Characterization of Nipah Virus from Outbreaks in Bangladesh, 2008–2010

Michael K. Lo

, Luis Lowe, Kimberly B. Hummel, Hossain M.S. Sazzad, Emily S. Gurley, M. Jahangir Hossain, Stephen P. Luby, David M. Miller, James A. Comer, Pierre E. Rollin, William J. Bellini, and Paul A. Rota

Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (M.K. Lo, L. Lowe, K.B. Hummel, D.M. Miller, J.A. Comer, P.E. Rollin, W.J. Bellini, P.A. Rota); International Centre for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh (H.M.S. Sazzad, E.S. Gurley, M.J. Hossain, S.P. Luby)

Abstract

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes fatal encephalitis in humans. The initial outbreak of NiV infection occurred in Malaysia and Singapore in 1998–1999; relatively small, sporadic outbreaks among humans have occurred in Bangladesh since 2001. We characterized the complete genomic sequences of identical NiV isolates from 2 patients in 2008 and partial genomic sequences of throat swab samples from 3 patients in 2010, all from Bangladesh. All sequences from patients in Bangladesh comprised a distinct genetic group. However, the detection of 3 genetically distinct sequences from patients in the districts of Faridpur and Gopalganj indicated multiple co-circulating lineages in a localized region over a short time (January–March 2010). Sequence comparisons between the open reading frames of all available NiV genes led us to propose a standardized protocol for genotyping NiV; this protcol provides a simple and accurate way to classify current and future NiV sequences.

Nipah virus (NiV) is a deadly paramyxovirus that was first described during 1998–1999 in Malaysia and Singapore, when a large epidemic of fatal encephalitis occurred in humans (283 cases, 109 deaths) (1). In this initial outbreak, most human cases were epidemiologically linked with activities involving close contact with sick pigs; the outbreak ended after >1 million pigs were culled and movement of pigs was stopped (2).

Although NiV infection has not been detected in Malaysia or Singapore since 1999, NiV has caused recurring (almost annual) outbreaks of fatal encephalitis in Bangladesh and sporadic outbreaks in India since 2001 (3–6). The outbreaks in Bangladesh have demonstrated human-to-human and foodborne transmission of NiV (7–9). Although the outbreaks in Bangladesh have been smaller, the case-fatality rates have been consistently higher (≈75%) than those from the initial outbreak in Malaysia and Singapore (≈40%) (8,10).
The clinical case definition used in Bangladesh differs from that used during the Malaysia outbreak and focuses on fatal or severe neurologic signs and symptoms. Sequence analysis of virus isolates and clinical samples obtained from persons affected by the outbreaks in Bangladesh and India indicated greater nucleotide heterogeneity than those from Malaysia (3,4,11).

Within 2 weeks in Bangladesh during February 2008, 2 clusters of human NiV infection resulted in 10 cases with 9 deaths (90% case-fatality rate). The locations of the clusters (Rajbari and Manikgonj districts) were ≈44 km apart, separated by the intersection of the Padma and Jamuna Rivers. The outbreak was linked to ingestion of raw date palm sap (12). From December 2009 through March 2010, an outbreak of NiV infection in Fardipur and Gopalganj districts was responsible for 17 cases and 15 deaths (88% case-fatality rate) (6).
In this study, we confirmed the suspected clinical cases of NiV infection from both outbreaks by using IgM and IgG ELISAs, real-time and conventional reverse transcription PCR (RT-PCR), and virus isolation. We characterized the complete genomic sequences of 2 identical NiV isolates from 2008 and 3 partial genomic sequences of isolates from 2010. Our results indicate the presence of multiple co-circulating lineages of NiV in a localized region over a short time in 2010. Phylogenetic and sequence analysis of all currently available full-length NiV gene open reading frames (ORFs) led us to propose a standardized protocol for genotyping NiV.