International Journal of Infectious Diseases 113 (2021) 65–73 Contents lists available at ScienceDirect International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid Chikungunya viruses containing the A226V mutation detected retrospectively in Cameroon form a new geographical subclade Bright Agbodzia , ∗, Francine Berlange Sado Yousseub , Fredy Brice Nemg Simob, Selassie Kumordjiea, Clara Yeboaha, Mba-Tihssommah Mosorea, Ronald E. Bentil a, Karla Prietoc , Sophie M. Colstond, Naiki Attrama , Shirley Nimo-Paintsil a , Anne T. Foxa , Joseph H.K. Bonneye, William Ampofoe, Heather G. Coatsworth f, Rhoel R. Dinglasanf, David M. a c b Wolfe , Michael R. Wiley , Maurice Demanou , Andrew G. Letiziaa a US Naval Medical Research Unit – No. 3, Ghana Detachment, Accra, Ghana b Virology Department, Centre Pasteur, Yaoundé, Cameroon c Department of Environmental, Occupational, and Agricultural Health, University of Nebraska Medical Center, Omaha, Nebraska, USA d Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington DC, USA e Virology Department, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana f University of Florida Emerging Pathogens Institute, Gainesville, Florida, USA a r t i c l e i n f o a b s t r a c t Article history: Background: Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with sporadic outbreaks Received 8 June 2021 in Cameroon since 2006. Viral whole genomes were generated to analyze the origins of evolutionary lin- Revised 31 August 2021 eages, the potential of emergence/re-emergence, and to infer transmission dynamics of recent Cameroon Accepted 22 September 2021 CHIKV outbreak strains. Methods: Samples collected between 2016 and 2019 during CHIKV outbreaks in Cameroon were screened KEYWORDS: for CHIKV using reverse transcription PCR (RT-PCR), followed by whole genome sequencing of positive Chikungunya virus (CHIKV) samples. Cameroon Results: Three coding-complete CHIKV genomes were obtained from samples, which belong to an emerg- New Central African Clade (nCAC) ing sub-lineage of the East/Central/South African genotype and formed a monophyletic taxon with pre- E1-A226V vious Central African strains. This clade, which we have named the new Central African clade, appears Aedes albopictus to be evolving at 3.0 × 10−4 nucleotide substitutions per site per year (95% highest posterior density (HPD) interval of 1.94 × 10−4 to 4.1 × 10−4 ). Notably, mutations in the envelope proteins (E1-A226V, E2- L210Q, and E2-I211T), which are known to enhance CHIKV adaptability and infectious potential in Aedes albopictus , were present in all strains and mapped to established high-density Ae. albopictus populations. Conclusions: These new CHIKV strains constitute a conserved genomic pool of an emerging sub-lineage, reflecting a putative vector host adaptation to Ae. albopictus , which has practically displaced Aedes aegypti from select regions of Cameroon. © 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) 1 v d a t s 2 s G A h 1 l . Introduction Arthropod-borne viral infections are a major public health hreat and continue to cause outbreaks worldwide ( Mayer et al., 017 ; Wilder-Smith et al., 2017 ). These infections are caused by ( ∗ aCorresponding author: Bright Agbodzi, US Naval Medical Research Unit – No. 3, r hana Detachment, Accra, Ghana. E-mail addresses: bagbodzi@noguchi.ug.edu.gh , bright.agbodzi@gmail.com (B. q gbodzi). ttps://doi.org/10.1016/j.ijid.2021.09.058 201-9712/© 2021 The Author(s). Published by Elsevier Ltd on behalf of International Soc icense ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) iruses mainly from the Peribunyaviridae, Flaviviridae , and Togaviri- ae families ( Gubler, 2001 ; Hughes et al., 2020 ). Hematophagous rthropods, primarily mosquitoes, are the principal vectors re- ponsible for the transmission of medically important viruses uch as Zika, West Nile, chikungunya, yellow fever, and dengue Gould et al., 2017 ; Gubler, 2001 ). The transmission dynamics nd epidemiology of these viruses are poorly understood in less esourced and endemic regions like sub-Saharan Africa. Conse- uently, critical data are lacking to inform public health decisions iety for Infectious Diseases. This is an open access article under the CC BY-NC-ND B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 t 2 2 R p a b l a E N S i C fi a o s a w T f d c S m T a a m f e B t c 2 d ( T M t s t p m ( a A f P p s b s g b 2 a y P 2 t 2 i G C d H A t c I i e 2 b t ( a o i U g o p 2 d w t r i w C t ( p 2 g P 2 s v b t b B i w o h to curb arboviral spread in Africa ( Braack et al., 2018 ; Gould