Molecular surveillance of arboviruses circulation and co-infection throughout a big chikungunya virus outbreak in Thailand, October 2018 to February 2020

On this research, we took benefit of our earlier chikungunya cohort research carried out from October 2018 to February 2020¹³ to look at proof of ZIKV, DENV, and co-infection amongst these three pathogens utilizing a nucleic acid amplification check. RT-PCR is the popular technique of prognosis as a result of its excessive specificity, excessive sensitivity, and low cross-reactivity. We screened serum samples from our earlier chikungunya cohort research collected throughout the giant outbreak of CHIKV an infection in Thailand for DENV and ZIKV. There have been 511 CHIKV-negative serum samples and 1295 CHIKV-positive samples. A complete of 36 sufferers have been discovered to be mono-infected with DENV among the many 511 sufferers with a CHIKV-negative check end result. Equally, different research additionally recognized DENV as a big virus in sufferers with undifferentiated fever in Thailand¹⁷. Between October 2018 and February 2020, a complete of 325 circumstances of Zika fever have been reported in Thailand by the Bureau of Epidemiology, Ministry of Public Well being¹⁰. Curiously, we discovered 29 circumstances of ZIKV mono-infection, representing roughly 10% of the Zika fever circumstances reported by the Bureau of Epidemiology throughout the identical interval. These circumstances recommend the potential of underdetection and underreporting of mosquito-borne virus infections in Thailand. Our findings additionally verify the excessive price of DENV, ZIKV, and CHIKV co-circulation in Thailand.

The medical manifestations of diseases attributable to mosquito-borne viruses overlap with these of different co-circulating viruses, significantly DENV, ZIKV, and CHIKV. An infection with CHIKV, DENV, and ZIKV could also be asymptomatic, undifferentiated acute febrile sickness and self-managed; therefore, the precise numbers of an infection circumstances are more likely to go underreported^18,19,20. Though pores and skin rash is usually noticed in circumstances of DENV, ZIKV, and CHIKV an infection, we discovered that rash was extra frequent in ZIKV-confirmed sufferers than in sufferers contaminated with DENV or CHIKV. Our discovering was in step with research from Brazil²¹ and Mexico²². RT-PCR analyses indicated that DENV- and ZIKV-positive samples tended to have been collected early after onset of sickness. The median time from onset of illness to DENV and ZIKV detection was 2 (vary 1–9) days and three (vary 1–7) days, respectively (Supplementary Tables 2 and three). Different research have reported that RT-PCR is extremely delicate for detecting these viruses within the early levels of an infection^23,24.

A complete of 444 samples that have been CHIKV adverse by RT-PCR and IgM testing have been additionally RT-PCR–adverse for DENV and ZIKV. The timing of the check relative to illness stage is one issue that influences the accuracy of the prognosis. The RT-PCR assay used right here would have low sensitivity for serum samples collected late within the illness development. Destructive viral RNA detection outcomes don’t rule out a prognosis of dengue or Zika fever as a result of the viremic part of DENV and ZIKV an infection is brief^25,26. Within the center and latter levels of an infection, serological assessments are more practical than viral RNA detection. Nonetheless, we didn’t carry out serological testing or plaque discount neutralization assessments (PRNTs) because of the insufficient quantity of serum samples. We additionally assumed that DENV- and ZIKV-negative check outcomes might be as a result of fever attributable to different pathogens. A latest acute undifferentiated fever etiology research in Thailand reported that along with DENV, acute undifferentiated fever may happen with murine typhus, leptospirosis, influenza, and bacteremia¹⁷.

An additional important discovering of this research was the detection of 13 circumstances of co-infection, which comprised eight circumstances of CHIKV/DENV co-infection, three circumstances of CHIKV/ZIKV co-infection, and two circumstances of DENV/ZIKV co-infection. Some research have reported that co-infection with mosquito-borne viruses can exacerbate illness severity^27,28. Mercado-Reyes et al. described seven fatalities associated to DENV/CHIKV and CHIKV/ZIKV co-infection throughout epidemiologic monitoring of the ZIKV outbreak in Colombia between October 2015 and December 2016. Among the many co-infection mortality circumstances, loss of life was associated to neurological manifestations, sepsis, and a number of organ failure in 5 circumstances, whereas the opposite two circumstances concerned fetal loss of life²⁸. Nonetheless, of the co-infection circumstances in our research, fever was the most typical medical function for all sufferers, and no extreme signs have been noticed. Many earlier research have additionally reported that the medical options of twin infections involving CHIKV/ZIKV²⁹, CHIKV/DENV³⁰, and DENV/ZIKV³¹ should not related to worse outcomes and don’t differ considerably by way of medical options in contrast with single infections with every virus. Furthermore, one systematic assessment reported that solely 9% of ZIKV–co-infected sufferers in cohort and cross-sectional investigations had problems³².

