Molecular Epidemiology, Evolution and Reemergence of Chikungunya Virus in South Asia

Chikungunya virus (CHIKV) is a vector (mosquito)-transmitted alphavirus (family Togaviridae). CHIKV can cause fever and febrile illness associated with severe arthralgia and rash. Genotypic and phylogenetic analysis are important to understand the spread of CHIKV during epidemics and the diversity of circulating strains for the prediction of effective control measures. Molecular epidemiologic analysis of CHIKV is necessary to understand the complex interaction of vectors, hosts and environment that influences the genotypic evolution of epidemic strains. In this study, different works published during 1950s to 2020 concerning CHIKV evolution, epidemiology, vectors, phylogeny, and clinical outcomes were analyzed. Outbreaks of CHIKV have been reported from Bangladesh, Bhutan, India, Pakistan, Sri Lanka, Nepal, and Maldives in South Asia during 2007–2020. Three lineages- Asian, East/Central/South African (ECSA), and Indian Ocean Lineage (IOL) are circulating in South Asia. Lineage, ECSA and IOL became predominant over Asian lineage in South Asian countries during 2011–2020 epidemics. Further, the mutant E1-A226V is circulating in abundance with Aedes albopictus in India, Bangladesh, Nepal, and Bhutan. CHIKV is underestimated as clinical symptoms of CHIKV infection merges with the symptoms of dengue fever in South Asia. Failure to inhibit vector mediated transmission and predict epidemics of CHIKV increase the risk of larger global epidemics in future. To understand geographical spread of CHIKV, most of the studies focused on CHIKV outbreak, biology, pathogenesis, infection, transmission, and treatment. This updated study will reveal the collective epidemiology, evolution and phylogenies of CHIKV, supporting the necessity to investigate the circulating strains and vectors in South Asia.

Chikungunya virus is a small (∼70 nm-diameter), enveloped virus with a linear, positive strand RNA genome of ∼11.8 kilo-bases (Khan et al., 2002;Kawashima et al., 2014). The RNA genome consists of one non-translated region (NTR) at 5 , two ORFs and another non-translated region (NTR) at 3 end. Two polyproteins are encoded by two major open reading frames (ORFs) in CHIKV (Simizu et al., 1984;Schlesinger and Schlesinger, 1986). The positive-sense 5 two-third RNA genome directly encodes a polyprotein containing four non-structural proteins (nsP1-4). The structural proteins are encoded by 3 one-third of the genome (Simizu et al., 1984). The structural polyprotein converts into a capsid protein, two major envelope surface glycoproteins (E1 and E2) as well as two small peptides, E3 and 6K (Simizu et al., 1984).
Transmission of CHIKV involves two major cycles depending on the region of circulation. In African regions, CHIKV circulates mainly in a sylvatic/enzootic cycle involving forest dwelling mosquitoes and non-human primates (NHP) (Silva et al., 2018;Simo et al., 2019). The viruses rely on NHP as reservoir (e.g., monkeys and other vertebrates) hosts during inter-epidemic periods and transmitted by Aedes (e.g., Aedes furcifer and Aedes africanus) mosquitoes from reservoirs to human during epidemic (Jupp et al., 1988;Jupp and McIntosh, 1990;Pialoux et al., 2007;Powers and Logue, 2007;LaBeaud et al., 2011). On the contrary in urban cycles, a mosquito to (and from) human transmission is maintained. The urban cycle of CHIKV has been associated with several large epidemics of CHIKV across different continents including Asia, Europe, and North America (Weaver and Lecuit, 2015;Silva et al., 2018). Two significant species of mosquitoes namely, Aedes aegypti and Aedes albopictus (the "tiger" mosquito) are mainly involved in urban transmission of the disease (Soekiman, 1987;McGill, 1995;Reiter et al., 2006;Weaver and Lecuit, 2015;Wahid et al., 2017;Silva et al., 2018;Vairo et al., 2019). In temperate climates, Ae. albopictus mosquitoes thrive in high density (Soekiman, 1987;Reiter et al., 2006). Ae. albopictus are expanding into and adapting to new areas with the potential transmission capability of CHIKV and have been involved in recent epidemics in Asia (Soekiman et al., 1986a,b;Soekiman, 1987;Reiter et al., 2006;Weaver and Lecuit, 2015).
