Infection, Genetics and Evolution 34 (2015) 402–409
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Phylogenetic study reveals co-circulation of Asian II and Cosmopolitan genotypes of Dengue virus serotype 2 in Nepal during 2013 q Sneha Singh a,1, Birendra P. Gupta b,1, Anoop Manakkadan a,2, Krishna Das Manandhar b,⇑, Easwaran Sreekumar a,⇑ a b
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud P.O., Thiruvananthapuram 695014, Kerala, India Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
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Article history: Received 5 June 2015 Received in revised form 3 July 2015 Accepted 5 July 2015 Available online 6 July 2015 Keywords: Dengue Phylogeny Mosquito cell infectivity Mutation Genotypes
a b s t r a c t The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Dengue is a major re-emerging mosquito-borne disease in the tropical and subtropical countries. For the last few decades it remains on top with respect to disease burden and mortality (Bhatt et al., 2013). The disease manifests as a mild febrile episode or as severe and life threatening haemorraghic syndrome. Dengue virus belongs to the genus Flavivirus, family Flaviviridae. Its genome consists of approximately 11 kb positive-sense, single stranded q A part of this work was presented as a poster in the 12th International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases (MEEGID), 11–13 December 2014, Bangkok, Thailand. ⇑ Corresponding authors. E-mail addresses: [email protected] (K.D. Manandhar), [email protected] (E. Sreekumar). 1 These authors contributed equally. 2 Present address: Department of Biotechnology, Cochin University of Science and Technology, Cochin-22, Kerala, India.
http://dx.doi.org/10.1016/j.meegid.2015.07.006 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
RNA molecule without a poly-A tail (Chambers et al., 1990). Among the three structural proteins (Capsid, Membrane and Envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4 and NS5) encoded by the genome, the envelope (E) protein plays a critical role in virus attachment and fusion to the host cell membrane, and hence infectivity. Three domains (I, II and III) have been identified in the E protein and the domain III interacts with the cellular receptor and mediates endosome fusion (Modis et al., 2004). The presence of four classical serotypes among the Dengue viruses (DENV-1, 2, 3, and 4) that have no cross-protective immunity in infected patients dramatically increases the complexity of this disease. DENV-2 has been attributed to cause a more severe disease even during primary infections (Rico-Hesse, 2003). At molecular genetic level, DENV-2 has been divided into 6 genotypes namely Cosmopolitan, Asian I, Asian II, American, American/Asian and Sylvatic. These genotypes have a 5% or more genetic divergence in their envelope as well as whole genome nucleotide sequences
S. Singh et al. / Infection, Genetics and Evolution 34 (2015) 402–409
(Weaver and Vasilakis, 2009). The Asian I genotype comprises of strains isolated from Thailand and Malaysia while Asian II genotype consists of strains circulating in China, Philippines, Sri Lanka, Taiwan and Vietnam. The strains circulating in China, Thailand, Vietnam, Brazil, Venezuela and the Caribbean belonged to American/Asian genotype while the isolates from Australia, Pacific Islands, South East Asia, Indian sub-continent, Middle East, Africa and Mexico fall in Cosmopolitan genotype (Twiddy et al., 2002; Mendez et al., 2012). Nepal, a Himalayan country, is surrounded by India on three sides and China to the North. It is an endemic nation for many vector borne diseases, like malaria, kala-azar, Japanese encephalitis and lymphatic filariasis (Dumre et al., 2013). The first report of dengue fever came in 2004 from a Japanese traveler in Nepal and it was reported to be of serotype 2 (Takasaki et al., 2008) with >99% similarity with viral strains from India. It was followed by a native report in 2006 with detection of four dengue serotypes (Malla et al., 2008). A few intermittent cases were reported nationwide from 2007 to 2009 with 2, 8 and 16 cases per year, respectively (Dumre et al., 2013; Griffiths et al., 2013). An outbreak occurred in 2010 with 350 (30%) confirmed cases out of 1215 clinically reported cases. The first isolation was done during the 2010 epidemic and all the strains belonged to serotype 1, genotype V closer to the Asian sub-cluster (Pandey et al., 2013). The year 2013 witnessed a total shift in the circulating serotype and DENV-2 outbreak was reported in the Terai belt of the country (Birendra et al., 2014; accepted manuscript). A large number of secondary infections are usually reported from the country and may be attributable to the circulation of multiple serotypes in the region (Malla et al., 2008; Dumre et al., 2013). There is lack of reliable dengue surveillance data from the country and inadequate response in accordance with international guidelines (Griffiths et al., 2013) that may hamper dengue control measures in the region. Adding to these is the lack of information on nucleotide sequences of DENV strains from the country (Malla et al., 2008; Pandey et al., 2013), which may also affect the epidemiological interventions. In the present study, DENV strains from an outbreak in Nepal in 2013 were characterized at the molecular level by sequencing of the complete envelope protein coding region and subsequent phylogenetic analysis. Strains representing different genotypes were studied by in vitro infection kinetics analysis in mosquito cells to understand the differences in infection competence and adaptation. The results shed light on the genotypes of dengue virus currently circulating in the region and add country specific information to the DENV sequence database.
