Partial Nucleotide Sequencing and Molecular Evolution of Epidemic Causing Dengue 2 Strains

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1 959 Partial Nucleotide Sequencing and Molecular Evolution of Epidemic Causing Dengue 2 Strains Urvashi B. Singh, 1 Arindam Maitra, 1 Shobha Broor, 1 Arvind Rai, 2 S. T. Pasha, 2 and Pradeep Seth 1 1 Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi; 2 Department of Biotechnology, National Institute of Communicable Diseases, Sham Nath Marg, Delhi, India To study the genetic variability and to detect evolutionary changes and movement of dengue 2 (DEN-2) strains, nucleotide sequencing of the envelope protein gene and the nonstructural protein 1 gene junction was performed for 9 isolates from the 1996 Delhi epidemic and 1 isolate from the 1967 Delhi epidemic. The epidemic strains had a divergence of 10% 11% from the 1967 strains, but were quite similar to DEN-2 isolates from Seychelles, Somalia, and Torres Strait. In addition, the sequence data were compared to the prototype DEN-2 strain, New Guinea C, and other published DEN-2 sequences from different parts of the world. The phylogenetic analysis by the Molecular Evolutionary Genetics Analysis program suggests that the 1996 Delhi isolates of DEN-2 were genotype IV. The 1967 isolate was similar to a 1957 isolate of DEN-2, P9-122, from India, and was classified as genotype V. This study indicates that earlier DEN-2 strains of genotype V have been replaced by genotype IV. Dengue viruses (family Flaviviridae, genus Flavivirus) occur as 4 antigenically distinct, although related, serotypes (on the basis of cross neutralization tests), which do not offer crossprotection. Infection with any of these leads to a mild, selflimiting febrile illness (dengue fever, DF). A more severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), is responsible for a high mortality rate, especially in children [1]. Dengue virus (DEN) is responsible for a growing health problem in the tropical and subtropical countries. The known geographic range of dengue includes the Americas, Africa, Asia, and the South Pacific, determined by the distribution of its mosquito vectors. The incidence of DF is on the increase and is spreading to geographic regions not previously affected. In terms of human morbidity, DF is currently one of the most important mosquito-transmitted viral diseases. It has been estimated that about 50 million cases of DF occur annually, with 10,000 infant deaths due to DHF [1]. DHF/DSS has been postulated to result from immune enhancement after a second het- Received 24 March 1999; revised 10 June 1999; electronically published 8 September Presented in part: 8th International Congress on Infectious Diseases, Boston, Massachusetts, May 1998 (abstract ). Short-term training (P.S.) under the Fogarty International Fellowship Program on Computational Analysis of Sequence Data at Centers of Disease Control and Prevention, Atlanta, Georgia, was kindly arranged by Dr. John L. Fahey (Department of Microbiology and Immunology, UCLA). Grant support: the Council of Scientific and Industrial Research, Government of India (to U.B.S.). Reprints or correspondence: Dr. Pradeep Seth, Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi , India (pseth@medinst.ernet.in; sethpradeep@hotmail.com). The Journal of Infectious Diseases 1999;180: by the Infectious Diseases Society of America. All rights reserved /1999/ $02.00 erologous DEN infection [2]. However, reports of primary infections resulting in DHF/DSS suggests that differences in virulence of DEN strains may also be involved [3]. It is presently not known whether specific virus genotypes are responsible for the more severe forms of the disease or for particular widespread epidemics. Movement of DEN between different geographic areas is an important element in the epidemiology of the disease. Molecular characterization of isolates may be used to define genetic variation between strains of the same serotype and follow geographic movement of virus strains. It is important to monitor the distribution and introduction of new DEN strains into areas where dengue activity is troublesome, thus facilitating identification of the source of virus strains in new outbreaks [4 8]. In India, DEN was first isolated in 1946, and many epidemics have since been reported [9 12]. DHF/DSS, although common in Southeast Asia, has rarely been reported in India except during dengue epidemics. DHF was first reported in Calcutta, West Bengal, in 1963 [13], again in 1964 [14], and subsequently in Visakhapatnam in 1969 [15] and in Jalore, Rajasthan, in 1985 [16]. The National Capital Territory of Delhi experienced its first major epidemic of DHF in 1996 because of dengue 2 (DEN-2) virus [17], although frequent outbreaks of DF have been reported since The epidemic began in mid-august and lasted until mid-november that year, during which time more than 10,000 patients suffered and 423 died [18]. In the present study, we analyzed genomic sequences from the envelope-nonstructural protein1 (E-NS 1 ) region of DEN-2 isolates from the New Delhi epidemic of 1996 and 1 isolate from the Delhi epidemic of 1967 and compared the data with the other published sequences of DEN-2 isolated from other parts of the world.