Persistent and larger epidemics of CHIKV infecting millions of people have been reported from Asia, but limited numbers of epidemiological research has been undertaken (Powers and Logue, 2007;Wahid et al., 2017;Vairo et al., 2019). South Asian regions are endemic for CHIKV epidemics. Continuous surveillance including phylogenetic, evolutionary, and epidemiologic analyses are required in these endemic regions to catch up the contemporary changes in the virus for developing effective diagnostics, treatments, and vaccines (Myers and Carey, 1967;Edwards et al., 2016;Rodriguez-Morales et al., 2016;Ng et al., 2018). In this review, updated epidemiology, evolution and phylogenomics of CHIKV in South Asia during 2004-2020 have been evaluated. Clinical features of chikungunya fever, transmission in temperate and tropical regions and the laboratory testing for the disease are also described in this study. Particularly, this study focuses on the recent trends of CHIKV epidemic in South Asia to create an integrated baseline for future studies.

MOLECULAR EPIDEMIOLOGY, PHYLOGENY AND EVOLUTION OF CHIKUNGUNYA VIRUS IN SOUTH ASIA AND REST OF THE WORLD
Starting from Africa, CHIKV has been transmitted globally. Recently, CHIKV infection has been detected from different countries on all continents, except Antarctica. Retrospective case studies have suggested that CHIKV epidemics have occurred during 1760s (Ross, 1956;Jupp et al., 1988). During early epidemics, they were inaccurately documented as dengue virus infection (Brighton et al., 1983;Silva and Dermody, 2017). CHIKV was first isolated and characterized from the serum of an infected patient with dengue like symptoms in Tanzania during 1952to 1953(Casals and Whitman, 1957Johnson et al., 1977). CHIKV was detected in South Asia in a short time after identification in East Africa (1952) (Ross, 1956;Powers and Logue, 2007;Silva and Dermody, 2017;Wahid et al., 2017;Simo et al., 2019). After the first identification, local and occasional outbreaks of CHIKV were recorded for the following ∼50 years before 2004 in many countries in Asia (Mason and Haddow, 1957;Peyrefitte et al., 2007;Silva and Dermody, 2017;Simo et al., 2019).
East-, Central-and South African lineage is considered as the ancestor and has circulated in South Asia from Africa at early 1960s. The second confirmed lineage, AUL, was first detected in outbreaks in Asian countries (Thailand, India, Cambodia, Vietnam, Malaysia, Taiwan, Myanmar, and Indonesia) during 1958 to 1973 and named as Asian lineage (Powers and Logue, 2007;Presti et al., 2016). Another distinct lineage called Indian Ocean Lineage (IOL) evolved from ECSA lineage Presti et al., 2016;Silva and Dermody, 2017;Pyke et al., 2018) was detected after 2004. Phylogenetic and mutational analysis revealed the presence of Asian lineage, IOL and Africa and Asia lineages in South Asian countries in recent time (Wahid et al., 2017;Deeba et al., 2020). Asian lineage was the most predominant during 1960s to 2000 in South Asian and South East Asian countries (Yadav et al., 2003;Powers and Logue, 2007;Wimalasiri-Yapa et al., 2019). After 2004, the IOL transmitted rapidly in the South Asian countries. After 2005 outbreaks, IOL lineage has been reported from most of the outbreaks (80%) in South Asian countries (Powers and Logue, 2007;Wimalasiri-Yapa et al., 2019). Since 2005, outbreak associated with IOL lineage have emerged and reemerged every year in South Asian countries (Wimalasiri-Yapa et al., 2019;Phadungsombat et al., 2020). Besides, ESCA, AUL, and AAL lineages are circulating in South Asia in a low frequency after 2005 outbreaks. Evolutionary analysis has revealed that during 2010 to 2020, outbreaks in Bangladesh, Bhutan, India, Pakistan, and Sri Lanka have been associated with ECSA-IOL lineage (Powers and Logue, 2007;Wahid et al., 2017;Melan et al., 2018;Deeba et al., 2020). During 2017 outbreaks in Bangladesh, only the ECSA-IOL lineage was reported (Supplementary Table 1). Other countries of South East Asia, namely, Bhutan, Myanmar, and Vietnam had reported the presence of IOL lineage during recent outbreaks (Powers and Logue, 2007;Staples et al., 2009;Pyke et al., 2018;Wimalasiri-Yapa et al., 2019).