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FBS (PAN Biotech, Germany) and was maintained at 37 °C in a 5% CO2 incubator (Sanyo, Japan). 2.3. Reverse transcriptase PCR and envelope (E) protein coding region sequencing Viral RNA was extracted from 150 ll of patient serum using viral RNA isolation kit (Macherey Nagel, Germany) in accordance with the manufacturer’s instructions. Initial dengue viral RNA detection in samples was done by a single-step RT-PCR using a set of generic primers (D1F-DencomR2) (Supplementary Table 1) that amplifies the Core-Pre-membrane (C-PrM) region of any of the four viral serotypes, as described previously (Anoop et al., 2010, 2012). Subsequently, serotype identification was done using a second step, multiplex, semi-nested PCR of this primary amplification by serotype-specific primers described previously (Lanciotti et al., 1992), with minor modifications in the primers (D1F-NTS1/NTS2/NTS3/nDen4) (Supplementary Table 1) (Anoop et al., 2012). Amplification of a 1616 bp region spanning the complete E gene was performed using a two-step RT-PCR with the primers D2Seq3F and D2Seq7R (Supplementary Table 1). Briefly, it consisted of a reverse transcription of 5 ll of the isolated RNA using AMV reverse transcriptase system (Fermentas) at 45 °C for 1 h, and denaturation of the enzyme at 98 °C for 5 min. This was followed by a PCR reaction for 35 cycles with cycling conditions of 98 °C for 30 s, 55 °C for 1 min, 72 °C for 2 min, and a final extension of 72 °C for 4 min. The amplified region was subjected to bi-directional sequencing using overlapping primers (Supplementary Table 1) and the Big Dye Terminator cycle sequencing kit in an ABI3730 Genetic Analyzer automated DNA sequencer (PE Applied Biosystems, Foster City, CA). 2.4. Virus isolation in C6/36 cells and in vitro infection kinetics analysis Briefly, the serum samples (1:10 diluted in phosphate buffered saline, PBS; pH 7.4) was allowed to adsorb on to the C6/36 cells for 2 h at 33 °C with slow shaking followed by a gentle wash with 1 PBS and addition of L-15 medium supplemented with 2% FBS and further incubation with shaking. The virus containing supernatant was harvested on the third day post-infection and stored in 80 °C as aliquots. For infection kinetics analysis, the C6/36 cells were infected at an MOI of 0.1. The supernatant was collected at 0 h, 6 h, 12 h and then every 24 h till five days post infection. The amount of virus in the supernatant was titrated by plaque assay on BHK-21 cells. 2.5. Infection of BHK-21 cells, immunofluorescence and plaque assay
2. Materials and methods 2.1. Ethical approval and patient samples Ethical approval for the study was obtained from the Nepal Health Research Council (NHRC) Ethical Review board (2071-04-15). A total of 52 venous blood samples from patients clinically diagnosed as dengue were obtained from the 2013 dengue outbreak in Nepal, as described earlier (Birendra et al., 2014; accepted manuscript). 2.2. Cell lines The Aedes albopictus mosquito cell line C6/36 was maintained in culture at 28 °C in L-15 medium (Invitrogen, New York) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech, Germany). Baby hamster kidney cells (BHK-21) were cultured in DMEM (Invitrogen, New York) supplemented with 10%
The BHK-21 cells were infected with DENV-2 isolates by adsorption of the virus for 2 h at 37 °C followed by two washes with 1 PBS. The cells were further grown in DMEM supplemented with 2% FBS for three days. Cells were fixed with 4% paraformaldehyde 72 h post-infection followed by washes with 1 PBS and permeabilization with 0.5% Triton X 100. The presence of dengue virus antigen was detected by in-house rabbit polyclonal anti-dengue envelope protein antibody as primary antibody and anti-rabbit Alexa Fluor 488 secondary antibody. Fixed cells were incubated with primary antibody at 4 °C overnight, followed by washes and incubation with secondary antibody at 37 °C for one hour. The cells were washed and counter stained with DAPI stain and visualized under an inverted fluorescent microscope (Nikon Eclipse Ti). For plaque assay and virus titration, 90% confluent BHK-21 cells were infected by adsorption of the virus for 2 h at 37 °C followed by two washes with 1 PBS (Sigma–Aldrich) and overlaying with 1 DMEM (Invitrogen, New York) containing a final concentration
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of 1% Carboxymethyl cellulose (CMC) (Sigma–Aldrich) and 1% heat-inactivated FBS. The cells were fixed with 30% formaldehyde on 5th day post infection for 2 h at 37 °C followed by counterstaining with crystal violet stain. 2.6. Envelope protein coding region sequence analysis The sequences generated from the present study were aligned using the Clustal W function of the Bio-Edit 6.0.7 software to obtain the complete coding region sequence of the envelope protein (1485 bp). For comparison and mapping of the changes, both the nucleotide and translated amino-acid sequences were used. The sequence identity was calculated by optimal global alignment between the selected strains. 2.7. Phylogenetic analysis Bayesian analysis was carried out using the BEAST software package (BEAST v1.6.1) (Drummond and Rambaut, 2007). Envelope protein coding region sequences of 155 DENV-2 strains from the Asia-Pacific region, strains identified from an initial on-line BLAST analysis of the obtained sequences and of reference strains of all the major genotypes retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov) along with the sequences of the Nepal strains were used as the data set. Using jModelTest 0.1.1, GTR + I + G (GTR is generalized time reversible, G is the gamma distribution and I is the proportion invariant) was identified as the best-fit model for Bayesian Markov Chain Monte Carlo (MCMC) analysis. The strict and relaxed molecular clock models and different tree priors (constant size, exponential growth and expansion growth) were used in the analysis. An effective sampling size of P200 was ensured by carrying out independent runs. Statistical convergence was obtained by doing MCMC chains for 10,000,000 generations and fixing a sampling frequency of 10,000 and burn-in of 1000 and 95% HPD (Highest Posterior Density) intervals. Tracer v1.5 program, Tree Annotator program and FigTree v1.4.0 were used to analyze the output from BEAST, and to generate and view the tree. 2.8. Recombination and selection pressure analysis SBP and GARD methods in the online software DATAMONKEY (http://www.datamonkey.org) were used to detect recombination. Selection pressure analysis employed SLAC, FEL and REL, FUMAR, MEME methods with default P-value/Bayes factor settings. 3. Results 3.1. Envelope protein (E) coding region sequence analysis from clinical samples Out of the 52 clinical samples that were initially subjected to a generic dengue virus pan-serotype specific RT-PCR, 15 samples were found to be positive (Supplementary Table 2). A serotype analysis by a second round serotype-specific PCR identified all of them to belong to Dengue virus serotype 2. This was later confirmed by the envelope coding region sequencing. Analysis of the complete nucleotide (1485 bp) sequences of the envelope coding region from these 15 samples (GenBank Accession Nos. KT232041-KT232055; Supplementary Table 2) showed substitutions, but no insertions/deletions in the sequences. Overall, at the nucleotide level, there were 92 point mutations in comparison to the prototype DENV-2 strain New Guinea C (NGC; GenBank Accession No. M29095). 85 of them were transversion mutations
