Discovery of Novel Alphacoronaviruses in European Rodents and Shrews

Abstract

Eight hundred and thirteen European rodents and shrews encompassing seven different species were screened for alphacoronaviruses using PCR detection. Novel alphacoronaviruses were detected in the species Rattus norvegicus, Microtus agrestis, Sorex araneus and Myodes glareolus. These, together with the recently described Lucheng virus found in China, form a distinct rodent/shrew-specific clade within the coronavirus phylogeny. Across a highly conserved region of the viral polymerase gene, the new members of this clade were up to 22% dissimilar at the nucleotide level to the previously described Lucheng virus. As such they might represent distinct species of alphacoronaviruses. These data greatly extend our knowledge of wildlife reservoirs of alphacoronaviruses.

Keywords: alphacoronavirus; coronaviruses; rodents; shrews.

Figure 1

(A) Geographical map of the East Midlands region of the United Kingdom. Twenty samples were collected from Site 1 (52°49'13.0" N 1°15'01.9" W), four of which were positive for coronaviruses. Four hundred and ninety three samples were collected from Site 2 (52.6528° N, 1.5297° W), six of which were positive for coronaviruses; (B) Geographical map of the Mazury Lake District region of NE Poland. A total of 300 samples were collected from three sites (Site 1: 53°47.7 N 21° 39.6 W, Site 2: 53°53.5 N 21° 32.4 W, Site 3: 53°42.3 N 21° 48.8 W), one of which was positive for coronaviruses.

Figure 2

Maximum likelihood phylogenetic analysis of coronavirus partial ORF1ab gene sequences, corresponding to positions 8331-8960 on the Lucheng Rn isolate (Genbank Accession number KF294380. Novel coronavirus sequences obtained from UK-resident Rattus norvegicus (UKRn), Microtus Agrestis (UKMa), Sorex araneus (UKSa) and Poland-resident (PLMg) or UK-resident (UKMg) were analysed alongside reference sequences representing the four different coronavirus genera. Reference sequences are indicated by their Genbank Accession Number. Branch lengths are drawn to scale: the bar indicates 0.10 nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only percentages >60 are shown.

Figure 3

Maximum likelihood phylogenetic analysis of partial rodent cytochrome b nucleotide sequences. Host cytochrome b sequences obtained from CoV positive UK-resident Rattus norvegicus (UKRn), Microtus Agrestis (UKMa), Sorex araneus (UKSa) and Poland-resident (PLMg) or UK-resident (UKMg) were analysed alongside reference sequences representing rodent and shrew cytochrome b sequences. Reference sequences are indicated by their Genbank Accession Number. Branch lengths are drawn to scale: the bar indicates 0.05 nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only percentages >60 are shown.

Figure 4

(Overleaf): Maximum likelihood phylogenetic analysis (rooted using the severe acute respiratory syndrome (SARS) bat HKU3 betacoronavirus as outgroup) of alphacoronavirus partial (394bp) ORF1ab gene sequences, corresponding to positions 8559-8952 on the Lucheng Rn isolate (Genbank Accession number KF294380. Novel coronavirus sequences obtained from UK-resident Rattus norvegicus (UKRn), Microtus Agrestis (UKMa), Sorex araneus (UKSa) and Poland-resident (PLMg) or UK-resident (UKMg) were analysed alongside bat coronaviruses that were recently discovered in the United Kingdom (GenBank Accession numbers JF440350–JF440366) and France (KT345294–KT345296) and other reference sequences representing the Alphacoronavirus genus. CoV sequences obtained from studies of bats performed in UK and France are highlighted. Reference sequences are indicated by their Genbank Accession Number. Branch lengths are drawn to scale: the bar indicates 0.05 nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only percentages >60 are shown.