Molecular evolutionary analysis confirmed the divergence of lineages from each other in previous studies and Nextstrain project and recently published phylogenies and evolutionary analysis (de Bernardi Schneider et al., 2019;Deeba et al., 2020;Nextstrain, 2021;Spicher et al., 2021). The single nucleotide variants of CHIKV can change the stability and fold of locally stable RNA structures. Besides, the 3 untranslated regions of CHIKV was found to contain non-structural RNA elements and evolutionary conserved regions (de Bernardi Schneider et al., 2019;Deeba et al., 2020;Spicher et al., 2021). Difference among lineages and origin of one lineage from other can be traced by analyzing duplication events and changes of architecture in 3 UTR (de Bernardi Schneider et al., 2019). An estimation of average evolutionary divergence over sequence pairs within CHIKV lineages was calculated in previous studies by following the maximum likelihood model (de Bernardi Schneider et al., 2019). The number of base substitutions per site was expressed from averaging over all sequence pairs within each group and found that AUL was most divergent (substitution per site was 0.0128) followed by MAL (0.0107)  (Nextstrain, 2021). Source: https://nextstrain.org/community/ViennaRNA/CHIKV. and WA (0.0102), while SAL was least divergent (0.003). In four countries, the available 157 whole genome of CHIKV in Nextstrain (India-88, Bangladesh-40, Sri Lanka-22, and Pakistan-7) had an estimated 2.63e −4 substitution per site per year (Figures 3A-C). The number of mutations including substitutions are higher in CHIKVgp1 within 5,000 bases to 6,000 bases position, while in CHIKVgp2 the frequency of mutation is about 1.9 per position within 8,500 bases to 11,000 bases.

TRANSMISSION OF CHIKV
Chikungunya virus is transmitted in humans by infected mosquitoes . CHIKV is an enzootic virus in tropical regions of Africa and Asia (Paul and Singh, 1968;Mourya, 1987;Silva et al., 2018). Emergence and reemergence of CHIKV is significantly regulated by the transmission of the virus through vectors. To understand the reemergence potential in South Asia, this review covered the transmission of CHIKV. Generally, an uninfected mosquito takes in CHIKV from infected viremic person during ingesting the blood (Mourya and Banerjee, 1987;Monteiro et al., 2019). The virus is replicated inside the mosquito midgut. When CHIKV carrying mosquitoes bite a healthy individual, the virus is transmitted inside his/her body (Silva et al., 2018). The virus also replicates inside newly infected person body (Silva and Dermody, 2017). If another uninfected mosquito bite the newly infected person after he has become viremic, the mosquito will take in CHIKV and start another cycle (Silva et al., 2018;Onyango et al., 2020). The complete transmission cycle from human to mosquito and back to humans can be completed within a week . Mosquitoes can act as vectors of CHIKV. Both vertical and horizontal transmissions of the virus can occurred in mosquitoes (Mavale et al., 2010;Jain et al., 2016). For successful transmission from arthropod vectors to a human, CHIKV must replicate inside the vectors and reach the salivary glands within 1 week (Lim et al., 2018).