and seven were transition mutations. Eleven non-synonymous mutations were observed in the amino acid sequence (Supplementary Fig. S1). Seven mutations (E47K, Q52H, D71A, K126E, V129I, H149 N, I164V) were falling in the domain I (aa positions 1–52, 133–193 and 281–296) and II (aa positions 53–132 and 194–280) of the envelope protein. The immunogenic domain – domain III (aa positions 297–493) had four mutations (N390S, I402F, I454T, I462V) (Table 1). The six strains that were closely related to the NGC strain had the E47K substitution differentiating them from the prototype strain. 3.2. Phylogenetic analysis We had to restrict the length of the sequences used in the phylogenetic analysis to 1097nt, instead of the complete 1485nt, to include the only DENV-2 sequence earlier reported from Nepal (GenBank Accession No. AB194885; indicated by an arrow in Supplementary Fig. S2). In the Bayesian analysis, the 2013 Nepal strains were found to fall into two distinct clusters within the phylogenetic tree, with a significant posterior probability (Fig. 1 and Supplementary Fig. S2). Out of the 15 strains, 9 strains were found to cluster with the Cosmopolitan Genotype IVa while the rest clustered along with the Asian II genotype. Among them, the nine Cosmopolitan Genotype IVa strains had 99.7–99.9% nucleotide level identity with each other whereas the six Asian II strains were 100% identical. Between the Asian II and Cosmopolitan Genotype IVa strains from Nepal, the identity varied from 93.7–99.9%.The Genotype IVa strains clustered with the strains from Malaysia (JF968005, JF968012) isolated during 2010. The Nepal strains that grouped within the Asian II genotype formed a distinct clade, and were found to be related genetically to the strains isolated from the United States and Papua New Guinea (FJ906968, FJ906959, GQ199901). The strain from Nepal identified earlier in 2004 (GenBank Accession No. AB194885) clustered with the Cosmopolitan Genotype IVb strains, with closer genetic relation to Indian and Singapore strains (Supplementary Fig. S2). 3.3. Molecular clock analysis Both strict and relaxed clock parameters were employed for carrying out the molecular clock analysis of the Envelope region of the Nepal DENV-2 strains. The best model was then selected based on the highest marginal likelihood value as obtained for the uncorrelated exponential molecular clock with exponential population size (Supplementary Table 3). This method was further carried out for determining the time to most recent common ancestor (tMRCA) and also the values for the nucleotide substitution rates for the datasets. The whole dataset was divided based on the presence of the strains isolated in the present study as Genotype IVa and Asian II groups for estimating the values. Accordingly, the mean tMRCA of the Asian II strains were found to be 78.98 years (95% HPD: 70.57, 90.15) while that of the Cosmopolitan Genotype IVa strains were found to be 48.65 years (95% HPD: 35.95, 64.14). The mean nucleotide substitution rate for all the isolates used in the study was found to be 7.85 10 4 substitutions per site per year (95% HPD: 5.54, 10.22), which was similar to the earlier reports (6 10 4 substitutions per site per year; Twiddy et al., 2003; Wang et al., 2000). 3.4. Recombination and selection pressure analysis No recombination events could be detected among the sequences in the data set used in the analysis. Selection pressure analysis was carried out by grouping the datasets into three as Asian II (n = 18), Cosmopolitan Genotype IVa (n = 44) and all DENV sequences (n = 155). Among the five different analyses,
T
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‘.’ mark indicates conserved residues; ‘–’ indicates that the sequence information is not available.
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We could successfully isolate only three strains from the 15 DENV-2 positive samples by infecting C6/36 mosquito cells. Two among them were of Cosmopolitan Genotype IVa and the third one was an Asian II strain. One isolate from each of the genotypes (DENV-2/NEP144_2013; Cosmopolitan Genotype IVa and DENV-2/NEP679_2013; Asian II genotype) (passage level 4), was selected for infectivity analysis. In BHK-21 cells that were used for virus quantification by plaque assay, infection was confirmed by immunofluorescence for the detection of viral envelope protein and also by observing cytopathic effect (Fig. 2A and B). The two virus strains formed plaques with similar morphology in infected BHK-21 cells (Fig. 2A). In infectivity analysis, the C6/36 cells infected with Cosmopolitan Genotype IVa or Asian II strains showed roundening whereas uninfected cells retained their spindle shape. However, the MTT assay showed that there was no significant reduction in the viability percentage of these cells when infected with either of the strains at identical MOIs (Fig. 3A and B). The in vitro virus replication kinetics in C6/36 cells revealed that the Cosmopolitan Genotype IVa had a marginally higher replication rate compared to the Asian II genotype in terms of progeny virus production (Fig. 3C). In the culture supernatants, at 24 h p.i the viral titre reached around 6.33 102 pfu/ml for the Cosmopolitan Genotype IVa strain, while for the Asian II strain it was 1.23 102 pfu/ml. At the 5th day post infection the Genotype IVa reached a titre of 3.2 105 pfu/ml and Asian II reached a titre of 1.3 105 pfu/ml.
V
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3.5. Virus isolation and in vitro infection kinetics assay in mosquito cells
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I 734 454
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namely, SLAC, FEL, REL, FUBAR and MEME, if at least two methods identified a site for positive selection, the result was taken into consideration. Accordingly, only the codon at position 360 was found to be under positive selection pressure by all the three methods, when the whole sequence dataset was used. No significant codon positions were found in other two datasets (Supplementary Table 4).
V
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F F F F F F F F . 682 402
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444 164
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429 149
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E E E E E E E E . 406 126
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A D 351
Q 332
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52
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Nepal New the on the
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NEP_11_2013 NEP_10_2013 NEP_10NA_2013 NEP_736_2013 NEP_718_2013 NEP_701_2013 NEP_144_2013 NEP_885_2013 NEP_798_2013 NEP_988_2013 NEP_9_2013 NEP_679_2013 NEP_96_2013 AB194885 M29095 Position on Position Gene
Table 1 Amino acid changes observed in DENV-2 strains of the Asian II and Cosmopolitan genotypes from Nepal compared to New Guinea Strain C reference strain (M29095) and Nepal DF 2004 strain (AB198445).
