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Isolation and molecular characterization of Banna virus from mosquitoes, Vietnam.

We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were isolated from field-caught mosquitoes at various locations; Banna virus was previously isolated from encephalitis patients in Yunnan, China, in 1987. Together, these findings suggest widespread distribution of this virus throughout Southeast Asia.

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Banna virus (BAV) is a 12-segment, double-stranded RNA virus that is classified in the family Reoviridae and the genus Seadornavirus (1). BAV was first isolated from encephalitis patients in southern China, Yunnan Province, Xishuangbanna Prefecture, in 1987 (2). In addition, antigenetically BAV-consistent viruses were isolated from pigs, cattle, and humans in Yunnan Province and from the sera of febrile patients in Xinjiang Province (3). Therefore, BAV is suspected to be a pathogen of vertebrates and an encephalitis pathogen for humans. BAV was also isolated from mosquitoes in China (3-5) and Indonesia (6), prompting an alert about a pathogenic arbovirus in these countries. Until now, no mammalian cell line has been reported to propagate BAV, and experimental infection with BAV has not resulted in clinical encephalitis in mice (7). However, another species of Seadornavirus, the Liao Ning virus, has been propagated in various mammalian cell lines. Although this virus has caused hemorrhaging in mice, it has not been associated with the development of encephalitis (7).

The pathogenicity and precise distribution of Seadornavirus spp., including BAV, are not well understood, and the scope and impact of BAV infection in Asia require further investigation. We report the circulation of BAV in Culex spp. mosquitoes in Vietnam and demonstrate that 2 new phylogenetically distinct types of BAVs are co-circulating. These findings suggest that this virus is widely distributed throughout Southeast Asia.

The Study

Mosquito samples were collected in Vietnam in 2002. Each species was separated and pooled, and each mosquito pool was then homogenized and centrifuged. The supernatant was filtrated through a 0.22-[micro]m filter and then inoculated onto a monolayer C6/36 mosquito cell culture and subsequently incubated at 28[degrees]C for 7 days. Virus amplification in C6/36 cells was repeated twice, and the culture fluids were stored at -80[degrees]C for further analysis. RNA was extracted from the second cell culture fluid by TRIZOL LS Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed by using Superscript III Reverse Transcriptase (Invitrogen) and random hexamers, after RNA denaturation at a temperature of 95[degrees]C for 5 minutes in the presence of 15% dimethyl sulfoxide. PCR was conducted by using TaKaRa LA Yaq DNA polymerase (Takara Bio, Inc., Otsu, Japan) with BAV targeting primers (for segment 5, 5'-CAGCTGCAGTGGTTATTGGA-3' and 5'ACCGTGCATCTTAACCCTTG-3'; for segment 8, 5'-TTGCAGTCGCTGAGCTTTTA-3' and 5-CGCATTTGATCGTATGCTTG-3'). These targeting primers were designed from sequences of BAV strains from China and Java (GenBank accession nos. AF134519-AF 134527,AY549307-AY549309,AF052024-AF052035, AY568287-AY568290,NC_004211,NC_004217-NC_004221, NC_004200-NC_004204, NC_004198, and AF052008-AF052013). The amplified cDNAs were sequenced in the 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The genomic sequences of all 12 genome segments for the 5 strains were determined (GenBank accession nos. EU265673-EU265727, EU312980).

The sequences were translated to protein sequences by using the Transeq program of the EMBOSS package (8), version 5.00. The protein sequences were aligned with the T-COFFEE program version 5.05 (9) with Chinese and Javanese BAVs. Nucleotide sequences were then aligned, on the basis of protein alignment, with the Tranalign program of the EMBOSS package. Maximum likelihood phylogenetic trees were constructed from the protein-guided alignments of nucleotide sequences with DNAML software of the PHYLIP package (10), version 3.67.

Five BAV strains were isolated in Ha Tay and Quang Bing Provinces (Figure 1) in Vietnam from Culex annulus and Cx. tritaeniorhunchus (Table 1). Phylogenetic analysis showed diversity within the phylogenetic clustering of each segment (Table 2). Segments 7 and 9 formed 5 independent clusters, segment 12 formed 3 clusters, and the remaining segments formed 4 clusters. Phylogenetic trees for some representative segments (segments 2, 6, 9, and 12) are shown in Figure 2. Javanese strains formed a single cluster for segments 1, 2, 6, 8, 10, and 12. For segments 7, 9, and 11, however, they diverged into 2 clusters, I and II (Figure 2, panel C). The Vietnamese strains were distributed within 2 independent clusters for all 12 segments. Segments 11 and 12 of the 2 Vietnamese strains 02VN178b and 02VN018b were included in cluster III with Chinese BAVs. Segments 6 and 9 of isolate 02VN178b belonged to cluster V, although segments 1, 2, 3, 4, and 8 belonged to cluster IV. Moreover, segments 5 and 7 of isolate 02VN178b and segments 8 and 11 of isolate 02VN009b showed a mixed electrogram pattern of clusters V and IV or III.

