Figure 1

Figure 1. Sites of collection and HEV PCR status of rats submitted by pest control professionals in southern Ontario, Canada, during November 2018–June 2021. Geographic administrative boundaries of the cities of Windsor,...

We conducted a study to investigate HEV infection in Norway rats from southern Ontario, Canada, and identify associations between host factors, season, land use, and year of collection. We obtained rat carcasses through collaboration with pest control professionals working in southern Ontario. Our rat and sample collection methods have been previously described and evaluated as a source of samples for zoonotic pathogen surveillance (9). We studied 372 Norway rats (species determined by external morphology) from 161 unique geographic coordinates within southern Ontario during November 2018–June 2021 (Figure 1). During necropsy, we recorded rat demographic characteristic data (Table) and collected liver samples aseptically. Most rats in our sample were sexually mature (65%), and there were more females (51.2%) than males (48.8%). We noted the body condition of rats to be poor (emaciated or underconditioned) in 69.1% and good (well conditioned or overconditioned) in 30.9%. We could not determine sex (3%), sexual maturity (3.2%), or body condition (2.4%) in a minority of rats because of poor carcass condition. We categorized rats by collection location as residential (52.4%), industrial (17.5%), institutional (15.6%), commercial (8.9%), and mixed (5.6%) land use. We collected most rats during the winter (36.8%) and fall (36.8%), followed by spring (21.8%) and summer (4.6%). To account for low sample size in the summer, we recategorized seasonal data as summer/fall (June–November) and winter/spring (December–May).

We screened liver RNA extracts for the presence of HEV by real-time PCR by using previously described primers and probes (10). Of 372 rats tested, 21 (5.6%, 95% CI 3.5%–8.5%) rats from 16 distinct locations in 7 cities/towns were positive for HEV (Figure 1). The odds of HEV infection were significantly higher in sexually mature rats (odds ratio 3.99, 95% CI 1.14–21.47; p = 0.025). By using exact logistic regression models, we observed no association with sex, body condition, land use, season, or year of collection (Table).

Figure 2

Figure 2. Phylogenetic tree based on the nucleotide alignment of hepatitis E virus (HEV) sequences from rats submitted by pest control professionals in southern Ontario, Canada, during November 2018–June 2021 (black dots),...

We amplified positive samples by using a previously described heminested PCR to generate an amplicon from the open reading frame (ORF) 1 region (11). We retrieved a 283-nt fragment of ORF1 from 17 samples and analyzed generated sequences with Lasergene software (DNASTAR, https://www.dnastar.com). We did not obtain sequence data for 4 rats. We aligned sequences with select GenBank reference sequences representing HEV genotypes currently known to infect rats, as well as rat HEV found in humans. Phylogenetic analysis of the partial ORF1-derived sequences showed that all PCR amplicons were rat HEV. We grouped rat HEV sequences from southern Ontario (GenBank accession numbers OQ617169–85) into 4 distinct clusters (Figure 2), with relatively low genetic divergence (14%). Sequences in our study had the highest nucleotide homology with rat HEV sequences from rats in the United States (83.3%), followed by Germany (82.2%), Vietnam (71.5%), and Indonesia (71.3%). Southern Ontario shares a border with 2 US states, New York and Michigan, and the westernmost samples from Windsor were collected directly adjacent to Detroit, Michigan. We noted Ontario rat HEV sequences to be genetically distinct (24.6% divergence) from rat HEV sequences reported in humans.

Laboratory analysis of samples taken from Norway rats in southern Ontario, Canada, revealed hepatitis E virus RNA in 21 (5.6%, 95% CI 3.5%–8.5%) of 372 rats, and phylogenetic analysis demonstrated that these sequences were closely related to those found in rats from other countries. Detection of rat HEV (R. ratti genotype C1) in Norway rats in our study shows that this virus is broadly distributed within southern Ontario, including 3 major cities (i.e., Toronto, Hamilton, and Windsor), and may be endemic in Norway rat populations. An absence of PCR-positive rats in some areas of southern Ontario may be the result of undersampling rather than an indication that HEV is absent in these populations.

We observed that sexually mature rats were at significantly greater odds of being infected with HEV than immature rats. This observation is in contrast to findings from previous studies of rats, which found no association with age and infection status (14,15). We concede that this disparity in findings might be owing to methodological differences in how age classes were defined (i.e., sexual maturity [open vaginal orifice in females, scrotal testes in males] vs. weight). The observed association in our study might be the result of cumulative exposure to HEV leading to increased risk for infection over time and behaviors in sexually mature rats that may increase transmission (e.g., exploratory and aggressive behaviors).

To date, 12 human cases of rat HEV have been reported in Hong Kong, Canada, and Spain (6–8). Although zoonotic transmission from rats to humans has been suggested, the exact source and route of transmission in these cases remains unclear. Notably, human hepatitis E caused by rat HEV may be underreported because of subclinical or mild infection, limited awareness, and diagnostic testing techniques for HEV that might not detect rat HEV. Further studies are needed to investigate potential modes and patterns of transmission and elucidate the zoonotic potential of rat HEV and associated public health risks.

This report of rat HEV (R. ratti genotype C1) in Canada provides further evidence that this virus has a broad geographic distribution globally and may be endemic in Norway rats. Our study highlights the importance of continued surveillance for HEV in rats and the need for additional research regarding the role of rats in human hepatitis E.