Transmission of CHIKV is maintained by sylvatic cycle in the African and urban cycle in the Asian regions (Jupp and McIntosh, 1990;Staples et al., 2009;Monteiro et al., 2019). In South Asia, the urban mosquito Ae. aegypti and Ae. albopictus have been reported to be the most significant vector (Soekiman et al., 1986a,b;Banerjee et al., 1988;Mourya et al., 1994;Diallo et al., 1999;Scolari et al., 2019). Regional large outbreaks in South Asia are caused by these urban and peridomestic mosquitoes (Soekiman et al., 1986a,b;Scolari et al., 2019;Wimalasiri-Yapa et al., 2019). Ae. albopictus have a great adaption capacity in new ecological niches, as a result it can expand its enzootic range globally (Silva et al., 2018). In urban cycles in South Asia, the onset of epidemics are dependent on environmental factors, viral genetics, mosquito ecology, human behavior, and presence of competent vectors (Soekiman et al., 1986a,b). During the 2005-2006 Indian Ocean Islands epidemic, a substitution point mutation originated at position 226 in the E1 glycoprotein (outer membrane protein) of CHIKV, replacing an Alanine to Valine (Weaver and Lecuit, 2015;Silva et al., 2018). This mutation in ECSA genotype of CHIKV enhanced the vector specificity and epidemic potential of CHIKV (Kumar et al., 2008). The new mutants of CHIKV namely, IOL of ECSA genotype became capable of surviving in and transmitting by Ae. albopictus (Tsetsarkin et al., 2016). The E1-A226V substitution increases viral infectivity in Ae. albopictus midgut cells without compromising viral replication. This mutant strain initiated autochthonous cases of CHIKV more rapidly through Ae. albopictus in South Asian countries (Soekiman et al., 1986a,b;Vazeille et al., 2007;Scolari et al., 2019). Further, Ae. furcifertaylori is the main group of vectors detected during epidemics associated with sylvatic cycle (Jupp and McIntosh, 1990;Scolari et al., 2019). Ae. furcifer, Aedes taylori, Aedes luteocephalus, Ae. africanus, and Aedes neoafricanus are the major species of vectors involved in sylvatic cycles for many years (Mourya and Banerjee, 1987;Jupp and McIntosh, 1990;Monteiro et al., 2019;Scolari et al., 2019). Numerous field and laboratory works have been undertaken on roles of mosquito vectors in the transmission of CHIKV, but less is known about the importance of vertebrate hosts in viral maintenance (Powers and Logue, 2007;Silva et al., 2018). Laboratory animal studies and serosurveys confirmed the presence of CHIKV specific antibodies in potential vertebrate reservoirs. Significant levels of antibody against CHIKV have been detected in wild non-human primates (Silva et al., 2018).
Vectors and vertebrates have significant roles in interepidemic periods both in the sylvatic and urban transmission. In sylvatic cycles, non-human primate (NHP) species including Guinea baboons, Chacma baboons, African green monkeys, patas monkeys, red-tail monkeys, guenons, bushbabies, and mandrills may have significant roles as amplifiers hosts or reservoirs of CHIKV (Silva et al., 2018). On the contrary, in urban cycle, the mosquitoes play main roles probably by trans-ovarian (vertical transmission) cycles (Silva et al., 2018).

CLINICAL FEATURES OF PATIENTS INFECTED WITH CHIKV
To understand the complete epidemiological prospects of CHIKV burden in South Asian countries, studies on the clinical manifestations in patients infected with CHIKV are required. Generally, the incubation period of CHIKV in human ranges from 3 days to 7 days (Munasinghe et al., 1966;Powers and Logue, 2007;Staples et al., 2009;Onyango et al., 2020). Most of the studies on CHIKV infection clinical presentation reported that about 70-93% of the patients develop symptoms, 3-25% seropositive patients may be asymptomatic, and 2-7% patients may develop atypical symptoms (Powers and Logue, 2007;Staples et al., 2009;Silva et al., 2018;Suhrbier, 2019;Wimalasiri-Yapa et al., 2019). The most reported triad of clinical signs and symptoms for CHIKV infection from documented epidemics and outbreaks includes fever, arthralgia (joint pain), and a rash (itchy rash) (Munasinghe et al., 1966;Riswari et al., 2016). Most of the time the triad is accompanied by other symptoms of the CHIKV infection. Generally, epidemics of CHIKV infection result in two clinical outcomes of illness including the acute phase and chronic phase (Silva et al., 2018;Suhrbier, 2019). In most of the cases, fever accompany with the joint pain and rash. Rash is reported from 50 to 60% cases (Silva and Dermody, 2017). The non-itchy rash becomes visible during 2-5 days of post-infection (Powers and Logue, 2007;Silva et al., 2018). After fever, the most significant clinical presentation of CHIKV infection is the severe joint pain (arthralgia) (Powers and Logue, 2007;Suhrbier, 2019). Arthralgia is reported from about 90 to 98% of CHIKV cases (Brighton and Simson, 1984;Silva and Dermody, 2017). Besides triad, weakness, malaise, headache, chills, retro-orbital pain, photophobia, lumbar back pain, conjunctivitis, pharyngitis, lymphadenopathy and myalgia are other common symptoms reported with CHIKV infection ( Table 1; Silva and Dermody, 2017;Silva et al., 2018;Suhrbier, 2019;Wimalasiri-Yapa et al., 2019). Most infections completely resolve within weeks or months but there have been documented cases of CHIKV-induced arthralgia persisting for several years with up to 12% of patients with CHIKV disease developing chronic joint problems (Powers and Logue, 2007;Silva et al., 2018). A comparison of clinical manifestations associated with CHIKV in South Asia and rest of the world is presented in Figure 4.