NEP_793_2013
NEP_140_2013
S. Singh et al. / Infection, Genetics and Evolution 34 (2015) 402–409
4. Discussion Analyzing dengue viral strains from outbreaks in various geographical locations adopting molecular approaches has significant epidemiological significance (Kyle and Harris, 2008). This is supported by the fact that the clinical spectrum of dengue disease has correlation with the viral serotypes, genotypes or sub-genotypic lineages prevalent in an area. (Messer et al., 2003; Rico-Hesse, 2003; Zhang et al., 2005; Ospina et al., 2010; Lee et al., 2012). In the present study, we analyzed DENV strains from Nepal. We observed that two different genotypes- the Asian II and Cosmopolitan Genotype IVa- were co-circulating (Fig. 1) in the dengue outbreak that occurred in the Terai as well as the hilly region in 2013. Though the severity of clinical disease and mortality was not high, the outbreak was more geographically widespread affecting a larger number of people. There is a genotype shift among the Cosmopolitan strains from Genotype IVb to IVa. In an earlier report, the DENV-2 strain acquired by a Japanese patient from the region was shown to belong to the Cosmopolitan Genotype IVb (referred to as Genotype I by Takasaki et al., 2008) with similarity to Indian strains. Genotype IVb is a predominant DENV-2 genotype in the neighboring country, India, and has been associated with larger and more severe dengue epidemics (Mishra et al., 2015). The Genotype IVa strains in the current outbreak shows genetic similarity to the strains from Malaysia and Singapore. Similarly, the closest relative for the Asian II strain identified was a 2009 strain from USA. The
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Fig. 1. Maximum clade credibility (MCC) tree showing the co-circulation of Cosmopolitan Genotype IVa and Asian II genotype in Nepal during 2013. MCC tree derived from the Bayesian analysis of the envelope protein of DENV-2 with the best fit model (strict and relaxed lognormal clock), showing the posterior probability at each principal node. The GenBank accession Nos. and the place and year of identification are shown. A few clades are collapsed to improve clarity and they are numbered. These clades are shown expanded in Supplementary Fig. S2 and correspondingly numbered. The strains from recent Nepal outbreak used in our study are shown in red and blue, representing the Asian II and Cosmopolitan Genotype IVa, respectively. The scale bar represents the time scale in years. The expanded MCC tree, indicating all the strains used in the study, is shown in Supplementary Fig. S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observations indicate that these DENV-2 strains might be of exotic introduction into Nepal. The country being a tourist destination, the chances of cross-border transmission of DENV strains cannot be ruled out as shown in many earlier studies from other parts of the world (Zaki et al., 2008; Shu et al., 2009; Jiang et al., 2012; Lee et al., 2012; Warrilow et al., 2012). Also, the DENV-1 strains identified in the recently reported dengue outbreak in Nepal in 2010 have a closer genetic relation to Indian DENV-1 strains (Pandey et al., 2013). Similarly, as indicated above, the virus strain from the first dengue fever case reported in Nepal from a Japanese traveler in 2004 also showed resemblance to strains from India and Singapore (Supplementary Fig. S2) (Takasaki et al., 2008). We focused on the envelope protein coding region for sequence analysis of these genotypes, as the changes in the envelope protein may have direct correlation with the infectivity and immunogenicity profile of the virus (Roehrig et al., 1994; Chen et al., 1997; Modis et al., 2004). The envelope protein is a dimeric protein in the pre-fusion stage with each monomer subunit containing 3 distinct domains – Domain I, II and III with the domain III located on the outer surface (Hung et al., 2004). It interacts with the receptors of the host-cell for virus entry inside the cell (Chen et al., 1997; Modis et al., 2004). As shown in Table 1 and mentioned in results, there were a few mutations that fell in these functional domains of the protein. The N-glycosylation sites at position 67 and 153 were conserved in both genotype strains and the type strain NGC. Purifying selection is more evident in dengue virus evolution, with only occasional occurrence of positive selection in certain sites
across the genome (Bennett et al., 2003). In some of the earlier studies, position 52, 126, 129, 390 and 402 were reported to be under positive selection (Kumar et al., 2010). We could identify changes in amino acid in all these positions in the Cosmopolitan strains from Nepal. However, none of these sites had statistically significant positive selection in our analysis. The only position (aa 360) found to be under positive selection in our data set was conserved in Nepal samples as in the NGC strain. The mutations at amino acid (aa) positions 71, 126, 390, 402 and 454 had been earlier reported to play functional role in virus infectivity phenotype (Kawano et al., 1993; Bray et al., 1998; Gualano et al., 1998). The position 126 has role in causing conformational change at low pH and has been reported to be under mutational pressure (Roehrig et al., 1994). The change of Glu (G) to Lys (K) at this site produced a neurovirulent virus. The amino-acid at the position 390 is predicted to be involved both in host-cell attachment and virulence in mice (Sanchez and Ruiz, 1996). The site 402 located in the stem-anchor region of the E protein structure also confer neurovirulence in mice (Bray et al., 1998). Most of these changes were observed in the Cosmopolitan genotype compared to the Asian II strain (Table 1), indicating differences in the functional properties among these virus strains. Analysis of the infectivity of DENV-2 strains from Nepal in mosquito cell lines identified that the Cosmopolitan Genotype IVa had a replication advantage over the Asian II strains (Fig. 3C). The observation was not statistically significant based on the comparison of only two isolates, one from each genotype; and will need
S. Singh et al. / Infection, Genetics and Evolution 34 (2015) 402–409
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Fig. 2. Infection of BHK-21 cells with the isolates of Asian II and Cosmopolitan Genotype IVa strains-cytopathic effect and visible plaques. (A) The BHK-21 cells were viewed 72 h post-infection using the phase contrast objective of a fluorescent microscope (NIKON Eclipse Ti) at 200 magnification to observe the cytopathic effect. The cytopathic effect could also be seen by the visible plaques using 1% carboxymethyl cellulose overlay with crystal violet counterstaining upon 6 days post-infection. (B) The immunofluorescence assay was done to detect the virus in BHK-21 cells at 72 h post-infection after fixing the cells with 4% paraformaldehyde and staining with in-house rabbit anti-DENV-2 envelope protein polyclonal serum followed by addition of anti-rabbit IgG Alexa fluor 488 conjugated secondary antibody. The nuclei were counter stained with DAPI and cells were viewed under a fluorescent microscope at 200 magnification.