[FIGURE 1 OMITTED]

Conclusions

We report isolation of BAV in Vietnam. The nucleotide sequences of the Vietnamese isolates' genomic RNA segments diverged into 2 phylogenetically distant clusters. Our data indicate that 2 BAV populations exist in the country and that both evolved independently. Two strains were clearly different from Chinese and Javanese BAVs (02VN078b and 02VN180b). Two other strains (02VN018b and 02VN178b) were phylogenetically close to Chinese BAVs when 2 shorter segments, segments 11 and 12, were analyzed but not when the remaining 10, longer segments were considered. This finding implies that a recent reassortment event has occurred and that segments of Chinese or Vietnamese strains were replaced. In addition, although some Vietnamese strains were phylogenetically distant, they could produce a viable progeny by reassortment of segments 5, 6, 7, 8, 9, and 11. This finding is clearly illustrated by the diversity in the phylogenetic clustering pattern and the mixed electrogram pattern of these segments (Table 2).

Analysis of segment 12 showed that cluster III (Table 2) contained 2 Vietnamese strains (02VN178b and 02VN018b) and the Chinese strains AF052030 and YN-6 (isolated in Yunnan Province) and BJ95-75 (isolated in Beijing). This finding suggests the possibility of wide circulation of BAVs containing segment 12 type III not only in Southeast Asia but also in East Asia.

A regionwide genotype shift of Japanese encephalitis virus (JEV), which is also carried by Culex spp. mosquitoes, from genotype III to genotype I, was witnessed in East Asia during the 1990s (11,12). These results and phylogenetic analysis of old JEV strains in Japan and Southeast Asia (11) strongly suggested that JEV could be transferred frequently from Southeast Asia to East Asia. Similarly, BAVs with segment 12 belonging to cluster type III were also distributed in both Southeast and East Asia. Therefore, we can speculate that BAVs are actively circulating within the Asian continent. To achieve a better understanding of the distribution dynamics of BAVs in Asia, more isolates are needed.

In Vietnam, a seasonal increase of viral encephalitis and meningitis cases is reported during the rainy season every year. Among these cases, 50%-70% are diagnosed as Japanese encephalitis, while the rest of them remain idiopathic (P.T. Nga et al., unpub, data). A clear association between BAV and human diseases, as well as the prevalence of BAV infection in humans, has not yet been established in Vietnam. This topic deserves further study. We also believe that BAV is a good candidate for the differential diagnosis of viral encephalitis and meningitis cases of unknown origin in tropical and subtropical Asia, where Culex mosquitoes are abundant.

[FIGURE 2 OMITTED]

This work was supported by the program of Founding Research Centers for Emerging and Reemerging Infectious Diseases (MEXT) Japan, the 21st Century Centers of Excellence Program (MEXT) Japan, Japan Society for the Promotion of Science Core University Program, and grant in Aid MEXT No. 18659131.

References

(1.) Attoui H, Billoir F, Biagini P, de Micco P, de Lamballerie X. Complete sequence determination and genetic analysis of Banna virus and Kadipiro virus: proposal for assignment to a new genus (Seadornavirus) within the family Reoviridae. J Gen Virol. 2000;81: 1507-15.

(2.) Xu P, Wang Y, Zuo J, Lin J. New orbiviruses isolated from patients with unknown fever and encephalitis in Yunnan province. Chin J Virol. 1990;6:27-33.

(3.) Tao SJ, Chen BQ. Studies of coltivirus in China. Chin Med J (Engl). 2005;118:581-6.

(4.) Attoui H, Mohd Jaafar F, de Micco P, de Lamballerie X. Coltiviruses and seadornaviruses in North America, Europe, and Asia. Emerg Infect Dis. 2005;11:1673-9.

(5.) Chen B, Tao S. Arbovirus survey in China in recent ten years. Chin Med J (Engl). 1996;109:13-5.

(6.) Brown SE, Gorman M, Tesh B, Knudson L. Coltiviruses isolated from mosquitoes collected in Indonesia. Virology. 1993; 196:363-7. DOI: 10.1006/viro. 1993.1490

(7.) Attoui H, Mohd Jaafar F, Belhouchet M, Tao S, Chert B, Liang G, et al. Liao ning virus, a new Chinese seadoruavirus that replicates in transformed and embryonic mammalian cells. J Gen Virol. 2006;87:199-208. DOI: 10.1099/vir.0.81294-0

(8.) Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16:276-7. DOI: 10.1016/S0168-9525(00)02024-2

(9.) Notredame C, Higgins G, Heringa J. T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000;302:205-17. DOI: 10.1006/jmbi.2000.4042

(10.) Felsenstein J. PHYLI--Phylogeny Inference Package (Version 3.2). Cladistics. 1989;5:164-6.

