Co-circulation of Chikungunya and Dengue viruses in Dengue endemic region of New Delhi,India during 2016

Co-circulation of Chikungunya and Dengue viral infections (CHIKV and DENV) have been reported mainly due to transmission by common Aedes vector. The purpose of the study was to identify and characterise the circulating strains of CHIKV and DENV in DENV endemic region of New Delhi during 2016. CHIKV and DENV were identified in the blood samples (n = 130) collected from suspected patients by RT-PCR. CHIKV was identified in 26 of 65 samples (40%). Similarly, DENV was detected in 48 of 120 samples (40%). Co-infection with both the viruses was identified in five (9%) of the samples. Interestingly, concurrent infection with DENV, CHIKV and Plasmodium vivax was detected in two samples. CHIKV strains (n = 11) belonged to the ECSA genotype whereas DENV-3 sequences (n = eight) clustered in Genotype III by phylogenetic analysis. Selection pressure of E1 protein of CHIKV and CprM protein of DENV-3 revealed purifying selection with four and two positive sites, respectively. Four amino acids of the CHIKV were positively selected and had high entropy suggesting probable variations. Co-circulation of both viruses in DENV endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance for implementation of effective control measures.Key words: Chikungunya virus, co-circulation, co-infection, Dengue virus, phylogenetic analysis     

Evolution and epidemiologic dynamics of dengue virus in Nicaragua during the emergence of chikungunya and Zika viruses

Arthropod-borne viruses (arboviruses) comprise a significant and ongoing threat to human health, infecting hundreds of millions annually. Three such arboviruses include circumtropical dengue, Zika, and chikungunya viruses, exhibiting continuous emergence primarily via Aedes mosquito vectors. Nicaragua has experienced endemic dengue virus (DENV) transmission involving multiple serotypes since 1985, with chikungunya virus (CHIKV) reported in 2014–2015, followed by Zika virus (ZIKV) first reported in 2016. In order to identify patterns of genetic variation and selection pressures shaping the evolution of co-circulating DENV serotypes in light of the arrival of CHIKV and ZIKV, we employed whole-genome sequencing on an Illumina MiSeq platform of random-amplified total RNA libraries to characterize 42 DENV low-passage isolates, derived from viremic patients in Nicaragua between 2013 and 2016. Our approach also revealed clinically undetected co-infections with CHIKV. Of the three DENV serotypes (1, 2, and 3) co-circulating during our study, we uncovered distinct patterns of evolution using comparative phylogenetic inference. DENV-1 genetic variation was structured into two distinct co-circulating lineages with no evidence of positive selection in the origins of either lineage, suggesting they are equally fit. In contrast, the evolutionary history of DENV-2 was marked by positive selection, and a unique, divergent lineage correlated with high epidemic potential emerged in 2015 to drive an outbreak in 2016. DENV-3 genetic variation remained unstructured into lineages throughout the period of study. Thus, this study reveals insights into evolutionary and epidemiologic trends exhibited during the circulation of multiple arboviruses in Nicaragua.     

Characterization of the 2013 dengue epidemic in Myanmar with dengue virus 1 as the dominant serotype

In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study aimed to determine the serological and molecular characteristics of dengue virus (DENV) infection among children with clinical diagnosis of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during this period. Single acute serum samples were collected from 300 children in Mandalay Children Hospital, Mandalay, Myanmar. Out of the 300 children, 175 (58.3%) and 183 (61%) were positive for anti-dengue IgM and anti-dengue IgG, respectively. Among the IgM positives, 41 (23.4%) had primary DENV infection. Thirty-nine DENV strains (23 DENV-1, 10 DENV-2 and 6 DENV-4) were successfully isolated after inoculation of the patient serum samples onto C6/36 cells. DENV 1 was the dominant serotype in the 2013 epidemic. There was no correlation between the infecting serotypes and clinical severities. The DENV-1 strains belonged to three lineages of the genotype 1; the DENV-2 strains were of the Asian I genotype and were separated into two lineages; and DENV-4 strains belonged to the same lineage of genotype I. It is of interest to note the diversity of DENV-1 and -2 circulating in the same location during June–August 2013. These DENV isolates were genetically close (98%–100%) to the other previously reported isolates from Myanmar and its neighboring countries, namely China, Thailand, Sri Lanka, Cambodia and Vietnam. Primary DENV infection was still high among the severe dengue cases. Different serotypes of DENV were co-circulating in 2013, however, genotype shift was not observed. Additionally, amino acid mutations were detected in the study strains not seen in the previously reported strains from other countries and Myanmar. This paper provided information on the circulating serotypes for the last 15 years and the recent dengue situation in Mandalay, Myanmar after 2006.     

