Banna Virus, China, 1987–2007

Banna viruses (BAVs) have been isolated from pigs, cattle, ticks, mosquitoes, and human encephalitis patients. We isolated and analyzed 20 BAVs newly isolated in China; this finding extends the distribution of BAVs from tropical zone to north temperate climates and demonstrate regional variations in BAV phylogeny and mosquito species possibly involved in BAV transmission.

B anna virus (BAV), the prototype species of genus Seadornavirus within the family Reoviridae, has a genome composed of 12 segments of double-stranded RNA (1). BAV was initially isolated from persons with encephalitis and fever in Xishuangbanna, Yunnan Province, People's Republic of China, in 1987 (2). Since then, BAV isolates have been obtained from pigs, cattle, and ticks in China (3,4) and from mosquitoes in Indonesia, China, and Vietnam. (5)(6)(7). BAV is a BioSafety Level 3 arboviral agent that is pathogenic to humans and may well be an emerging pathogen or undiagnosed cause of human viral encephalitis in some areas (1). Our objective was to describe new BAV isolates from China and to defi ne the geographic distribution and the phylogenetic relationships of these isolates with reference to the previously described isolates.

The Study
In this study, 20 new BAV isolates were obtained from mosquitoes collected from July through September during 2006 to 2007 at sites in Gansu Province (latitude 32°-35°N, 104°-107°E), Liaoning Province (39°-41°N, 123°-125°E), Shanxi Province (37°-38°N, 111°-113°E), and Inner Mongolia Province (41°-43°N, 121°-123°E) (Table, Figure 1). Mosquito samples were collected by using 12 V, 200 mA mosquito-trapping lamps (Wuhan Lucky Star Environmental Protection Tech Co., Ltd., Hubei, China) and by collecting mosquitoes from 8:00 PM to 11:00 PM at nearby cow barns, a piggery, and fi sh pond sites where human activity was frequent. Mosquitoes were put into a -20°C freezer for 30 min and then were rapidly sorted into pools of 50 to 100 specimens according to species. The pools were put into labeled tubes and stored in liquid nitrogen.
Viruses were isolated and BAV isolates were identifi ed using described procedures (8). Trizol reagent category no. 10296-028 (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA. cDNA was prepared by using Ready-to-Go You-Prime First-Strand Beads Kit (Amersham Pharmacia Biotech, Piscatawy, NJ, USA) according to the manufacturer's protocol. An 850-bp gene fragment from the 12th segment, which codes for the double-stranded RNA binding protein, was amplifi ed from the cDNA of the BAV isolates by using previously published primers (9). PCR products were recovered by using purifi cation kits (QIA-GEN, Valencia, CA, USA), and then were inserted into pGEM-T easy vector (Promega, Madison, WI, USA). The insert sequence was determined by using M13 universal primers and an ABI Prism 3730 sequence analyzer (ABI, Shirley, NY, USA).
The genomic sequences of the 12th segment for the 20 new BAV strains were determined (GenBank accession nos. GQ331954-GQ331973). Phylogenetic trees were constructed from the amplifi ed region of the 12th segment sequence by using the molecular evolutionary genetics analysis (MEGA) version 4 software (www.megasoftware. net) from aligned nucleotide sequences. We used neighborjoining algorithms with 1,000 replicates for bootstrap support of tree groupings.
In this study, 38 BAV strains isolated during 1987-2007 were analyzed, which included 30 strains isolated in China (including 20 new BAV isolates fi rst reported in this study and 10 previously described isolates from China (8,10-12), 3 strains from Indonesia, and 5 strains from Vietnam) (Table). Initial BAVs were isolated from Indonesia and Yunnan Province of China, which belong to tropical and subtropical zones (2,5).The new BAV isolates in our study were observed in Gansu, Shanxi, Liaoning, and Inner Mongolia provinces of China (northern China), which belong to the northern temperate zone. These strains represent a geographic distribution ranging from near the equator to latitude 45°N, extending from the tropical zone to the northern temperate zone ( Figure  1). These data show that the distribution of BAVs is not limited to Southeast Asia but that it extends into northeast Asia as well.
Phylogenetic analysis based on the complete coding sequence (624 nt) of the 12th segment of the BAV genome  (Figure 1).

Conclusions
Our results demonstrate that BAV strains are distributed from the tropics of Southeast Asia to the northern temperate regions of China. These observations suggest that the distribution of BAV is wider than previously recognized and may be increasing. Consistent with previous observations (9), we report that BAV isolates from China cluster in group A and separate into subgroups mainly according to the geographic origin of the isolate; subgroup A1 is found in the north and subgroup A2 in the south. However, 2 isolates from northern China grouped in subgroup A2 (south), and 3 isolates from Vietnam grouped in subgroup A1 (north).
Considering that group A isolates are geographically located across the monsoon climate zone, where south-to-   tively circulating in areas where Japanese encephalitis virus (JEV) is endemic (14) and where C. tritaeniorhynchus, which is the main vector of JEV, is active. This mosquito also appears to be a common vector of BAV. The clinical symptoms of disease caused by the 2 viruses is similar, and BAV cases may be undetected during a JE outbreak. It has been reported that ≈14% of clinically diagnosed JE cases are BAV immunoglobulin (Ig) M positive (15), indicating that BAV epidemics may have occurred but have been clinically misdiagnosed as Japanese encephalitis. The apparent active transmission of BAV over a large geographic area, genetic variation between geographic regions, and the potential to cause severe disease underscore the need for additional surveillance, further characterization, and improved diagnostic systems worldwide.