THE RISK OF CHIKV EPIDEMICS IN SOUTH ASIA IN FUTURE
The previous epidemiological studies support for a larger outbreak of CHIKV in South Asian countries in future. Due to the cyclic nature of CHIKV infection, the epidemics reappear in every 3-4 years in the endemic regions. Further, high density of vectors and co-circulation of CHIKV-DENV during the same seasons are the major concerns in South Asian countries (Thaung et al., 1975;Furuya-Kanamori et al., 2016;Villamil-Gómez et al., 2016). There are various monitoring systems for CHIKV in South Asian countries, but they are only applied during epidemics. As a result, the real disease burden of CHIKV in South Asia still remains underrated. Further, the number of existing genotypic characterization of CHIKV is not enough to point out the diversity in South Asia. The evolutionary and epidemiologic analysis in this article supports for a severe and prolonged epidemics of CHIKV in south Asian countries in near future. To manage such an epidemic in future, this study suggests to conduct routine genotypic surveillance and genomic characterizations of CHIKV to assess the actual diversity of the virus in South Asia. Further, to mitigate the risk of larger epidemic, integrated approaches including epidemiologic characterizations, vector surveillance, evolutionary analysis and effective routine diagnosis are required to reduce the risk of future outbreaks and associated health burden.

CONCLUSION
In conclusion, this study finds that CHIKV has become a consistent health burden in South Asia. Tropical regions of South Asia and South America have been the main focal point of CHIKV transmission after 2007. Recently, larger epidemics of CHIKV involving millions of people have been reported from India, Bangladesh, Nepal, Bhutan, and Pakistan. Three lineages of CHIKV namely, Asian, ECSA and IOL are circulating in South Asia during 2011-2020. After 2011, ECSA lineage and IOL lineage of genotype ECSA has become predominant in South Asian countries. Prevalence of E1-A226V mutants and density of vectors namely, Aedes aegypti and Aedes albopictus remain high in South Asian countries. This study provides a comprehensive analysis on the updated phylogenomic, evolution and molecular epidemiology of CHIKV in South Asian countries, which will not only provide exact scenario of CHIKV but also help in developing better treatment, diagnosis and preventive measures. Further, this study adds integrated knowledge on recent diagnosis, clinical characteristics and transmission of CHIKV in South Asia. The rapid spread of CHIKV in recent years urges the utmost need to take control measures, as well as to search for options to develop vaccines. In future, more studies focusing the molecular characterizations and evolution of CHIKV, as well as vectorpathogen interaction should be conducted to understand the CHIKV infection in depth. This study will work as an updated database for future studies focusing molecular epidemiology, evolution, phylogeny, diagnosis, vaccine development and prevention of CHIKV in South Asia.

AUTHOR CONTRIBUTIONS
NS performed the systematic and data collection, provided with the illustrations, and was a major contributor in writing the manuscript. MS performed the data analysis and was a major contributor in revising the manuscript. RF performed the data analysis and was a minor contributor in revising the manuscript. SA performed the data minor analysis. MB performed the minor revision. AT performed the critical evaluation and verification of the manuscript. MZ was a major contributor in revising the manuscript. SD conceptualized the review article and provided oversight, critical evaluation, and verification of the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
The authors wish to thank the faculty members of Department of Microbiology, Jahangirnagar University.