et al., 017 ). Chikungunya virus (CHIKV) is a positive-sense, single-stranded NA Alphavirus belonging to the family Togaviridae . CHIKV has genome size of approximately 11.8 kb and has been evo- utionarily classified into three genotypes: West African (WA), ast/Central/South African (ECSA), and Asian ( Rahman et al., 2019 ; chuffenecker et al., 2006 ). However, between 2005 and 2006, HIKV outbreaks in the Indian Ocean region caused by CHIKV with n alanine to valine mutation in the envelope protein gene at po- ition 226 (E1-A226V) gave rise to the Indian Ocean Lineage (IOL), sub-lineage of ECSA ( Weaver, 2014 ; Schuffenecker et al., 2006 ). his virus has caused several outbreaks in Africa, Asia, Europe, In- ian/Pacific Oceans, and the Americas ( Petersen and Powers, 2016 ; taples et al., 2009 ). Transmitted primarily by the mosquito vectors Aedes aegypti nd Aedes albopictus, CHIKV infection often presents with only ild fever and a rash in humans and is usually not fatal. How- ver, CHIKV infection can be associated with chronic morbidity due o debilitating polyarthralgia ( Gubler, 1998 ). Additionally, it has re- ently been linked to numerous other complications including car- iomyopathies, as well as autoimmune and neurological diseases Alvarez et al., 2017 ; Bonifay et al., 2018 ; Pinheiro et al., 2016 ; anay, 2017 ). Currently, there are no licensed antiviral therapies to reat CHIKV infection ( Chang et al., 2014 ; Erasmus et al., 2016 ) and here is no licensed vaccine. CHIKV first posed a problem to US ilitary members in Thailand from 1962 to 1964, then in Vietnam, nd more recently in Puerto Rico and other Western countries rom 2010 to 2016 ( Frickmann and Herchenröder, 2019 ). Troops de- loyed to austere environments for extended periods are suscepti- le to CHIKV, highlighting the need to better understand regional enomes of the pathogen to inform mitigation strategies. The de- ilitating impact of CHIKV on deployed forces degrades readiness nd force health protection in tropical environments. Cameroon, a region endemic for arboviruses, has a history of ellow fever, dengue, and chikungunya outbreaks ( Demanou et al., 014 ; Fokam et al., 2010 ; Nemg Simo et al., 2019 ; Peyrefitte et al., 007 ; Vicens et al., 1993 ; Yousseu et al., 2018 ). The southern uinean subequatorial region of the country provides an ideal wet- ense tropical ecology for sylvatic and urban populations of both e. aegypti and Ae. albopictus . Several seroprevalence and molecular haracterization studies have been conducted on CHIKV outbreaks n Cameroon since 2006 ( Demanou et al., 2010 ; Demanou et al., 015 ; Kuniholm et al., 2006 ; Peyrefitte et al., 2007 ). However, hese approaches are limited in their ability to completely char- cterize the viruses, resolve the evolutionary history of circulat- ng strains, and inform public health policy. Recent viral whole enome sequencing (WGS) efforts have revealed the emergence f evolutionarily distinct sub-lineages within the ECSA genotype, rompting the need for constant genomic surveillance beyond tra- itional molecular or serological diagnostics. In this study, viral hole genomes were generated to analyze the origins of evolu- ionary lineages, the potential of emergence/re-emergence, and to nfer transmission dynamics of recent CHIKV outbreak strains in ameroon. . Methodology .1. Ethical review This non-human subject study was reviewed and approved by he Naval Medical Research Center (NMRC) Institutional Review oard (IRB), approval number NAMRU3.PJT.19.01, in compliance ith all applicable Federal regulations governing the protection of uman subjects. 66 .2. Samples This retrospective study used de-identified human plasma sam- les originally collected as part of dengue and chikungunya out- reak responses in Cameroon. The samples were stored at −80 °C t the Centre Pasteur Cameroon in Yaoundé prior to WGS at the oguchi Memorial Institute for Medical Research (NMIMR) located n Accra, Ghana. Samples sent to NMIMR lacked personally identi- able information, and instead samples were identified using lab- ratory identification codes. Samples were collected from individuals in all age categories ho met the World Health Organization definition for CHIKV in- ection. Samples were collected as part of sentinel site surveillance, linical case evaluation, and outbreak investigations occurring at edical facilities throughout Cameroon between 2016 and 2019. he specific facilities were Medico-Social Health Center of Yaoundé nd Mfou District Hospital in the Centre region, community-based acilities and Londji Health