2 960 Singh et al. JID 1999;180 (October) Table 1. Dengue 2 virus isolates from 1996 and 1967 Delhi epidemics used for genome sequence analysis. Virus isolate ID no. Date of receipt of sample Age (years)/sex Diagnosis Genbank accession no. 838/96 09/16/96 7/F DF AF /96 09/16/96 7/M DHF AF /96 09/26/96 16/F DF AF /96 09/26/96 16/F DF AF /96 09/30/96 9/M DHF AF /96 09/30/96 18/M DHF AF /96 10/24/96 20/F DHF AF /96 10/24/96 20/F DF AF /96 10/31/96 58/M DHF AF Unknown DF AF Materials and Methods Virus isolates and cell line. Nine isolates of DEN-2 from the 1996 epidemic of Delhi and 1 from the 1967 epidemic of Delhi were selected for sequence analysis (table 1). These viruses were isolated from the serum samples of patients on the Aedes albopictus cell line, C6/36, with the standard technique [19]. Two isolates, 838/ 96 and 841/96, were from 2 children who had DF and DHF, respectively, during the initial part of the epidemic. Three isolates (979/96, 980/96, and 1029/96) were from patients who were infected with the virus during the peak of epidemic, and 4 isolates (1430/ 96, 1432/96, 1436/96, and 1451/96) were from patients who were admitted during the last phase of the epidemic RNA extraction. Extraction of RNA was done when 150% of the cells showed specific immunofluorescence in an indirect immunofluorescence assay with dengue virus type-specific monoclonal antibodies. Viral RNA was isolated by the method described by Chomczynski and Sacchi [20]. Virus-infected cells were mixed with an equal volume of guanidine isothiocyanate lysis buffer. The solution was sequentially mixed with a buffer containing a 1 : 10 volume of 2 M sodium acetate, ph 4, an equal volume of water equilibrated phenol, and a 1 : 5 volume of chloroform. The mixture was kept at 4 C for 15 min, followed by centrifugation at 16,000 g for 15 min. The aqueous phase was removed, mixed with an equal volume of isopropanol, and kept at 20 C overnight to precipitate the RNA. The RNA was pelleted at 16,000 g for 15 min, washed with 75% ethanol, and finally dissolved in diethylpyrocarbonate treated water-containing RNAsin. Viral RNA was reverse transcribed to cdna prior to enzymatic DNA amplification by use of MoMLV reverse transcriptase. For reverse transcription (RT) of viral RNA to cdna prior to Taq polymerase amplification, primer D 2 RPO 1 was used. Subsequent amplification was performed on the cdna with DEN-2 consensus primers designed in our laboratory to amplify a 455-bp region in the E-NS1 gene [forward primer, envfpo1 ( ), 5 - CCAGTCAACATAGAAGCAGAACCTC-3 ; reverse primer, D 2 RPO 1 ( ) 5 -ATCCCACAACCACACTTCAGTTCTT- TATTTTTCCAGCTCA-3 ] (Commonwealth Biotechnologies, Richmond, VA). RT and polymerase chain reaction (PCR) were performed sequentially in the same tube with the help of the Gene Amp RNA PCR kit (Perkin Elmer, Norwalk, CT) according to manufacturer s instructions. The RT was allowed to proceed at 42 C for 1 h, followed by inactivation of reverse transcriptase at 99 C for 5 min. For PCR amplification, 30 cycles were done after the initial denaturation at 95 C for 2 min. Each cycle was composed of denaturation (95 C for 30 s), annealing (56 C for 2 min), and extension (72 C for 2 min). The final extension was for 7 min at 72 C. Positive displacement pipettes and cotton-plugged disposable tips were used to avoid carryover contamination. Each test included a positive control, RNA extracted from uninfected C6/36 cells, and a minimum of 3 negative controls containing distilled water in place of sample. The amplified product was electrophoresed into 1.2% agarose gel. The gel was stained with ethidium bromide and visualized in an ultraviolet transilluminator. Cloning. The PCR products thus amplified were purified by the Wizard PCR preps purification system (Promega, Madison, WI).The final concentration and integrity of the purified amplicon was checked by running a 1.5% agarose gel electrophoresis along with a known quantity of HindIII (New England Biolabs, Beverly, MA) digest of lambda DNA. After purification, the PCR products were cloned in pgem-t vector (Promega). The ligation mixture was used for transforming JM109 competent cells, and the transformed cells were plated on YT agar plates containing X-gal, IPTG, and ampicillin (Sigma, St. Louis). The inserts were selected by bluewhite screening after overnight incubation at 37 C. DNA sequencing. Each clone with a 455-bp insert was reamplified by each of the primers envfpo1 and D2RPO1. The use of both the forward