Along with diagnostic testing, investigations of viral genetic variations are important for an infection prevention and understanding how a virus spreads is critical for additional improvement of antiviral brokers. On this research, phylogenetic analyses demonstrated that in Thailand, ZIKV belongs to the Asian genotype however varieties two principal clades. Most isolates clustered with earlier Thai and Singapore strains remoted in 2016–2017. Isolates within the different clade have been intently associated to a ZIKV pressure remoted in a Chinese language traveler getting back from Myanmar in 2019 in addition to strains reported beforehand in French Polynesia and the Americas. Our findings are much like these of a earlier research that categorized ZIKV from mosquitoes in 2016 in Thailand into two clades intently associated to the Thai pressure of 2013–2017 and the opposite clade associated to viruses recognized within the Americas³³. Primarily based on variations within the amino acid residues in three proteins−place 139 (localized in residue 17 of prM protein), 982 (188 within the NS1 protein), and 2634 (114 in NS5 protein)−the Asian lineage ZIKV isolates have been categorized into 4 main genotypes, SAM, SVM, NVM, and NVV¹⁶. Modifications in a single amino acid residue at positions 139 and 982 are reportedly related to more-severe illness and enhanced viral transmission^34,35.

ZIKV reveals a serine to asparagine substitution at place 139 (S139N) in prM, which arose throughout the ZIKV outbreak that occurred in early 2013 in French Polynesia earlier than spreading all through the Americas³⁶. Yuan et al.³⁴ reported that the S139N substitution in prM enhances ZIKV replication in human and mouse neural progenitor cells, leading to more-severe microcephaly in contaminated mouse fetuses. The A982V substitution within the NS1 protein will increase ZIKV NS1 secretion into the host circulation and considerably enhances transmission of ZIKV in mosquito vectors³⁵. Moreover, the NS1 A982V mutation enhances ZIKV replication through inhibition of interferon-β induction³⁷. A change of methionine or threonine to valine at place 2634 (M//T2634V) from the beginning codon of the genome or residue 114 within the NS5 protein is a novel molecular hallmark of all American strains remoted in 2015–2016^36,38.

In distinction, strains from Southeast Asia, the Pacific, and Africa harbor 2634M, and a ZIKV Malaysian pressure remoted in 1966 harbored 2634T³⁶. It might be hypothesized that the M2634V substitution within the American strains is chargeable for the elevated outbreak potential of the mutant virus within the Americas. Nonetheless, latest research have proven that the insignificant M2634V mutation within the NS5 protein enhances viral replication and transmission potential, indicating that the M2634V substitution is just not chargeable for enhancing the ZIKV epidemic potential^39,40. Right here, we discovered that the ZIKV Thai strains remoted in 2019–2020 have been SVM, much like most beforehand remoted Thai and different Southeast Asian strains. Though the SVM strains don’t carry the S139N mutation, which is related to elevated neurovirulence of ZIKV³⁴, it was reported that an infection with SVM ZIKV variants can result in congenital Zika syndrome and microcephaly. Nonetheless, one essential issue within the improvement of microcephaly in a fetus or toddler is the timing of ZIKV an infection throughout being pregnant. A excessive threat of microcephaly with ZIKV an infection is noticed within the first trimester⁴¹.

Amongst DENV-positive samples, all 4 serotypes have been detected on this research, indicating that every one 4 DENV serotypes co-circulated in Thailand in 2019–2020. The commonest serotype was DENV-2, in step with a 2020 report from the Bureau of Vector-Borne Illnesses, Division of Illness Management, Ministry of Public Well being, Thailand. On this research, six totally different DENV genotypes have been recognized among the many 4 serotypes. DENV-1 contains genotypes I, II, III, IV, and V⁴². All DENV-1 isolates detected on this research belonged to genotype I, which was first documented in Thailand in 1981⁴³. Since that point, DENV-1 genotype I has been routinely remoted in Thailand and several other different international locations in Southeast Asia^44,45. Curiously, our DENV-1 phylogenetic tree confirmed that earlier and current genotype I Thai strains didn’t cluster in the identical clade. Many of the Thai strains in every clade have been intently associated to genotype I strains from neighboring international locations, demonstrating intra-genotypic variation of DENV-1 genotype I strains in Thailand and a genetic hyperlink between DENV-1 circulating in Thailand and different international locations in Southeast Asia. We additionally noticed geographic growth of DENV-1 genotype I strains from Southeast Asia to East Asia on account of the shut relationship between Thai and Cambodian with DENV-1 remoted from China. A earlier report additionally confirmed that an epidemic involving DENV-1 in China was associated to contaminated Chinese language vacationers getting back from Thailand⁴⁶.