Divergence of the dengue virus type 2 Cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in Surabaya,Indonesia, 2008–2014

Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1–3, dominated by Surabaya strains, were defined as the “Surabaya lineage”. Clusters 4–6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the “South East Asian lineage”. The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013–14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170 V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013–14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries.     

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses

During 1960–80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences.All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A–D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782–1934) and DENV-4 (1719–1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938–1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India.The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.     

Molecular epidemiology of dengue virus strains from Finnish travelers

We characterized 11 dengue virus (DENV) isolates obtained from Finnish travelers during 2000-2005 using monoclonal antibodies and phylogenetic analysis. The analysis of DENV isolated from travelers contributes to the global picture of strain distribution and circulation. The isolates included all serotypes, including a DENV-2 isolate from Ghana.     

Evolutionary history and spatiotemporal dynamics of dengue virus type 1 in Asia

Previous studies showed that DENV-1 transmitted from monkeys to humans approximately 125 years ago. However, there is no comprehensive analysis about phylogeography and population dynamics of Asian DENV-1. Here, we adopt a Bayesian phylogeographic approach to investigate the evolutionary history and phylogeography of Asian DENV-1 using envelope (E) protein gene sequences of 450 viruses isolated from 1954 to 2010 throughout 18 Asian countries and regions. Bayesian phylogeographic analyses indicate that the high rates of viral migration possibly follows long-distance travel for humans in Southeast Asia. Our study highlights that Southeast Asian countries have acted as the main viral sources of the dengue epidemics in East Asia. The results reveal that the time to the most recent common ancestor (TMRCA) of Asian DENV-1 is 1906 (95% HPD, years 1897–1915). We show that the spatial dissemination of virus is the major source of DENV-1 outbreaks in the different localities and leads to subsequent establishment and expansion of the virus in these areas.     

Continuous dengue type 1 virus genotype shifts followed by co-circulation,clade shifts and subsequent disappearance in Surabaya,Indonesia, 2008–2013

Four serotypes of dengue virus (DENV-1 to DENV-4) and their genotypes are distributed in tropical and subtropical regions. Indonesia has been recently suggested as the origin of some dengue virus genotypes. In Surabaya, the second biggest city of Indonesia, we previously reported a shift of the predominantly circulating serotype from DENV-2 to DENV-1 in November 2008, followed by a genotype shift of DENV-1 from genotype IV (GIV) to genotype I (GI) in September 2009, based on nucleotide sequences in the envelope protein coding region. Since then, GI strains had predominantly circulated until December 2010. In this report, we investigated further DENV-1 transitions in Surabaya during 2011–2013 in order to comprehend dengue dynamics during 2008–2013 in more detail. From January 2011 through December 2011, only GIV strains were isolated, indicating that a genotype shift again took place from GI to GIV. In January 2012, GI and GIV strains started co-circulating, which continued until June 2013. To further investigate this phenomenon, analysis was performed at a clade level. GI and GIV strains isolated in Surabaya formed four and three distinct clades, respectively. Concomitant with co-circulation, new clade strains appeared in both genotypes. In contrast, some previously circulating clades were not isolated during co-circulation, indicating clade shifts. Among our Surabaya isolates, nucleotide and amino acid differences in the E region were, respectively, 1.0–2.3% and 0.2–1.0% for GI isolates and 2.0–6.3% and 0.0–1.8% for GIV isolates. Several characteristic amino acid substitutions in the envelope ectodomain were observed in some clades. After July 2013, DENV-1 strains were not isolated and were replaced with DENV-2. This study showed that continuous shifts of more than one genotype resulted in their co-circulation and subsequent disappearance and suggested the relevance of clade replacement to genotype co-circulation and disappearance in Surabaya.     

Molecular investigation of the dengue outbreak in Karnataka,South India,reveals co-circulation of all four dengue virus serotypes

The growing incidence of dengue outbreaks in the state of Karnataka prompted us to study the circulating dengue virus (DENV) and their proportion among the suspected cases of dengue patients during the disease outbreak at Mysuru district of Southern India. The presence of the DENV in a patient's serum sample was identified by RT-PCR using previously published primer pairs targeting CprM gene. DENV serotyping was carried out by semi-nested multiplex PCR using serotype-specific primers and nucleotide sequencing. Three hundred fifty-five samples of serum from suspected dengue cases were collected, and 203 samples (57.18%) were found positives. In 2016, DENV-4 (97.87%) was found to be the most dominant DENV serotype either alone or as co-infection, followed by DENV-2 (8.51%) and DENV-3 (4.25%). In 47 positive cases, co-infection with more than one serotype was detected in 4 cases (8.51%). The analysis of the dengue cases in 2017, DENV-4 was dominating serotype (33.97%), followed by the emergence of DENV-2 (32.05%), DENV-3 (25.64%), and DENV-1 (25.00%). Our study also reports the circulation of all four DENV serotypes in the Mysuru district of Southern India, with concurrent infections rate of 16.66% in 2017. The present study provides information regarding the genetic variation among the circulating DENV serotype in an Indian state of Karnataka. The need for the studying genetic diversity of DENV will be useful during the continuous monitoring for disease burden as well as the development of appropriate prophylactic measures to control the spread of dengue infection.     