and the reverse primers enabled the sequencing of both the strands of the double-stranded PCR amplicons. The amplified product was similarly purified by Wizard PCR preps DNA purification kit and was sequenced on the ABI 373 Automated Sequencer with the help of an ABI Prism, Dye Terminator cycle sequencing kit (Perkin Elmer) by following the manufacturer s recommendations. The sequences thus obtained were edited and analyzed by the DNASIS program (Hitachi Software Engineering, Yokahama, Japan). A minimum of 4 5 clones per isolate was sequenced to reduce misincorporation error by Taq polymerase on the sequencing ladder. The sequences obtained were aligned by Eyeball SEquencing Editor (ESEE) program [21]. The phylogenetic analysis, including estimation of DNA distances by Kimura two-parameter method, bootstrapping, and generation of a neighbor-joining tree, were performed with the Molecular Evolutionary Genetics Analysis package [22]. Sequences were compared with those of DEN-2 strains from different geographic areas that have been previously published and downloaded from Genbank (table 2). Results Sequences of cdna of the E-NS1 region of all the viruses listed in table 1 are shown in figure 1. The sequences were aligned to the New Guinea C strain (NGC), a prototype strain of DEN-2. Analysis of sequence data shows a divergence of 0% 3% (mean, 1%) among the 1996 Delhi epidemic strains. The nucleotide differences were scattered throughout the length of the gene, and there appears to be no particular region of hypervariability. The majority of mutations occurred at the third position of the codon and was mostly silent. No base insertions or deletions were found among the DEN-2 gene sequences. In this genomic region, transitions (i.e., changes from

3 Figure 1. cdna sequence alignment of envelope protein gene and the nonstructural protein 1 gene junction (E-NS1) region of 10 dengue 2 (DEN-2) isolates from Delhi (see table 1 for details) with the prototype DEN-2 strain, New Guinea C (NGC, genotype I). Sequences of representative strains belonging to other genotypes (see table 2 for details), obtained from Genbank, were included for comparison.

4 962 Singh et al. JID 1999;180 (October) Figure 1. (Continued.) cdna sequence alignment of envelope protein gene and the nonstructural protein 1 gene junction (E-NS1) region of 10 dengue 2 (DEN-2) isolates from Delhi (see table 1 for details) with the prototype DEN-2 strain, New Guinea C (NGC, genotype I). Sequences of representative strains belonging to other genotypes (see table 2 for details), obtained from Genbank, were included for comparison. purine to purine, G to A or vice versa, or pyrimidine to pyrimidine, C to T or vice versa) were predominant. Only a few transversions (i.e., changes between purine and pyrimidine) were noticed. The transition : transversion ratio was 21 : 2. The isolates were taken from patients who were infected in the beginning, the peak, and toward the end of the epidemic and were suffering from DF/DHF/DSS. No significant difference was observed at the genome level to correlate with the severity of disease in these patients. When these strains were compared with 2 earlier DEN-2 isolates, 1 from the 1967 epidemic of DF in Delhi and another from the 1957 Indian epidemic of DF (P9-122), there was a divergence of 10.7% (range, 10% 11%) and 9.7% (range, 8% 10%), respectively. However, these 2 earlier isolates from India were quite similar (divergence, 3%). A comparison of 1996 Delhi strains with other global DEN- 2 strains (table 2, figure 1) shows that these strains are quite similar to strains isolated from Seychelles (S-4452; divergence, 3.5%), Somalia (strain 10; divergence, 4.8%), and Torres Straits (strain Torres Straits 1; divergence, 5.3%). They are divergent from prototype strain NGC, Malaysian strains P7-843 and P7-863, 1993 Thai strains (ThNH-p14/93, ThNH-p16/93, and ThNH-p36/93), and strains from Cook Islands (Cook Islands 1) and Jamaica (ARAC ). Comparison of 360-bp long alignable nucleotide fragments by using the neighbor-joining analysis generated phylogenetic trees (figure 2) that closely resemble those published earlier by Lewis et al. [23]. Five genotypic groups of DEN-2 serotype were quite evident, with little relationship between their position in dendrogram and date of virus isolation. Group I is represented by prototype strain NGC; group II includes 1993 strains from Thailand and Malaysian strain P7-863; group III consti-

