Bat coronaviruses and experimental infection of bats, the Philippines

Abstract

Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription-PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9-1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.

Figure 1

Phylogenetic tree based on deduced amino acid sequences of partial RNA-dependent RNA polymerase of coronaviruses (CoVs), the Philippines. The 2 new viruses detected in this study are underlined. Percentage of replicate trees in which the associated taxa clustered in the bootstrap test (1,000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed by using the Poisson correction method and are shown as number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset. The final dataset included 120 positions. Phylogenetic analyses were conducted in MEGA4 (14). Coronaviruses used for comparisons and their GenBank accession numbers are human coronavirus (HCoV) 229E (HCoV-229E) (NC_002645), porcine epidemic diarrhea virus (PEDV) (NC_003436), transmissible gastroenteritis virus (TGEV) (NC_002306), feline infectious peritonitis virus (FIPV) (AY994055), human coronavirus NL63 (HCoV-NL63) (NC_005831), bat-CoV/A512/2005 (NC_009657), bat-CoV/A515/2005 (DQ648822), bat-CoV/A620/2005 (DQ648828), bat-CoV/A911/2005 (DQ648850), bat-CoV/GhanaKwan/19/2008 (FJ710046), bat-CoV/GhanaKwan/20/2008 (FJ710047), bat-CoV/M.das/Germany/D3.3/2007 (EU375854), bat-CoV/USA/RM-11 (EF544563), bat-CoV HKU2 (EF203064), HKU4 (NC_009019), HKU5 (NC_009020), HKU6 (DQ249224), HKU8 (DQ249228), HKU9 (NC_009021), CoV-HKU1 (NC_006577), human coronavirus (HCoV-OC43) (NC_005147), murine hepatitis virus (MHV) (NC_001846), bovine coronavirus (BCoV) (NC_003045), porcine hemagglutinating encephalomyelitis virus (PHEV) (NC_007732), human severe acute respiratory syndrome coronavirus (SARS) (SARS-human) (NC_004718), civet SARS-like coronavirus (SARS-civet) (AY304488), bat-SARS-like coronavirus HKU3 (bat-SARS-CoV HKU3) (NC_009694), infectious bronchitis virus (IBV) (NC_001451), and turkey coronavirus (AF124991).

Figure 2

Comparison of mRNA sequences of bat coronavirus (BatCoV) with viral genomic sequences. Read 1 was obtained by using reverse transcription–PCR and HKU9-Leader42–64 and N468–448r primers. Read 2 was obtained by using HKU9-Leader42–64 and Ns7a440–420r primers. Asterisks indicate sequence identity for read and virus genome sequences. TRS, transcription regulatory sequence; N, nucleocapsid; NS, nonstructural.

Figure 3

Bat coronavirus/Philippines/Dilliman1525G2/2008 mRNA in experimentally infected fruit bats, the Philippines. Reverse transcription–PCR results for small intestines of bats A and B. Lane M, 100-bp DNA ladder; lane –, nontemplate control.