Detection of dengue-4 virus in pune, western india after an absence of 30 years - its association with two severe cases
© Cecilia et al; licensee BioMed Central Ltd. 2011
Received: 15 November 2010
Accepted: 1 February 2011
Published: 1 February 2011
Difference in severity of dengue outbreaks has been related to virus serotype, genotype and clades within genotypes. Till the 1980 s, India and Sri Lanka reported low number of dengue hemorrhagic fever (DHF) cases despite circulation of all four serotypes of dengue virus (DENV). Since the 1990 s the occurrence of DHF has increased. The increase has been attributed to changes in virus lineage especially with regard to DENV-2 and DENV-3. DENV-1 has been associated with dengue fever (DF) outbreaks and DENV-4 reports have been rare. The emergence of DENV-4 was reported recently in 2003 in Delhi and in 2007 in Hyderabad. The last report of DENV-4 from Maharashtra was in 1975 from Amalner.
We report on the detection of DENV-4 in Pune, Maharashtra after an absence of almost 30 years. Two cases were detected in 2009-10, serotyped by multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Both the cases were recorded as severe dengue (Category 3) requiring intensive care unit (ICU) level of treatment. Depending on the hemagglutination inhibiting (HI) antibody titres the 2009 case was characterized as a primary infection and the 2010 case as a secondary infection. Both the cases presented plasma leakage and neither showed any kind of haemorrhage. The 2009 case survived while the 2010 case was fatal. An isolate was obtained from the 2009 case. Based on envelope (E) gene sequence analysis, the virus belonged to genotype I of DENV-4, and clustered with isolates from India and Sri Lanka and was distant from the isolates from Thailand. The nucleotide and amino acid diversity of the E gene of the Indian isolates increased from 1996 to 2007 to 2009 in context of the E gene sequences of other isolates belonging to genotype I.
The increasing diversity in the circulating DENV-4 calls for close monitoring of the DENV-4 serotype.
The National Institute of Virology is the WHO Collaborating Centre For Arbovirus And Hemorrhagic Fever Reference And Research. We work in close collaboration with clinicians in providing dengue diagnosis. Samples from suspected dengue cases are tested for dengue specific IgM, using NIV MAC-ELISA kit , viral RNA using dengue-specific real time RT-PCR  and serotyped by multiplex nested RT-PCR test . As a gold standard, virus isolation is attempted by infecting C6/36 cells (Aedes albopictus mosquito cell line) with patient sera. The infected cells are examined for the presence of virus by immunofluorescence assay (IFA) and RT-PCR. Sequencing of viral RNA is carried out using big dye terminator kit (Applied Biosystems, Foster city, CA, USA). The infection is characterized as primary or secondary based on the HI antibody response.
Clinical profile of patients infected with DENV-4
Case1 - 2009 (0952326)
Case2 - 2010 (1014847)
80-901 , 60-702
24000, 12000, 17000, 42000, 92000
124000, 100000, 34000, 11000
Serum albumin (g%)
Serum ALT (normal up to 40 U/L)
Serum AST (normal up to 30 U/L)
Serum bilirubin (normal <1 mg%)
DENV-specific IgM antibodies were assessed in serum samples collected on 5th day post onset of illness for Case 1 and 4th day post onset of illness for Case 2. Case 1 was positive for IgM while Case 2 was negative. The titre of HI antibodies in the serum was determined to define whether the individuals had suffered primary or secondary infections. HI antibody titre of >1:2560 in the acute phase of infection is considered confirmatory of secondary infection . Case 1 had very low levels of HI antibodies (1:40 to 1:80) indicating a primary infection. Case 2 had HI titre of 1:2560 indicating a secondary infection. The serum samples were also tested in the dengue IgG capture ELISA (Panbio Ltd.) and showed the presence of 31 units of IgG in Case 1 and 78 units in Case 2.
The high degree of diversity in the envelope gene observed for the DENV-4 viruses circulating on the subcontinent indicates that the serotype is evolving and that close monitoring of the serotype is needed.
The authors wish to thank the Indian Council of Medical Research for providing the funds and the Director, National Institute of Virology for the support.
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