Fig. 3. C6/36 cells infection of the Asian II strain and Cosmopolitan Genotype IVa strains: (A) The C6/36 cells were observed for 5 days post infection under bright field at 200 magnification under a Nikon Eclipse Ti microscope to visualize the morphological changes and cytopathic effect caused by the viral strains, if any. (B) MTT assay was performed on C6/36 cells to quantitate the percentage viability of the mosquito cells upon infection till 5 days (C) In vitro replication kinetics of Asian II and Cosmopolitan Genotype IVa strains was compared in C6/36 mosquito cells. Virus containing supernatant collected from infected C6/36 cells till 5 days post infection were subjected to plaque assay on BHK-21 cells. Each data point represents mean value of plaque counts from three biological replicates, each done in multiple dilutions. Dilutions with countable number of plaques (
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Phylogenetic study reveals co-circulation of Asian II and Cosmopolitan genotypes of Dengue virus serotype 2 in Nepal during 2013 q Sneha Singh a,1, Birendra P. Gupta b,1, Anoop Manakkadan a,2, Krishna Das Manandhar b,⇑, Easwaran Sreekumar a,⇑ a b
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud P.O., Thiruvananthapuram 695014, Kerala, India Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
a r t i c l e
i n f o
Article history: Received 5 June 2015 Received in revised form 3 July 2015 Accepted 5 July 2015 Available online 6 July 2015 Keywords: Dengue Phylogeny Mosquito cell infectivity Mutation Genotypes
a b s t r a c t The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Dengue is a major re-emerging mosquito-borne disease in the tropical and subtropical countries. For the last few decades it remains on top with respect to disease burden and mortality (Bhatt et al., 2013). The disease manifests as a mild febrile episode or as severe and life threatening haemorraghic syndrome. Dengue virus belongs to the genus Flavivirus, family Flaviviridae. Its genome consists of approximately 11 kb positive-sense, single stranded q A part of this work was presented as a poster in the 12th International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases (MEEGID), 11–13 December 2014, Bangkok, Thailand. ⇑ Corresponding authors. E-mail addresses: [email protected] (K.D. Manandhar), [email protected] (E. Sreekumar). 1 These authors contributed equally. 2 Present address: Department of Biotechnology, Cochin University of Science and Technology, Cochin-22, Kerala, India.
http://dx.doi.org/10.1016/j.meegid.2015.07.006 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
RNA molecule without a poly-A tail (Chambers et al., 1990). Among the three structural proteins (Capsid, Membrane and Envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4 and NS5) encoded by the genome, the envelope (E) protein plays a critical role in virus attachment and fusion to the host cell membrane, and hence infectivity. Three domains (I, II and III) have been identified in the E protein and the domain III interacts with the cellular receptor and mediates endosome fusion (Modis et al., 2004). The presence of four classical serotypes among the Dengue viruses (DENV-1, 2, 3, and 4) that have no cross-protective immunity in infected patients dramatically increases the complexity of this disease. DENV-2 has been attributed to cause a more severe disease even during primary infections (Rico-Hesse, 2003). At molecular genetic level, DENV-2 has been divided into 6 genotypes namely Cosmopolitan, Asian I, Asian II, American, American/Asian and Sylvatic. These genotypes have a 5% or more genetic divergence in their envelope as well as whole genome nucleotide sequences
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(Weaver and Vasilakis, 2009). The Asian I genotype comprises of strains isolated from Thailand and Malaysia while Asian II genotype consists of strains circulating in China, Philippines, Sri Lanka, Taiwan and Vietnam. The strains circulating in China, Thailand, Vietnam, Brazil, Venezuela and the Caribbean belonged to American/Asian genotype while the isolates from Australia, Pacific Islands, South East Asia, Indian sub-continent, Middle East, Africa and Mexico fall in Cosmopolitan genotype (Twiddy et al., 2002; Mendez et al., 2012). Nepal, a Himalayan country, is surrounded by India on three sides and China to the North. It is an endemic nation for many vector borne diseases, like malaria, kala-azar, Japanese encephalitis and lymphatic filariasis (Dumre et al., 2013). The first report of dengue fever came in 2004 from a Japanese traveler in Nepal and it was reported to be of serotype 2 (Takasaki et al., 2008) with >99% similarity with viral strains from India. It was followed by a native report in 2006 with detection of four dengue serotypes (Malla et al., 2008). A few intermittent cases were reported nationwide from 2007 to 2009 with 2, 8 and 16 cases per year, respectively (Dumre et al., 2013; Griffiths et al., 2013). An outbreak occurred in 2010 with 350 (30%) confirmed cases out of 1215 clinically reported cases. The first isolation was done during the 2010 epidemic and all the strains belonged to serotype 1, genotype V closer to the Asian sub-cluster (Pandey et al., 2013). The year 2013 witnessed a total shift in the circulating serotype and DENV-2 outbreak was reported in the Terai belt of the country (Birendra et al., 2014; accepted manuscript). A large number of secondary infections are usually reported from the country and may be attributable to the circulation of multiple serotypes in the region (Malla et al., 2008; Dumre et al., 2013). There is lack of reliable dengue surveillance data from the country and inadequate response in accordance with international guidelines (Griffiths et al., 2013) that may hamper dengue control measures in the region. Adding to these is the lack of information on nucleotide sequences of DENV strains from the country (Malla et al., 2008; Pandey et al., 2013), which may also affect the epidemiological interventions. In the present study, DENV strains from an outbreak in Nepal in 2013 were characterized at the molecular level by sequencing of the complete envelope protein coding region and subsequent phylogenetic analysis. Strains representing different genotypes were studied by in vitro infection kinetics analysis in mosquito cells to understand the differences in infection competence and adaptation. The results shed light on the genotypes of dengue virus currently circulating in the region and add country specific information to the DENV sequence database.