5 JID 1999;180 (October) Molecular Evolution of Dengue Table 2. Virus isolate ID no. Global dengue-2 viruses used for genome sequence analysis. Code (genotype) Genbank accession no. Country Year NGC (Prototype) D 2 NG1 (I) AF New Guinea 1944 P7-863 P7-863 (II) U Malaysia ThNH-P36/93 ThNH36 (II) AF Thailand 1993 ThNH-P16/93 ThNH16 (II) AF Thailand 1993 ThNH-P14/93 ThNH14 (II) AF Thailand 1993 ARAC D2-JAM (III) M Jamaica 1982 Cook Island-1 COOK-IS (III) AF Cook Islands 1997 P7-843 P7-843 (III) U Malaysia S-4452 SEY-4452 (IV) L Seychelles 1977 S-10 SOMAL-10 (IV) L Somalia 1984 Torres Straits-1 TORRESST-1 (IV) AF Torres Straits 1996 P9-122 P9-122 (V) L India 1957 tutes the Jamaican strain of 1981, Malaysian strain P7-843, and a strain from Cook Islands; and group IV includes strains from Seychelles, Somalia, Torres Strait, and all the strains of 1996 Delhi epidemic. The last group, group V, includes the older Indian strains of 1957 and The genetic relationships were independent of the type of phylogenetic analysis algorithms used (maximum parsimony, UPGMA; data not shown). Discussion The epidemiology of DEN is of continuing health importance, as the incidence of DF and DHF/DSS is increasing worldwide and is appearing in areas where it was previously unreported. This has recently led to the classification of this viral infection as an important emerging infection. In Delhi, although frequent outbreaks of DF have been occurring since 1967, no major outbreak of DHF/DSS had ever been reported until The present study of natural field isolates from patients representing the full spectrum of DEN infections during the epidemic has allowed us to determine the evolutionary origin of DEN associated with the epidemic. A short nucleotide sequence of 396 bp from a functional domain of the DEN genome, the E-NS1 gene junction, of 9 strains isolated from 1996 Delhi epidemic and 1 isolate from the 1967 epidemic in Delhi were explored by RT-PCR sequencing and compared with some of the global isolates. The worldwide transmission of DEN has been followed by comparing nucleotide sequences by several researchers [23 29]. Because the rate of variation of DEN in nature seems to be lower than that seen in other RNA viruses [26, 30, 31], a lower arbitrary value for the definition of a genotype was chosen. A genotype was defined as a group of DEN having no more than 6% sequence divergence within the chosen interval [25]. A 0% 3% divergence among 9 isolates of the 1996 Delhi epidemic shows that this epidemic was caused by a single genotype of DEN-2. A divergence of 10% 11% with the 1967 Delhi strain provides the evidence that thecurrent virus type was different from the earlier DEN-2 isolate and not a reemerging strain derived from a mutation of an earlier virus through silent transmission. As mentioned previously (see results), transitions were predominant among the isolates with a few transversions only. The transition-transversion ratio was 21 : 2. This shows that the 1996 strains of DEN-2 from the Delhi epidemic are in the early evolutionary phase. There is evidence from many studies (e.g., [32]) that transitions occur more frequently, especially in third silent codon positions, during early evolutionary divergence of nucleotide sequence. Transversion occurs much less frequently, but when it occurs, especially in second codon positions, it invariably causes amino acid changes in the encoded proteins. Further, as the phylogenetic trees gave evidence of previously unknown evolutionary relationships among viruses over a vast geographic range and time period, the sequence data were further analyzed phylogenetically (figure 2). It can be seen that DEN-2 strains isolated from India in 1957 and 1967 belong to the same genotype (genotype V), probably representing the circulating genotype during that period. This group of viruses apparently had been evolving independently for some time in this part of the world. These DEN-2 strains seem to have been replaced by the genotype IV from Seychelles, Somalia, or Torres straits, probably because of trade links between the Indian subcontinent and these areas [33 35]. A similar approach has been shown to reflect the geographic origin of DEN-2 strains in other parts of the world. In Sri Lanka, earlier isolates of DEN-2 ( ), which clustered with the prototype strain, NGC (genotype I) [25], were maintained for a 24-year period and were then replaced by genotype IV strains [23]. Also, southeast Asia has been identified as a source of DEN-2 strains that