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Journal of Virology, June 2006, p. 5627-5630, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02448-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Batai and Ngari Viruses: M Segment Reassortment and Association with Severe Febrile Disease Outbreaks in East Africa

Thomas Briese,1* Brian Bird,2,3 Vishal Kapoor,1 Stuart T. Nichol,2 and W. Ian Lipkin1

Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York, New York 10032,1 Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,2 School of Veterinary Medicine, University of California, Davis, California 956163

Received 21 November 2005/ Accepted 13 March 2006


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ABSTRACT
 
Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.


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TEXT
 
Viruses with segmented genomes have the capacity to expand their genetic diversity not only through slow genetic drift, resulting from an accumulation of individual point mutations, but also through sudden genetic shift, resulting from a new arrangement of whole genome segments in progeny viruses produced during a coinfection event. Although reassortment of the tripartite orthobunyaviral genome, comprising segments designated small (S), medium (M), and large (L), has been shown experimentally to occur easily between genetically related viruses (2, 3, 10, 19), examples of natural reassortment have not frequently been described for Bunyamwera or California serogroup viruses (16). Recently, Ngari virus (NRIV) was recognized as a natural reassortant virus associated with a hemorrhagic fever outbreak in Kenya and Somalia (4, 11). Sequence analysis of isolates from this outbreak revealed that their S, M, and L segments genetically matched those of NRIV prototype DakArD28542 (21) and also that their L and S segment sequences were closely related to Bunyamwera virus (BUNV), whereas the M segment sequences (i.e., NRIV, SBUN MUNIQUE LBUN) were unique. Analysis of partial M segment sequences of 45 members of the genus Orthobunyavirus failed to reveal the progenitor of the NRIV M segment (11). Here we present additional sequence information for members of this genus and identify the potential origin of NRIV M segment sequence.

We analyzed M segments of Batai virus (BATV), an orthobunyavirus first reported from Malaysia (14) that has not been isolated from humans, and Ilesha virus (ILEV), an African virus described as an antigenic variant or subtype of BATV (12) that like NRIV has been reported in association with hemorrhagic fevers (14, 17). Viral sequences were amplified from reverse-transcribed RNA extracts using broad-range PCR primers targeting the S, M, and L segment sequences of Bunyamwera and California serogroup viruses (4, 5). Sequence analysis indicated only 68% nucleotide and 70% deduced amino acid identity between BATV and ILEV M segments (GenBank accession no. AY772534/DQ375394 and AY859372, respectively), consistent with ILEV being a distinct virus and genetically less closely related to BATV than might have been expected based on earlier serologic analysis. M segment sequence comparisons between ILEV and NRIV did not reveal obvious sequence similarities relating to a potentially unique pathogenicity of these viruses (Table 1 and Fig. 1). Surprisingly, the prototype BATV (MM2222) M segment closely matched that of NRIV, showing only 11% and 5% differences in nucleotide or deduced amino acid sequence, respectively. Gn, which is conserved among Bunyamwera serogroup and California serogroup viruses, was highly conserved between BATV and NRIV. More remarkable NSm, which usually differs considerably between species, was also well conserved (Fig. 1A; approximate position 350 to 450). Limited divergence was observed in the N-terminal portion of Gc (Fig. 1A; approximate position 600 to 900), mainly around the conserved potential trypsin cleavage site that had been characterized in California serogroup snowshoe hare and La Crosse viruses (8, 15).


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  		TABLE 1. M segment sequence conservation between Batai and Ngari virus isolates and selected other orthobunyavirusesa


Figure 1
Figure 1
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  		FIG.1. Analysis of M segment sequence conservation between BATV, NRIV, and ILEV. (A) Sliding-window distance analysis between BATV (MM2222) and NRIV (Kenya), ILEV, CVV, BUNV, Guaroa virus (GROV; GenBank accession no. AY380581), and California encephalitis virus (CEV; AF123483) (window, 60 amino acids [aa]; step 10 amino acids). (B) Alignment of terminal M segment sequences of BATV (MM2222), NRIV (Kenya), and ILEV. Coding sequence for the polyprotein is omitted, and only the ochre codon and initiation codon are indicated (underlined and overlined UUA and CAU, respectively; sequence given in genomic antisense orientation). Sequences used in PCR priming are indicated in lowercase italics.

A close relationship between the BATV M segment sequence and that of NRIV is also evident at the nucleotide level, including the terminal untranslated sequences. A high degree of conservation was observed not only for the 11 terminal nucleotides that are invariant among members of the genus, but also throughout the 5'- and 3'-untranslated regions of BATV and NRIV (Fig. 1). The S segment sequence of BATV, obtained from the same cDNA used for M segment amplification, matched the published sequence (6). Furthermore, S and partial L segment sequences of BATV (GenBank accession no. AY822469) were different from those of BUNV and NRIV. These data indicate that BATV is a genetically distinct virus within the Bunyamwera serogroup with an M segment that shares a common ancestor with the M segment of NRIV (SBUN MBAT LBUN).