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FBS (PAN Biotech, Germany) and was maintained at 37 °C in a 5% CO2 incubator (Sanyo, Japan). 2.3. Reverse transcriptase PCR and envelope (E) protein coding region sequencing Viral RNA was extracted from 150 ll of patient serum using viral RNA isolation kit (Macherey Nagel, Germany) in accordance with the manufacturer’s instructions. Initial dengue viral RNA detection in samples was done by a single-step RT-PCR using a set of generic primers (D1F-DencomR2) (Supplementary Table 1) that amplifies the Core-Pre-membrane (C-PrM) region of any of the four viral serotypes, as described previously (Anoop et al., 2010, 2012). Subsequently, serotype identification was done using a second step, multiplex, semi-nested PCR of this primary amplification by serotype-specific primers described previously (Lanciotti et al., 1992), with minor modifications in the primers (D1F-NTS1/NTS2/NTS3/nDen4) (Supplementary Table 1) (Anoop et al., 2012). Amplification of a 1616 bp region spanning the complete E gene was performed using a two-step RT-PCR with the primers D2Seq3F and D2Seq7R (Supplementary Table 1). Briefly, it consisted of a reverse transcription of 5 ll of the isolated RNA using AMV reverse transcriptase system (Fermentas) at 45 °C for 1 h, and denaturation of the enzyme at 98 °C for 5 min. This was followed by a PCR reaction for 35 cycles with cycling conditions of 98 °C for 30 s, 55 °C for 1 min, 72 °C for 2 min, and a final extension of 72 °C for 4 min. The amplified region was subjected to bi-directional sequencing using overlapping primers (Supplementary Table 1) and the Big Dye Terminator cycle sequencing kit in an ABI3730 Genetic Analyzer automated DNA sequencer (PE Applied Biosystems, Foster City, CA). 2.4. Virus isolation in C6/36 cells and in vitro infection kinetics analysis Briefly, the serum samples (1:10 diluted in phosphate buffered saline, PBS; pH 7.4) was allowed to adsorb on to the C6/36 cells for 2 h at 33 °C with slow shaking followed by a gentle wash with 1 PBS and addition of L-15 medium supplemented with 2% FBS and further incubation with shaking. The virus containing supernatant was harvested on the third day post-infection and stored in 80 °C as aliquots. For infection kinetics analysis, the C6/36 cells were infected at an MOI of 0.1. The supernatant was collected at 0 h, 6 h, 12 h and then every 24 h till five days post infection. The amount of virus in the supernatant was titrated by plaque assay on BHK-21 cells. 2.5. Infection of BHK-21 cells, immunofluorescence and plaque assay
2. Materials and methods 2.1. Ethical approval and patient samples Ethical approval for the study was obtained from the Nepal Health Research Council (NHRC) Ethical Review board (2071-04-15). A total of 52 venous blood samples from patients clinically diagnosed as dengue were obtained from the 2013 dengue outbreak in Nepal, as described earlier (Birendra et al., 2014; accepted manuscript). 2.2. Cell lines The Aedes albopictus mosquito cell line C6/36 was maintained in culture at 28 °C in L-15 medium (Invitrogen, New York) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech, Germany). Baby hamster kidney cells (BHK-21) were cultured in DMEM (Invitrogen, New York) supplemented with 10%
The BHK-21 cells were infected with DENV-2 isolates by adsorption of the virus for 2 h at 37 °C followed by two washes with 1 PBS. The cells were further grown in DMEM supplemented with 2% FBS for three days. Cells were fixed with 4% paraformaldehyde 72 h post-infection followed by washes with 1 PBS and permeabilization with 0.5% Triton X 100. The presence of dengue virus antigen was detected by in-house rabbit polyclonal anti-dengue envelope protein antibody as primary antibody and anti-rabbit Alexa Fluor 488 secondary antibody. Fixed cells were incubated with primary antibody at 4 °C overnight, followed by washes and incubation with secondary antibody at 37 °C for one hour. The cells were washed and counter stained with DAPI stain and visualized under an inverted fluorescent microscope (Nikon Eclipse Ti). For plaque assay and virus titration, 90% confluent BHK-21 cells were infected by adsorption of the virus for 2 h at 37 °C followed by two washes with 1 PBS (Sigma–Aldrich) and overlaying with 1 DMEM (Invitrogen, New York) containing a final concentration
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of 1% Carboxymethyl cellulose (CMC) (Sigma–Aldrich) and 1% heat-inactivated FBS. The cells were fixed with 30% formaldehyde on 5th day post infection for 2 h at 37 °C followed by counterstaining with crystal violet stain. 2.6. Envelope protein coding region sequence analysis The sequences generated from the present study were aligned using the Clustal W function of the Bio-Edit 6.0.7 software to obtain the complete coding region sequence of the envelope protein (1485 bp). For comparison and mapping of the changes, both the nucleotide and translated amino-acid sequences were used. The sequence identity was calculated by optimal global alignment between the selected strains. 2.7. Phylogenetic analysis Bayesian analysis was carried out using the BEAST software package (BEAST v1.6.1) (Drummond and Rambaut, 2007). Envelope protein coding region sequences of 155 DENV-2 strains from the Asia-Pacific region, strains identified from an initial on-line BLAST analysis of the obtained sequences and of reference strains of all the major genotypes retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov) along with the sequences of the Nepal strains were used as the data set. Using jModelTest 0.1.1, GTR + I + G (GTR is generalized time reversible, G is the gamma distribution and I is the proportion invariant) was identified as the best-fit model for Bayesian Markov Chain Monte Carlo (MCMC) analysis. The strict and relaxed molecular clock models and different tree priors (constant size, exponential growth and expansion growth) were used in the analysis. An effective sampling size of P200 was ensured by carrying out independent runs. Statistical convergence was obtained by doing MCMC chains for 10,000,000 generations and fixing a sampling frequency of 10,000 and burn-in of 1000 and 95% HPD (Highest Posterior Density) intervals. Tracer v1.5 program, Tree Annotator program and FigTree v1.4.0 were used to analyze the output from BEAST, and to generate and view the tree. 2.8. Recombination and selection pressure analysis SBP and GARD methods in the online software DATAMONKEY (http://www.datamonkey.org) were used to detect recombination. Selection pressure analysis employed SLAC, FEL and REL, FUMAR, MEME methods with default P-value/Bayes factor settings. 3. Results 3.1. Envelope protein (E) coding region sequence analysis from clinical samples Out of the 52 clinical samples that were initially subjected to a generic dengue virus pan-serotype specific RT-PCR, 15 samples were found to be positive (Supplementary Table 2). A serotype analysis by a second round serotype-specific PCR identified all of them to belong to Dengue virus serotype 2. This was later confirmed by the envelope coding region sequencing. Analysis of the complete nucleotide (1485 bp) sequences of the envelope coding region from these 15 samples (GenBank Accession Nos. KT232041-KT232055; Supplementary Table 2) showed substitutions, but no insertions/deletions in the sequences. Overall, at the nucleotide level, there were 92 point mutations in comparison to the prototype DENV-2 strain New Guinea C (NGC; GenBank Accession No. M29095). 85 of them were transversion mutations