Phylodynamics of DENV-1 reveals the spatiotemporal co-circulation of two distinct lineages in 2013 and multiple introductions of dengue virus in Goiás,Brazil

Dengue virus type 1 (DENV-1) was the first serotype introduced in Brazil, during in the 1980s. Since then, this virus has spread in the Brazilian territory, causing several outbreaks. In 2013 the highest number of dengue cases was notified, when compared to the previous years in Brazil, and the state of Goiás reported over 160 thousand cases. In this study, we aimed to present the Phylodynamics of DENV-1 isolates from the state of Goiás, Brazil, during 2013 outbreak, based on the envelope gene (E) sequences. Phylogenetic analysis revealed that Brazilian DENV-1 isolates are grouped together with viruses from genotype V in two distinct lineages (lineage I and lineage II) reflecting co-circulation. Phylogeographic analyses showed that these lineages were introduced in different moments in Goiás, Brazil, using distinct routes, likely originated from the Caribbean. Lineage I was first introduced coming from Rio de Janeiro (2007–2012), followed by the introduction from Argentina (2010 − 2013). Lineage II was introduced in a single moment from Rio de Janeiro and this clade has existed since 2007–2010. The different viral introduction events demonstrate the viral dispersion process with neighboring regions, which is essential for the maintenance of outbreaks and introduction of new emerging viruses. In conclusion, obtained data reveals the importance of continuous molecular surveillance of this virus in different regions, providing a better understanding of DENV-1 circulation, considering the evolutionary and virus spread patterns.     

Genotype replacement of dengue virus type 3 and clade replacement of dengue virus type 2 genotype Cosmopolitan in Dhaka,Bangladesh in 2017

Dengue is a mosquito-borne disease that has spread to >100 countries and is caused by the dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to -4), and each serotype is further divided into distinct genotypes. In India, it is reported that all 4 serotypes of DENV co-circulate. Although Bangladesh is a neighboring country of India, very few reports have published DENV sequence data for the country, especially after 2012. To understand the current distribution of DENV genotypes in Bangladesh, we determined the nucleotide sequences of envelope regions obtained from 58 DENV-positive patients diagnosed at Apollo Hospitals Dhaka during the period between September 2017 and February 2018. We found 5 DENV-1, 47 DENV-2, and 6 DENV-3 serotypes. A phylogenetic analysis of the obtained viral sequences revealed that DENV-3 genotype I was present instead of DENV-3 genotype II, which was predominant in Bangladesh between 2000 and 2009. Furthermore, we found two distinct lineages of the Cosmopolitan genotype of DENV-2, one of which was closely related to strains from Southeast Asia and has never been reported previously in Bangladesh. These results indicated that DENVs in Bangladesh have increased in genotypic diversity and suggest that the DENV genotypic shift observed in other Asian countries also might have been taking place in Bangladesh.     

Origin, evolution, and phylogeography of recent epidemic CHIKV strains

Chikungunya virus (CHIKV) is an arthropod-borne virus of the Alphavirus genus, which is transmitted to humans by Aedes spp. mosquitoes and was firstly identified in Tanzania in the mid 1950s. In this article, the findings of a phylogenetic and phylogeographic analysis of the recent CHIKV pandemic are reported. We estimated time of origin of the ancestral virus, time and place of occurrence of A226V mutation, and the flow of viral strains from an area to the other. The Bayesian phylogenetic and phylogeographic analysis was performed on the whole dataset, which consisted of 195 E1 (envelope 1) CHIKV sequences, and on a subset (D2), including 146 of the 195 previous sequences. Using the relaxed clock model, we estimated a CHIKV E1 mean evolutionary rate (in the whole dataset) of 1.4 × 10(-3)substitution/site/year (95% highest posterior density interval HPD 6.4 × 10(-4)-2.5 × 10(-3)), and of 2.2 × 10(-3) (95% HPD 9.6 × 10(-4)-3.8 × 10(-3)) in the D2 subset, including only the strains involved in the recent Indian Ocean epidemic. The phylogeographical analysis suggested an African origin of CHIKV with a tMRCA of 146 years corresponding to 1863 (95% HPD 1741-1941). Moreover D2 subset most probably originated in Kenya, with a tMRCA corresponding to the year 2002 (95% HPD 2000-2004), then spread following two distinct routes: one throughout the Indian Ocean (Reunion, Comoros) and the other moving from India then scattered in the South East Asia and reached Italy. In conclusion, we reconstructed the geographic spread of CHIKV during the last epidemic wave, which showed an eastward path from Africa to Indian Ocean island to India, and from there to other South East Asian countries. Whether A226V variants followed the same migration path remains undefined, since local independent mutations, followed by fixation due to selective advantage conferred by better adaptation to local vectors of infection, cannot be excluded.     