Six distinct genotypes of DENV-2 have been recognized⁴⁷. The sylvatic genotype circulates in West Africa and Southeast Asia and ceaselessly reveals enzootic cycles between forest mosquitoes and nonhuman primates. The American genotype is current in South and Central America. Over the previous three many years, the Asian/American genotype circulating within the Americas has prompted a number of epidemics in South and Central America⁴⁸. This genotype was beforehand the dominant circulating genotype in Southeast Asia, however Asian I genotype viruses started to switch the Asian/American genotype in 1992⁴⁹. The Asian I genotype of DENV-2 is ceaselessly detected in lots of Asian international locations^50,51. The Asian II genotype contains strains from Asia, amongst which the DENV-2 Cosmopolitan genotype is essentially the most prevalent globally⁵². A phylogenetic evaluation of DENV-2 isolates primarily based on envelope area sequence indicated that each the Asian I and Cosmopolitan genotypes co-circulated in Thailand throughout 2019–2020, particularly in Bangkok. Earlier research additionally discovered that each genotypes of DENV-2 co-circulated in Southern Thailand in 2015–2016⁴⁵.

5 DENV-3 genotypes (I-V) have been recognized to this point⁵³. DENV-3 genotype II was the predominant genotype in Thailand within the Nineteen Seventies⁵⁴. Displacement of genotype II by genotype III in Thailand was reported in 2008⁵⁵. DENV-3 genotype I used to be first documented in 1988⁵⁶. In 2015, DENV-3 genotype I re-emerged and co-circulated with DENV-3 genotype III in southern Thailand, however genotype III was detected extra ceaselessly than genotype I⁴⁵. In distinction, different research have reported that solely genotype III circulated in Thailand in 2016–2017⁵⁷. The current research discovered that DENV-3 genotypes I and III co-circulated in Bangkok in 2019–2020, with genotype I detected at larger frequency than genotype III. This proof suggests there was a genotypic shift in DENV-3 in Thailand. A research in Myanmar, a neighboring nation of Thailand, additionally reported co-circulation of the rising genotype I in that nation from 2017 to 2019⁵⁸. Our phylogenetic evaluation confirmed that DENV-3 genotype I is intently associated to DENV-3 strains from Myanmar, indicating the potential of the virus spreading throughout international locations. As well as, our DENV-3 genotype III fashioned two phylogenetic clusters, one intently associated to strains from Myanmar and the opposite associated to Indian strains, indicating the unfold of the virus between international locations in Southeast Asia and South Asia.

DENV-4 was not often remoted on this research. Phylogenetic evaluation revealed that the DENV-4 Thai strains remoted in 2019–2020 have been predominantly of genotype I and intently associated to the DENV-4 isolate from Myanmar and a Chinese language traveler who visited Myanmar suggesting the unfold of the virus to a close-by nation and wider geographic growth of the virus from Southeast Asia to East Asia.

In conclusion, we discovered that circumstances of dengue fever and Zika an infection have been underestimated and underreported throughout the giant CHIKV outbreak that occurred in Thailand between October 2018 and February 2020. A potential cause for this underestimation and underreporting is that infections involving CHIKV, DENV, and ZIKV could cause related however extensively various medical displays. As such, correct laboratory prognosis is crucial. We thus spotlight the co-infection and co-circulation of those three mosquito-borne viruses in Thailand. ZIKV circulation in Thailand throughout 2019–2020 was the Asian genotype divided into two clades: one group associated to earlier circulating Thai and Singaporean strains, and the opposite intently associated to viruses recognized in China, French Polynesia, and the Americas. 4 DENV serotypes have been detected, together with six genotypes consisting of 13 DENV-1 genotype I, 10 DENV-2 Asian I, 10 DENV-2 Cosmopolitan, 6 DENV-3 genotype I, 2 DENV3 genotype III, and 5 DENV-4 genotype I. Notably, a lot of the genotypes have been additionally categorized into a number of clades. Most confirmed circumstances occurred in Bangkok, the capital and most populous metropolis in Thailand. As well as, molecular characterization of the viral genomes revealed the diploma of genetic variation between ZIKV and the 4 DENV serotypes. Bangkok undoubtedly serves because the epicenter for mosquito-borne virus range in Thailand. Steady monitoring of viral genetics, along with improvement of a surveillance database, might improve our understanding of viral genotype modifications and their affiliation with epidemiology, thereby serving to forestall or management outbreaks involving these viruses.