Molecular characterization of two hantavirus strains from different rattus species in Singapore

Abstract

Background: Hantaviruses cause human disease in endemic regions around the world. Outbreaks of hantaviral diseases have been associated with changes in rodent population density and adaptation to human settlements leading to their proliferation in close proximity to human dwellings. In a parallel study initiated to determine the prevalence of pathogens in Singapore's wild rodent population, 1206 rodents were trapped and screened. The findings established a hantavirus seroprevalence of 34%. This paper describes the molecular characterization of hantaviruses from Rattus norvegicus and Rattus tanezumi, the predominant rodents caught in urban Singapore.

Methodology: Pan-hanta RT-PCR performed on samples of Rattus norvegicus and Rattus tanezumi indicated that 27 (2.24%) of the animals were positive. sequence analysis of the S and M segments established that two different hantavirus strains circulate in the rodent population of Singapore. Notably, the hantavirus strains found in Rattus norvegicus clusters with other Asian Seoul virus sequences, while the virus strains found in Rattus tanezumi had the highest sequence similarity to the Serang virus from Rattus tanezumi in Indonesia, followed by Cambodian hantavirus isolates and the Thailand virus isolated from Bandicota indica.

Conclusions: Sequence analysis of the S and M segments of hantavirus strains found in Rattus norvegicus (Seoul virus strain Singapore) and Rattus tanezumi (Serang virus strain Jurong TJK/06) revealed that two genetically different hantavirus strains were found in rodents of Singapore. Evidently, together with Serang, Cambodian and Thailand virus the Jurong virus forms a distinct phylogroup. Interestingly, these highly similar virus strains have been identified in different rodent hosts. Further studies are underway to analyze the public health significance of finding hantavirus strains in Singapore rodents.

Figure 1

Hantavirus positive rodents. Scatter plot displaying along the horizontal axis the individual rodents captured in Singapore between 2006-2008 and along the vertical axis, absorbance units determined by ELISA using serum samples of indicated rodents. Dark blue triangles indicate R. norvegicus and open circles R. tanezumi. Other rodent species are pointed out as grey squares. Individual dots are shown as Large or Small data points indicating PCR positive or negative rodents, respectively. The calculated cut-off value (0.135) is indicated by a red arrow.

Figure 2

Nucleotide sequence identity by SimPlot comparison. SimPlot analysis of selected hantavirus genes encoding the nucleocapsid protein. The nucleotide sequences were analyzed in a window of 200 nucleotides and steps of 20 nucleotides. The complete open reading frames of the Singaporean hantavirus strain Jurong TJK/06 (This study) were chosen as query and Seoul SR11 (M34881), Hantaan virus (AB027111), Dobrava-Belgrade (L41916), Seoul Singapore (This study) and Thailand virus (AM397664) as references. SimPlot comparison of the partial sequences of the Serang virus (AM998808) and Cambodian strains (AJ427511) are shown in dark green and yellow curves in addition to the above sequences. Before analysis these two sequences were truncated at the 5' and 3' ends into a 486 nt overlapping region of the Serang virus and the Camb117 and Camb132 strains and compared against the corresponding Jurong TJK/06 cDNA sequence.

Figure 3

Phylogenetic trees of hantavirus S sequences. Phylogenetic trees of hantaviruses based on the complete open reading frames (upper top figure) and partial S segment sequences (bottom left and right figures). In trees, the results after 1000 bootstrap replicates are indicated. (Upper top figure), Tula virus (Z69991); Puumala virus Umea/hu AY526219); Puumala Sotkamo (X61035); Dobrava-Belgrade virus (L41916); Dobrava/Saaremaa/160v (AJ009773); Da Bie Shan virus NC167 (AB027523); Amur virus Solovey/AP61/1999 (AB071183); Hantaan virus A9 (AF329390); Hantaan virus Hu (AB027111); Hantavirus Z5 (EF103195); Hantavirus 76-118 (AF321095); Seoul virus IR461 (AF329388); Hantaan Maaji (M14626); Jurong TJK/06 (this study); Thailand virus (Nakhon Ratchasima/Bi0017/2004) (AM397664); Thailand virus 741 (AB186420); Seoul virus Gou3 (AF184988); Seoul virus Singapore (this study). (Bottom left) Thailand virus (Nakhon Ratchasima/Bi0017/2004) (AM397664); Thailand virus 741 (AB186420); Cambodia 96 (AJ427512); Cambodia 174 (AJ427513); Cambodia 117 (AJ427511); Serang virus/Rt60/2000 (AM998808); Jurong TJK/06 (This study). (Bottom right) Seoul virus IR461 (AF329388); Seoul virus Z37 (AF187082); Seoul virus Pf26 (AY006465); Seoul virus Singapore (this study); Seoul virus Camb41 (AJ427501); Seoul virus Camb180 (AJ427506); Seoul virus Camb32 (AJ427508); Seoul virus Camb58 (AJ427510); Seoul virus SR11 (M34881); Seoul virus Tchoupitoulas (AF329389); Seoul virus L99 (AF288299); Seoul virus Gou3 (AF184988).

Figure 4

Phylogenetic trees of hantavirus M sequences. Phylogenetic trees of hantaviruses based on the complete open reading frames (upper top figure) and partial M segment sequences (bottom left and right figures). In the trees, the results after 1000 bootstrap replicates are indicated. (Upper top figure) Thottapalayam VRC-66412 (EU001329); New York hantavirus (U36802); Sin Nombre virus (NC_005215); Black Creek Canal virus (L39950); Bayou virus (L36930); Andes virus (NC_003467); Laguna Negra virus (AF005728); Puumala virus Umea/hu (AY526218); Puumala Sotkamo (NC_005223); Tula_Moravia 5302v/95 (NC_005228); Soochong virus SC-1 (AY675353); Amur virus JilinAP06 (EF371454); Hantaan virus (NC_005219); Hantaan virus Z5 (EU074224); Hantaan virus TJJ16 (EU074672); Jurong TJK/06 (This study); Thailand virus (L08756); Hantavirus Gou3 (AF145977); Seoul virus, Singapore (this study); Dobrava virus DOB/Saaremaa/160V (AJ009774); Dobrava virus SK/Aa (AY961616); Dobrava-Belgrade virus (L33685); Dobrava-Belgrade virus Ap1584/Sochi-01 (EU188450). (Bottom left) Thailand virus (L08756); Serang virus Rt60/2000 (AM998807); Jurong TJK/06 (This study). (Bottom right) Hantavirus IR461 (AF458104); Hantavirus L99 (AF288298); Seoul virus, Singapore (this study); Seoul virus China (EU163437); Hantavirus Z37 (AF187081); Seoul virus (NC_005237); Seoul virus KI-83-262 (D17592); Hantavirus Gou3 (AF145977); Seoul virus Gou3-e5 (AF288650).