Delineating the serotype-specific neutralizing antibody response to a live attenuated tetravalent dengue vaccine

The dengue virus (DENV) vaccines that are licensed or in clinical development consist of DENV serotype 1–4 tetravalent formulations given simultaneously and are not acquired sequentially like natural infections. It is unclear what effect this has on development of protective levels of immunity to all four serotypes. Serotype-specific neutralizing antibody (NAb) is considered the most relevant correlate of protection from dengue disease. Here we assessed levels of serotype-specific and cross-reactive NAb in immune sera from 10 subjects vaccinated with a live attenuated tetravalent DENV vaccine developed at the Walter Reed Army Institute of Research. The majority of subjects NAb responses to DENV-2 and DENV-4 were type-specific, while their NAb responses to DENV-1 and DENV-3 were primarily cross-reactive. Vaccine virus RNAemia has been most frequently detected for DENV-2 and DENV-4 in vaccinated subjects, strongly suggesting that replication is important for eliciting serotype-specific immunity.     

Molecular epidemiology of American/Asian genotype DENV-2 in Peru

During the past decade, countries in South America have reported dengue hemorrhagic fever (DHF) associated with American/Asian genotype of dengue virus serotype 2 (DENV-2). DENV-2 strains have been associated with large outbreaks of dengue fever and DHF in numerous regions of Peru since the mid-1990s, but studies to address the origins, distribution, and genetic diversity of DENV-2 strains have been limited. To address this knowledge gap, we sequenced the envelope gene region of DENV-2 isolates from Peru, Ecuador, Paraguay, and Bolivia. Sequences were aligned and compared to a global sample of DENV-2 viruses. Phylogenetic analysis confirmed the circulation of two DENV-2 genotypes in Peru: American (prior to 2001) and American/Asian (2000 to present). American/Asian genotype variants can be classified into two lineages, and these were introduced into Peru from the north (Ecuador, Colombia, and/or Venezuela) and the east (Brazil and Bolivia). American/Asian lineage II replaced lineage I after 2009. We estimate the time to the most recent common ancestor for American/Asian DENV-2 genotype in the Americas was in 1980, and 1984 and 1989 for lineages I and II, respectively. In light of evidence for increased virulence of lineage II of American/Asian DENV-2, our results support the need for continuous monitoring for the emergence of new DENV genotypes that may be associated with severe disease.     

Molecular characterization of the E gene of dengue virus type 1 isolated in Guangdong province, China, in 2006

We determined the genetic relationships and origin of the dengue virus (DENV) responsible for an outbreak of dengue fever (DF) in Guangdong province, China, in 2006. Five DENV type 1 (DENV-1) isolates were obtained from human serum samples collected from DF patients during the outbreak. The nucleotide sequences of the E (envelope) gene were compared with those of 48 previous DENV-1 isolates: 18 from Guangdong province, one from Fujian province, one from Zhejiang province, and 28 from other countries in the South Asian region. The results suggested that four DENV-1 isolates identified in Guangdong province in 2006 might be in general circulation there, although these DENV-1 viruses may have been originally introduced into the province from other countries. In contrast, one isolate from Guangzhou city in 2006, may have been introduced by a recently imported case from Cambodia.     

The evolution of dengue-2 viruses in Malindi,Kenya and greater East Africa: Epidemiological and immunological implications

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses shows spatial structure at the country level in Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region.     

Rapid spread of chikungunya virus following its resurgence during 2006 in West Bengal, India

Re-emergence of chikungunya virus (CHIKV) in West Bengal was detected after almost 40 years when an outbreak of fever occurred in Baduria village (West Bengal, India) in October 2006. The symptoms of CHIKV infection are similar to those of dengue virus (DENV) infection. Serum samples were tested for detection of IgM antibody to CHIKV and DENV and the aetiological agent was detected as CHIKV. RT-PCR was carried out for confirmation of CHIKV infection. By 2009, CHIKV had spread rapidly within ten districts of West Bengal. Middle-aged women (age group 31-40 years) were predominantly affected. Here we report the analysis of 2134 serum samples collected during 2006-2009 from the different districts of West Bengal, among which IgM antibody to CHIKV and DENV was detected in 403 and 199 samples, respectively. This report highlights the gradual dominating activity of CHIKV with dengue-like clinical features in dengue-endemic regions such as West Bengal.