(11.) Nga PT, del Carmen Parquet M, Cuong D, Ma P, Hasebe F, Inoue S, et al. Shift in Japanese encephalitis virus (JEV) genotype circulating in northern Vietnam: implications for frequent introductions of JEV from Southeast Asia to East Asia. J Gen Virol. 2004;85:1625-31. DOI: 10.1099/vir.0.79797-0

(12.) Ma SP, Yoshida Y, Makino Y, Tadano M, Ono T, Ogawa M. Short report: a major genotype of Japanese encephalitis virus currently circulating in Japan. Am J Trop Med Hyg. 2003 ;69:151-4.

Takeshi Nabeshima, * Phan Thi Nga, ([dagger]) Posadas Guillermo, * Maria del Carmen Parquet, * Fuxun Yu, * Nguyen Thanh Thuy, ([dagger]) Bui Minh Trang, ([dagger]) Nguyen Tran Hien, ([dagger]) Vu Sinh Nam, ([dagger]) Shingo Inoue, * Futoshi Hasebe, * and Kouichi Morita *

* Nagasaki University, Nagasaki City, Japan; and ([dagger]) National Institute of Hygiene and Epidemiology, Hanoi, Vietnam

DOI: 10.3201/eid 1408.080100

Dr Nabeshima is a virologist at Nagasaki University. His current research interest is the evolutional history of viruses.

Address for correspondence: Kouichi Morita, Department of Virology, Institute of Tropical Medicine, Nagasaki University, Sakamoto-machi 1-12-4, Nagasaki City, Japan 852-8523; email: moritak@net.nagasaki-u. ac.jp
Table 1. Mosquito samples collected in Vietnam, 2002

 Month No.
Sample Location collected Origin mosquitoes

02VN009b Ha Tay Jan Culex annulus 25
02VN018b Quang Binh Mar Cx. annulus 170
02VN078b Ha Tay May Cx. tritaeniorhunchus 100
02VN178b Quang Binh Aug Cx. tritaeniorhunchus 102
02VN180b Quang Binh Aug Cx. tritaeniorhunchus 83

Table 2. Phylogenetic clustering of Banna virus genomic RNA segments *

 Isolation Sediments

Strain Location Year 1 2 3 4 5

Length (bp) 3747 3048 2400 2038 1716
Aligned area 990- 211- 108- 570- 110-
 3227 2541 2183 1748 760
JKT-6423 Java, 1980 I I I I I
 Indonesia
JKT-6969 Java, 1981 I I NA NA NA
 Indonesia
JKT-7043 Java, 1981 I I NA NA NA
 Indonesia
Chinese Yunnan, 1987 III III III III III
 China
YN-6 Yunnan, 2000 NA NA NA NA NA
 China
BJ95-75 Beijing, 1995 NA NA NA NA NA
 China
02VN018b Quang 2002 IV IV IV IV IV
 Binh,
 Vietnam
02VN178b Quang 2002 IV IV IV IV IV/V
 Binh,
 Vietnam
02VN009b Ha Tay, 2002 V V V V V
 Vietnam
02VN078b Ha Tay, 2002 V V V V V
 Vietnam
02VN180b Quang 2002 V V V V V
 Binh,
 Vietnam

 Sediments

Strain 6 7 8 9 10 11 12

Length (bp) 1671 1136 1119 1101 977 867 862
Aligned area 106- 145- 129- 597- 86- 75- 44-
 1386 981 913 872 751 609 664
JKT-6423 I I I I I I I

JKT-6969 I II I II I II I

JKT-7043 I II I II I II I

Chinese III III III III III III III

YN-6 NA NA NA III NA NA III

BJ95-75 NA NA NA III NA NA III

02VN018b IV IV IV IV IV III III

02VN178b V IV/V IV V IV III III

02VN009b V V IV/V V V V/III V

02VN078b V V V V V V V

02VN180b V V V V V V V

* Length, length of each segment in the reference sequence of
Banna virus (BAV) from RefSeq (NC_004198, NC_004200-NC_004204,
NC_004211, NC_004217-NC_004221); aligned area, the fragment used to
make the alignment and phylogenetic tree. The numbers are set
following the sequences of strain JHT-6423, which is the reference
strain in National Center for Biotechnology Information Reference
Sequences. I, the cluster including the Javanese JKT-6423 strain;
II, the cluster specific to the JKT-6969 and JKT-7043 strains; III,
the cluster including Chinese BAVs; IV, the cluster including the
Vietnamese 02VN018b strain; V, the cluster includin, the Vietnamese
02VN180b strain. NA not available.
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Article Details
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Title Annotation:DISPATCHES
Author:Nabeshima, Takeshi; Nga, Phan Thi; Guillermo, Posadas; Parquet, Maria del Carmen; Yu, Fuxun; Thuy, N
Publication:Emerging Infectious Diseases
Geographic Code:9VIET
Date:Aug 1, 2008
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