and seven were transition mutations. Eleven non-synonymous mutations were observed in the amino acid sequence (Supplementary Fig. S1). Seven mutations (E47K, Q52H, D71A, K126E, V129I, H149 N, I164V) were falling in the domain I (aa positions 1–52, 133–193 and 281–296) and II (aa positions 53–132 and 194–280) of the envelope protein. The immunogenic domain – domain III (aa positions 297–493) had four mutations (N390S, I402F, I454T, I462V) (Table 1). The six strains that were closely related to the NGC strain had the E47K substitution differentiating them from the prototype strain. 3.2. Phylogenetic analysis We had to restrict the length of the sequences used in the phylogenetic analysis to 1097nt, instead of the complete 1485nt, to include the only DENV-2 sequence earlier reported from Nepal (GenBank Accession No. AB194885; indicated by an arrow in Supplementary Fig. S2). In the Bayesian analysis, the 2013 Nepal strains were found to fall into two distinct clusters within the phylogenetic tree, with a significant posterior probability (Fig. 1 and Supplementary Fig. S2). Out of the 15 strains, 9 strains were found to cluster with the Cosmopolitan Genotype IVa while the rest clustered along with the Asian II genotype. Among them, the nine Cosmopolitan Genotype IVa strains had 99.7–99.9% nucleotide level identity with each other whereas the six Asian II strains were 100% identical. Between the Asian II and Cosmopolitan Genotype IVa strains from Nepal, the identity varied from 93.7–99.9%.The Genotype IVa strains clustered with the strains from Malaysia (JF968005, JF968012) isolated during 2010. The Nepal strains that grouped within the Asian II genotype formed a distinct clade, and were found to be related genetically to the strains isolated from the United States and Papua New Guinea (FJ906968, FJ906959, GQ199901). The strain from Nepal identified earlier in 2004 (GenBank Accession No. AB194885) clustered with the Cosmopolitan Genotype IVb strains, with closer genetic relation to Indian and Singapore strains (Supplementary Fig. S2). 3.3. Molecular clock analysis Both strict and relaxed clock parameters were employed for carrying out the molecular clock analysis of the Envelope region of the Nepal DENV-2 strains. The best model was then selected based on the highest marginal likelihood value as obtained for the uncorrelated exponential molecular clock with exponential population size (Supplementary Table 3). This method was further carried out for determining the time to most recent common ancestor (tMRCA) and also the values for the nucleotide substitution rates for the datasets. The whole dataset was divided based on the presence of the strains isolated in the present study as Genotype IVa and Asian II groups for estimating the values. Accordingly, the mean tMRCA of the Asian II strains were found to be 78.98 years (95% HPD: 70.57, 90.15) while that of the Cosmopolitan Genotype IVa strains were found to be 48.65 years (95% HPD: 35.95, 64.14). The mean nucleotide substitution rate for all the isolates used in the study was found to be 7.85 10 4 substitutions per site per year (95% HPD: 5.54, 10.22), which was similar to the earlier reports (6 10 4 substitutions per site per year; Twiddy et al., 2003; Wang et al., 2000). 3.4. Recombination and selection pressure analysis No recombination events could be detected among the sequences in the data set used in the analysis. Selection pressure analysis was carried out by grouping the datasets into three as Asian II (n = 18), Cosmopolitan Genotype IVa (n = 44) and all DENV sequences (n = 155). Among the five different analyses,
T
V
T
V V V
‘.’ mark indicates conserved residues; ‘–’ indicates that the sequence information is not available.
T T
V V
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We could successfully isolate only three strains from the 15 DENV-2 positive samples by infecting C6/36 mosquito cells. Two among them were of Cosmopolitan Genotype IVa and the third one was an Asian II strain. One isolate from each of the genotypes (DENV-2/NEP144_2013; Cosmopolitan Genotype IVa and DENV-2/NEP679_2013; Asian II genotype) (passage level 4), was selected for infectivity analysis. In BHK-21 cells that were used for virus quantification by plaque assay, infection was confirmed by immunofluorescence for the detection of viral envelope protein and also by observing cytopathic effect (Fig. 2A and B). The two virus strains formed plaques with similar morphology in infected BHK-21 cells (Fig. 2A). In infectivity analysis, the C6/36 cells infected with Cosmopolitan Genotype IVa or Asian II strains showed roundening whereas uninfected cells retained their spindle shape. However, the MTT assay showed that there was no significant reduction in the viability percentage of these cells when infected with either of the strains at identical MOIs (Fig. 3A and B). The in vitro virus replication kinetics in C6/36 cells revealed that the Cosmopolitan Genotype IVa had a marginally higher replication rate compared to the Asian II genotype in terms of progeny virus production (Fig. 3C). In the culture supernatants, at 24 h p.i the viral titre reached around 6.33 102 pfu/ml for the Cosmopolitan Genotype IVa strain, while for the Asian II strain it was 1.23 102 pfu/ml. At the 5th day post infection the Genotype IVa reached a titre of 3.2 105 pfu/ml and Asian II reached a titre of 1.3 105 pfu/ml.
V
T
3.5. Virus isolation and in vitro infection kinetics assay in mosquito cells
V
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– I 742 462
I 734 454
405
namely, SLAC, FEL, REL, FUBAR and MEME, if at least two methods identified a site for positive selection, the result was taken into consideration. Accordingly, only the codon at position 360 was found to be under positive selection pressure by all the three methods, when the whole sequence dataset was used. No significant codon positions were found in other two datasets (Supplementary Table 4).
V
T
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F F F F F F F F . 682 402
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.
.
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.
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S S S S S S S S . 670 390
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444 164
I
429 149
V 409 129
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E E E E E E E E . 406 126
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.
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.
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A D 351
Q 332
71
E 327
52
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DF04 Guinea C polyprotein protein
47 E
Nepal New the on the
.
K
H
NEP_11_2013 NEP_10_2013 NEP_10NA_2013 NEP_736_2013 NEP_718_2013 NEP_701_2013 NEP_144_2013 NEP_885_2013 NEP_798_2013 NEP_988_2013 NEP_9_2013 NEP_679_2013 NEP_96_2013 AB194885 M29095 Position on Position Gene
Table 1 Amino acid changes observed in DENV-2 strains of the Asian II and Cosmopolitan genotypes from Nepal compared to New Guinea Strain C reference strain (M29095) and Nepal DF 2004 strain (AB198445).