Implicit in the concept of natural reassortment is geographic coincidence. In this context, it is noteworthy that BATV is one of the most widespread of the orthobunyaviruses (12). In addition to the initial BATV isolates from Southeast Asia (14), Calovo (from Slovakia) (1), Chittoor (from India) (20), and Olkya (from Russia) (9) have been serologically characterized as isolates of BATV. The presence in Africa of viruses serologically related to BATV was reported in 1967 in Uganda (7, 12) and in 1988 in Sudan (18). Based on the geographic origin of these viruses, the question was raised as to whether the M segment of the Ugandan isolate UgMP-6830 and the Sudanese isolate KV-141 may be a closer match to that of the Kenyan NRIV than those of BATV isolates from more distant geographic locales. Thus, sequences of the UgMP-6830, KV-141, Calovo, and Chittoor isolates were analyzed, and the KV-141 and UgMP-6830 M segments (GenBank accession no. DQ375393 and DQ436460, respectively) were found to be the closest match to NRIV, with only 2.7% and 5.1% nucleotide differences and 1.5% and 2.4% deduced amino acid differences, respectively. Whereas partial S and L segment sequences of UgMP-6830, Chittoor, and Calovo viruses were related to those of MM2222, examination of partial S and L segment sequence of KV-141 revealed that its genotype was SBUN MBAT LBUN, identical to that of NRIV (Kenya). These results indicate that the Batai-related virus isolate from mosquitoes in Uganda (7) is an isolate of BATV, whereas the isolates from the 1988 Sudan febrile disease outbreak are actually NRIV. Plaque-reduction neutralization test results obtained previously with BATV prototype MM2222, BATV UgMP-6830, and what we now know to be NRIV KV-141 show these viruses to be essentially indistinguishable (12, 18). Neutralizing antibodies target the surface glycoproteins encoded by the virus M segment. Thus, the combination of genetic and serologic criteria confirms that the M segment of NRIV is that of BATV, represented in Africa by UgMP-6830 (12). The biological potential for formation of a BUNV/BATV chimera had previously been demonstrated experimentally (13). However, unless we understand the phylogenetic relationships of all viruses in this serogroup and of all their genome segments, assignments of reassortants will remain arbitrary. BATV may represent a reassortant of NRIV with an as yet not identified third virus, as BUNV might represent a reassortant of NRIV with an unidentified M segment donor. Further phylogenetic analyses of complete S, M, and L segments will be necessary to comprehensively assess the evolution of these viruses.

Recognition of KV-141 as another isolate of NRIV points to interesting similarities between the severe febrile disease outbreaks observed in Sudan in 1988 and in Kenya and Somalia in 1997 and 1998. In both cases, the rather arid regions experienced unusually heavy rains and extensive flooding and outbreaks of human febrile illness ensued. In the 1988 Sudan outbreak, 77,500 patients were recorded, of which 14,395 were diagnosed with malaria based primarily on clinical presentation (18). However, two apparently identical virus isolates were obtained from patient sera (NRIV KV-141 and KV-66), and immunoglobulin M (IgM) antibody reactive with these isolates was detected in 7% of 195 sera tested, indicating recent infection. These results suggest that NRIV was associated with a portion of the febrile disease observed. During the 1997-1998 outbreak in Kenya and Somalia, an estimated 89,000 human infections occurred with over 250 deaths. Of 231 febrile patients for which clinical records existed, 50% met the case definition of hemorrhagic fever (4). The outbreak was diagnosed as a Rift Valley fever outbreak; however, of the hemorrhagic fever cases investigated, 23% had evidence of acute Rift Valley fever virus infection (based on IgM, virus isolation, PCR, and/or immunohistochemistry), whereas 27% had evidence of acute NRIV infection (based on PCR and/or IgM antibody). Identification of the virus isolates from both these outbreaks as NRIV indicates that this reassortant virus can be associated with large outbreaks of severe febrile illness in East Africa and that NRIV should be considered in the differential diagnosis of such outbreaks throughout sub-Saharan Africa.


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ACKNOWLEDGMENTS
 
We thank Robert B. Tesh and the late Robert E. Shope, University of Texas, Galveston, and Robert Lanciotti, CDC, Fort Collins, CO, for providing Ilesha, Batai, Chittoor, Calovo, UgMP-6830, and KV-141 virus stocks, and we thank Jack Woodall for information on UgMP-6830. We are also indebted to our reviewers for their valuable comments.

This work was supported by awards from the Veterinary Science Training Program of the School of Veterinary Medicine, University of California at Davis to B.B., and by awards from the Ellison Medical Foundation and NIH (AI056118-02) to T.B. and W.I.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York, NY 10032. Phone: (212) 342-9035. Fax: (212) 342-9044. E-mail: thomas.briese{at}columbia.edu. Back


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Journal of Virology, June 2006, p. 5627-5630, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02448-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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