virus. From a public health perspective it is important to clarify the role of rats for infection of humans. Rats often live close to humans and are therefore of special interest to public health. Rats live of waste and inside the sewage system and may become infected. Reports of hepatitis E virus in rats have been published but not from France. The possibility that rats in an urban area in France were Hepatitis E virus infected, with which type and relationship to other strains was investigated. This study provides information important to public health and better understanding the occurrence of hepatitis E virus in the environment.Eighty one rats (Rattus Norvegicus) were captured, euthanized, sampled (liver and faeces) and analyzed by real-time RT-PCR's, one specific for Hepatitis E virus in rats and one specific for genotype 1-4 that that is known to infect humans. Positive samples were analyzed by a nested broad spectrum RT-PCR, sequenced and compared with sequences in Genbank.

Findings: Twelve liver and 11 faeces samples out of 81 liver and 81 faeces samples from 81 captured rats were positive in the PCR specific for Hepatitis E virus in rats and none in the PCR specific for genotype 1-4. Comparison by nucleotide BLAST showed a maximum of 87% similarity to Hepatitis E virus previously detected in rats and significantly less to genotype 1-4.

Conclusions: This is the first study demonstrating that rats in France carries hepatitis E virus and provide information regarding its relation to other virus strains previously detected in rats and other host animals world-wide. Genotype 1-4 was not detected.

Figure 1

Multiple sequence alignment of deduced amino acid sequences from nucleotide sequences used for phylogeny (Figure2). The multiple alignment was constructed using MEGA5. It corresponds to the same fragment used for phylogeny (Figure 2).

Figure 2

Phylogenetic tree depicting the relation between the HEV sequences from French rats to a selection of other HEV sequences. Phylogenetic tree of sequences corresponding to a 254 nt long fragment from the nested PCR product of the amplified RdRp fragment. The sequence corresponds to nucleotide position 4127 to 4371 of the rat HEV strain rat/Mu/0685/DEU2010, accession number JN167537.1. The tree was constructed by the Neighbor joining method using MEGA 5.05. The tree is depicting the relationship of the French HEV strains from 12 rats, here called “Rat HEV Ly, sample number, and Fr 2012” and described in this article, with selected HEV sequences from rat, ferret, avian HEV and genotype 1-4. The bar indicates the evolutionary distance as number of base substitutions per site. The bootstrap consensus was generated using 1000 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown. The tree is drawn to scale. The evolutionary distances were computed using the Tamura-Nei method (number of base substitutions per site). The rate variation among sites was modeled with a gamma distribution (shape parameter = 6). All positions containing gaps and missing data were eliminated.