Examine on the ecology of viruses in city rodents with particular emphasis on SARS-CoV-2

In a study recently published on the bioRxiv* preprint server, researchers examined whether commensal rodent pests such as rats and mice derived from sewers and urban environments could transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They also studied viruses present in commensal rodents.

Animals contract viral diseases naturally, and some causative viral organisms may have zoonotic transmission potential. The 2019 coronavirus disease (COVID-19) pandemic has alerted researchers to the possibility of human-to-rodent retransmission of SARS-CoV-2. Rodent species are known to transmit virus to humans and have been implicated as likely SARS-CoV-2 (and other CoV) reservoirs.

Study: The ecology of viruses in urban rodents with a focus on SARS-CoV-2. Photo credit: Rudmer Zwerver/Shutterstock

About the study

In the present study, researchers examined and described the range of viruses found in rats and mice, and examined the exposure and infection of commensal rodent species with SARS-CoV-2.

The team collected samples of mice (Apodemus sylvaticus) and rats (Rattus norvegicus) from urban environments in 2020 during the SARS-CoV-2 human pandemic. The main catch occurred between June 15, 2020 and November 20, 2020 in and around the city of Liverpool, England. The team captured 45 and 35 R. norvegicus rats alive, respectively, from Greenbank Park and a sewage treatment plant near Liverpool city centre. Sixty-nine A. sylvaticus mice were trapped in several Liverpool municipal parks such as Princes Park, Sefton Park, Wavertree Botanic Gardens and Calderstones Park. Samples of lung and intestinal tissue and feces from the rodents were obtained.

Ribonucleic acid (RNA) extracted from the tissues was subjected to metagenomic sequence analysis for viruses and polymerase chain reaction (PCR) analysis for the detection of SARS-CoV-2. The number of viruses, the prevalence of different viruses, the number of infections caused by different viruses, the viral load of different viruses and the tissue specificity were evaluated. Liquid extracts of heart, liver, and lung tissue were subjected to enzyme-linked immunosorbent assay (ELISA) to measure anti-SARS-CoV-2 spike (S) protein immunoglobulin G (IgG) and IgA titers.

In vitro neutralization assays using SARS-CoV-2 enveloped PVPs (pseudotyped virus particles) encoding Wuhan Hu-1 strain S were performed to determine inhibition of SARS-CoV-2 infection by cardiac and wild rat lung fluid to evaluate human embryonic kidney (HEK)293T angiotensin converting enzyme 2 (ACE2) transmembrane serine protease 2 (TMPRSS2) cells. For comparison, SPF laboratory rats and Mus musculus mice with high (108 virus particles/gram tissue) and low (104 virus particles/gram tissue) doses of inactivated SARS-CoV-2 were included as negative controls.

Wister rats and C57BL/6J immunized intramuscularly with SARS-CoV-2 S were used as positive controls. In addition to the captured rodents, 41 southern England rats were provided by pest controllers and removed from a residential area in Reading and a household waste disposal plant in Basingstoke. Rodents provided by pest controllers were used for serological analysis only.

Results

A total of 6.0 x 109 and 1.0 x 1010 reads were obtained for mouse and rat samples, respectively, of which 4.0% and 2.0% of the reads from the corresponding animals were identified as viral in origin. Among the samples, 297 viruses were detected in the samples, of which 264 viruses, 13 viruses and 20 viruses were detected only in rats, only in mice and rodent species, respectively. In rats, 97 virus organisms were detected exclusively in lung tissue, seven viruses exclusively in intestinal tissue and four viruses exclusively in rat faeces, with < 50% of the viruses showing tissue specificity. Most of the viruses detected in the rodents belonged to four families of viruses, ie Baculoviridae, Picornaviridae, Astroviridae or Poxviridae. No virus of the family Coronaviridae was detected in mice; However, the family led to 1.0% of viral diseases in rats.

In mice, Simbu orthobunyavirus, Shamonda orthobunyavirus (both of the Peribunyaviridae family), and Choristoneura fumiferana granulovirus (of the Baculoviridae family) had >50% prevalence. In rats, 23 viral organisms were found with a prevalence >50%, belonging to the families Picornaviridae, Virgaviridae and Nodaviridae. In rats, the viruses with >0.50% sequence reads were Boolarra virus, Rosavirus B, Cardiovirus C, and Hunnivirus. In mice, the viruses accounting for >1.0% of reads were Choristoneura fumiferana granulovirus, Shamonda orthobunyavirus, and Simbu orthobunyavirus.

Three rats showed SARS-CoV-2 readings greater than 104 SARS-CoV-2 particles/gram of tissue. In addition, three positive samples were obtained from a pool of lung tissue from three rats and two pools of intestinal tissue, each from three different live-captured rats. Eight, 72, and 39 reading pairs in the positive samples were aligned with the SARS-CoV-2 genome, representing low-level contamination. None of the mouse samples had more SARS-CoV-2 reads than the positive controls.

Metagenomic CoV reads have been identified in rats, specifically Ferret CoV (alpha-CoV) in seven samples, Rhinolophus bat-CoV HKU2 (alpha-CoV) in one sample, and MERS (Middle East Respiratory Syndrome)-CoV (beta-CoV) in seven samples rat samples. However, no CoVs were detected above the cut-off in mice and PCR analysis showed no presence of SARS-CoV-2 in the rodents. Most rodents had very low anti-S-IgA and IgG titers, although seven rats had optical densities (OD) comparable to the positive control for pulmonary humoral responses. Three (out of 15) rat heart samples showed 36% to 70% inhibition of SARS-CoV-2 infection and four rat lung samples showed 39% to 61% inhibition of SARS-CoV-2 infection.

Overall, study results showed no molecular evidence of SARS-CoV-2 infection in the rodents, although pulmonary anti-SARS-CoV-2 S-IgA titers and partial SARS-CoV-2 neutralization were observed in rats. Results showed that exposure of rats to SARS-CoV-2 or other viruses resulted in titers of cross-reactive antibodies.

*Important NOTE

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health behavior, or be treated as established information.