are new to the Americas [26]. The native American genotype appears to have been replaced by the imported southeast Asian genotype. The period during which the displacement of genotype V DEN-2 strains in this subcontinent with genotype IV strains would have taken place is not clear at present. However, there have been several epidemics of DF/DHF in India in the intervening period ( ) [18]. The virus isolates from these epidemics need to be studied in detail to know the true interplay of transmission and evolution of DEN-2 in India. It remains to be determined whether there is a direct association between specific genotypes and severity of disease. The cause(s) of the more severe form of DHF/DSS is (are) not fully understood. It has been postulated that DHF/DSS results from immune enhancement caused by infection by a second DEN serotype [2]. It has also been suggested that DEN may vary in virulence and that more severe disease may be associated with specific strains [36]. Halstead [37] proposed that there are biotypes of DEN that cause DHF at a high rate when they infect permissive hosts [37]. Recent observations by Rico-Hesse and colleagues [26] that southeast Asian DEN-2 strains were directly responsible for DHF cases in the Americas suggest that there is a direct association between introduction of an imported strain and severe form of DEN infection. Similarly, DEN-2 isolates from the 1981 Cuban epidemic of DHF were found to be similar to contemporaneous Jamaican isolates circulating in

6 964 Singh et al. JID 1999;180 (October) Figure 2. Phylogenetic analysis of cdna sequences of envelope protein gene and the nonstructural protein 1 gene junction (E-NS1) region of 10 denuge 2 (DEN-2) isolates from 1996 and 1967 epidemics of dengue fever/dengue hemorrhagic fever in Delhi (see table 1). The analysis is based on 360 alignable positions (positions in alignment containing one or more gaps were excluded from analysis) in the E-NS1 region. Sequences of DEN-2 isolates from other parts of the world (obtained from Genbank) were added for comparison (see table 2). The numbers at the branch points indicate the percentage bootstrap values in 500 bootstrap replications. Genotypes of DEN-2 viruses are indicated by Roman numerals I V. the Caribbean region during that period [25]. Also, emergence of DHF in Delhi in 1996 and in Sri Lanka in early 1980s, caused by genotype IV strains where multiple serotypes of DEN have circulated silently for decades, give credence to this hypothesis. However, additional work is needed to validate this concept. References 1. World Health Organization. Dengue and Dengue Haemorrhagic Fever. Fact sheet. 1996: Halstead SB. Pathogenesis of dengue: challenge to molecular biology. Science 1988;239: Rosen L. The pathogenesis of dengue hemorrhagic fever. South Am J Med 1986;(Suppl.)11: Trent DW, Grant JA, Rosen L, Monath TP. Genetic variation among dengue 2 viruses of different geographic origin. Virology 1983;128: Trent DW, Grant JA, Monath TP, et al. Genetic variation and microevolution of dengue 2 virus in South East Asia. Virology 1989;172: Monath TP, Wands JR, Hill LJ, et al. Geographic classification of dengue 2 virus strains by antigen signature analysis. Virology 1986;154: Repik PM, Dalrymple JM, Brandt WE, McCown JM, Russell PK. RNA fingerprinting as a method for distinguishing dengue 1 virus strains. Am J Trop Med Hyg 1983;32: Walker PJ, Henchal EA, Blok J, et al. Variation in dengue type 2 viruses isolated in Bangkok during J Gen Virol 1988;69: Balaya S, Paul SD, D Lima LV, Pavri KM. Investigation of an outbreak of dengue in Delhi in Indian J Med Res 1969;57: Rodrigues FM, Patankar MR, Banerjee K, et al. Etiology of the 1965 epidemic of febrile illness in Nagpur city, Maharashtra State, India. Bull WHO 1972;46: Padbidri VS, Dandawate CN, Goverdhan MK, et al. An investigation of the etiology of the 1971 outbreak of febrile illness in Jaipur city, India. Indian J Med Res 1973;61: Karamchandani PV. Study of 100 cases of dengue fever in Madras Penitentiary. Indian Med Gazette 1973;72: Aikat BK, Konar NR, Banerjee G. Haemorrhagic fever in the Calcutta area. Indian J Med Res 1964;152: Sarkar JK, Chakravorty SK Sarkar RK. Sporadic cases of hemorrhagic and/ or shock during dengue epidemics. Trans Royal Soc Trop Med Hyg 1972;66: Ghosh SN, Pavri KM, Singh KRP, et al. Investigation on the outbreak of dengue fever in Ajmer city, Rajasthan state in Part 1. Epidemio-

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