NEP_793_2013
NEP_140_2013
S. Singh et al. / Infection, Genetics and Evolution 34 (2015) 402–409
4. Discussion Analyzing dengue viral strains from outbreaks in various geographical locations adopting molecular approaches has significant epidemiological significance (Kyle and Harris, 2008). This is supported by the fact that the clinical spectrum of dengue disease has correlation with the viral serotypes, genotypes or sub-genotypic lineages prevalent in an area. (Messer et al., 2003; Rico-Hesse, 2003; Zhang et al., 2005; Ospina et al., 2010; Lee et al., 2012). In the present study, we analyzed DENV strains from Nepal. We observed that two different genotypes- the Asian II and Cosmopolitan Genotype IVa- were co-circulating (Fig. 1) in the dengue outbreak that occurred in the Terai as well as the hilly region in 2013. Though the severity of clinical disease and mortality was not high, the outbreak was more geographically widespread affecting a larger number of people. There is a genotype shift among the Cosmopolitan strains from Genotype IVb to IVa. In an earlier report, the DENV-2 strain acquired by a Japanese patient from the region was shown to belong to the Cosmopolitan Genotype IVb (referred to as Genotype I by Takasaki et al., 2008) with similarity to Indian strains. Genotype IVb is a predominant DENV-2 genotype in the neighboring country, India, and has been associated with larger and more severe dengue epidemics (Mishra et al., 2015). The Genotype IVa strains in the current outbreak shows genetic similarity to the strains from Malaysia and Singapore. Similarly, the closest relative for the Asian II strain identified was a 2009 strain from USA. The
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Fig. 1. Maximum clade credibility (MCC) tree showing the co-circulation of Cosmopolitan Genotype IVa and Asian II genotype in Nepal during 2013. MCC tree derived from the Bayesian analysis of the envelope protein of DENV-2 with the best fit model (strict and relaxed lognormal clock), showing the posterior probability at each principal node. The GenBank accession Nos. and the place and year of identification are shown. A few clades are collapsed to improve clarity and they are numbered. These clades are shown expanded in Supplementary Fig. S2 and correspondingly numbered. The strains from recent Nepal outbreak used in our study are shown in red and blue, representing the Asian II and Cosmopolitan Genotype IVa, respectively. The scale bar represents the time scale in years. The expanded MCC tree, indicating all the strains used in the study, is shown in Supplementary Fig. S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observations indicate that these DENV-2 strains might be of exotic introduction into Nepal. The country being a tourist destination, the chances of cross-border transmission of DENV strains cannot be ruled out as shown in many earlier studies from other parts of the world (Zaki et al., 2008; Shu et al., 2009; Jiang et al., 2012; Lee et al., 2012; Warrilow et al., 2012). Also, the DENV-1 strains identified in the recently reported dengue outbreak in Nepal in 2010 have a closer genetic relation to Indian DENV-1 strains (Pandey et al., 2013). Similarly, as indicated above, the virus strain from the first dengue fever case reported in Nepal from a Japanese traveler in 2004 also showed resemblance to strains from India and Singapore (Supplementary Fig. S2) (Takasaki et al., 2008). We focused on the envelope protein coding region for sequence analysis of these genotypes, as the changes in the envelope protein may have direct correlation with the infectivity and immunogenicity profile of the virus (Roehrig et al., 1994; Chen et al., 1997; Modis et al., 2004). The envelope protein is a dimeric protein in the pre-fusion stage with each monomer subunit containing 3 distinct domains – Domain I, II and III with the domain III located on the outer surface (Hung et al., 2004). It interacts with the receptors of the host-cell for virus entry inside the cell (Chen et al., 1997; Modis et al., 2004). As shown in Table 1 and mentioned in results, there were a few mutations that fell in these functional domains of the protein. The N-glycosylation sites at position 67 and 153 were conserved in both genotype strains and the type strain NGC. Purifying selection is more evident in dengue virus evolution, with only occasional occurrence of positive selection in certain sites
across the genome (Bennett et al., 2003). In some of the earlier studies, position 52, 126, 129, 390 and 402 were reported to be under positive selection (Kumar et al., 2010). We could identify changes in amino acid in all these positions in the Cosmopolitan strains from Nepal. However, none of these sites had statistically significant positive selection in our analysis. The only position (aa 360) found to be under positive selection in our data set was conserved in Nepal samples as in the NGC strain. The mutations at amino acid (aa) positions 71, 126, 390, 402 and 454 had been earlier reported to play functional role in virus infectivity phenotype (Kawano et al., 1993; Bray et al., 1998; Gualano et al., 1998). The position 126 has role in causing conformational change at low pH and has been reported to be under mutational pressure (Roehrig et al., 1994). The change of Glu (G) to Lys (K) at this site produced a neurovirulent virus. The amino-acid at the position 390 is predicted to be involved both in host-cell attachment and virulence in mice (Sanchez and Ruiz, 1996). The site 402 located in the stem-anchor region of the E protein structure also confer neurovirulence in mice (Bray et al., 1998). Most of these changes were observed in the Cosmopolitan genotype compared to the Asian II strain (Table 1), indicating differences in the functional properties among these virus strains. Analysis of the infectivity of DENV-2 strains from Nepal in mosquito cell lines identified that the Cosmopolitan Genotype IVa had a replication advantage over the Asian II strains (Fig. 3C). The observation was not statistically significant based on the comparison of only two isolates, one from each genotype; and will need
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Fig. 2. Infection of BHK-21 cells with the isolates of Asian II and Cosmopolitan Genotype IVa strains-cytopathic effect and visible plaques. (A) The BHK-21 cells were viewed 72 h post-infection using the phase contrast objective of a fluorescent microscope (NIKON Eclipse Ti) at 200 magnification to observe the cytopathic effect. The cytopathic effect could also be seen by the visible plaques using 1% carboxymethyl cellulose overlay with crystal violet counterstaining upon 6 days post-infection. (B) The immunofluorescence assay was done to detect the virus in BHK-21 cells at 72 h post-infection after fixing the cells with 4% paraformaldehyde and staining with in-house rabbit anti-DENV-2 envelope protein polyclonal serum followed by addition of anti-rabbit IgG Alexa fluor 488 conjugated secondary antibody. The nuclei were counter stained with DAPI and cells were viewed under a fluorescent microscope at 200 magnification.
Fig. 3. C6/36 cells infection of the Asian II strain and Cosmopolitan Genotype IVa strains: (A) The C6/36 cells were observed for 5 days post infection under bright field at 200 magnification under a Nikon Eclipse Ti microscope to visualize the morphological changes and cytopathic effect caused by the viral strains, if any. (B) MTT assay was performed on C6/36 cells to quantitate the percentage viability of the mosquito cells upon infection till 5 days (C) In vitro replication kinetics of Asian II and Cosmopolitan Genotype IVa strains was compared in C6/36 mosquito cells. Virus containing supernatant collected from infected C6/36 cells till 5 days post infection were subjected to plaque assay on BHK-21 cells. Each data point represents mean value of plaque counts from three biological replicates, each done in multiple dilutions. Dilutions with countable number of plaques (