Center in the South region, and New ell District Hospital in the Littoral region. .3. PCR detection of chikungunya virus Viral RNA extraction was done using the QIAamp Viral RNA ini Kit (Qiagen, Hilden, Germany) following manufacturer’s in- tructions. CHIKV detection and data interpretation/validation were er the United States Centers for Disease Control and Prevention US CDC) Trioplex real-time RT-PCR protocol ( CDC, 2017 ) on an BI 7500 system using the SuperScript III Platinum One-step qRT- CR Enzyme Kit (Invitrogen, Waltham, MA, USA). The PCR-positive amples were selected for whole genome library preparation and equencing. .4. Whole genome sequencing (WGS) Sequencing libraries were prepared using the KAPA Hyper rep Kit (Kapa Biosystems, Wilmington, MA, USA) according to he manufacturer’s instructions. Viral enrichment was done us- ng custom target capture probes (Twist Bioscience, San Francisco, A, USA). In brief, extracted RNA was fragmented, spiked with ELA RNA to increase ligation efficiency, and reverse-transcribed o cDNA. Dual indexing of cDNA libraries was achieved using DT unique dual indexes (IDT, Coralville, IA, USA). Libraries were nriched using the 1-plex pooling strategy described previously y quartering the amount of reagents for the enrichment step Blackley et al., 2016 ). Barcoded pooled libraries were sequenced n an Illumina MiSeq with v3 reagent kits (Illumina, San Diego, CA, SA). .5. Whole genome sequencing analysis Illumina sequence adaptors were removed from the sequencing eads using Cutadapt v1.9 (dev1), and low-quality reads or bases ere filtered using Prinseq-lite v0.20.3. Reads were aligned to he non-redundant National Center for Biotechnology Information NCBI) database using BLAST. Viral hits were screened for appro- riate references and viral reads were aligned to the reference enome using Bowtie2 v2.0.6. Duplicates were removed with icard ( http://broadinstitute.github.io/picard ), and a new consen- us sequence was generated using a combination of Samtools 0.1.18 and custom scripts ( https://github.com/jtladner/Scripts/ lob/master/reference _b ased _a ssembly/consensus _f asta.py ). Only ases with Phred quality scores ≥20 were used in consensus call- ng, and a minimum of three times read-depth coverage, in support f the consensus, was required to make a call; positions lacking his depth of coverage were treated as missing (that is, called as B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 ‘ 2 u D t f 2 d j K o a c ( C d s e 3 w m T i p D t l p S l a u M a 3 b i ( a s g s ( A 2 w t f t ( a i W p e F w m a w t p v s l C p T u t g a M p t s / s s t w t d i T a ( 3 C c A g o A n C m c t a s v a o u 1 i c t 1 i r d aN’). The final consensus sequence was submitted for annotation sing the VIGOR4 Genome Annotation tool at the Virus Pathogen atabase and Analysis Resource (ViPR) ( https://www.viprbrc.org/ ). .6. Phylogenetic analysis The sequences from the current study were submitted to the nline Genome Detective virus tool ( https://www.genomedetective. om/ ) for genotyping. The strains were aligned to complete HIKV genomic sequences retrieved from NCBI. The sequences elected for phylogenetic analysis covered the three major lin- ages of CHIKV. Genome alignments and phylogenetic construction ere conducted using MUSCLE ( https://www.ebi.ac.uk/Tools/msa/ uscle/ ) and Mega X software ( Kumar et al., 2018 ), respectively. o correct for the effects of ambiguous alignments due to polymor- hisms in ′ ′ the 5 and 3 untranslated regions, the sequences were rimmed to the open reading frames (ORFs), and all subsequent hylogenetic analyses were conducted on the ORFs. A maximum ikelihood phylogenetic analysis was conducted on the sequences sing the GTR + G4 nucleotide substitution model predicted by odelFinder ( Kalyaanamoorthy et al., 2017 ) as the best fit model nd implemented in IQ-TREE 1.6.11 ( http://www.iqtree.org ). The ro- ustness of each node of the phylogenetic tree was ascertained us- ng the bootstrap method with 10 0 0 replicates. To investigate the origins and better understand the evolution- ry dynamics of the sequenced strains, a Bayesian inference phylo- enetic approach based on the Bayesian Markov chain Monte Carlo MCMC) method was implemented in BEAST v1.10.4 ( Suchard et al., 018 ) to determine the time of the most recent common ances- or (tMRCA) of study strains and the evolutionary rates of geno- ypes/clades. A total of 70 CHIKV genomes were used in this nalysis, comprising 38 ECSA (18 African, 20 IOL), 17 Asian, 12 est African, and three strains from the current study. The pres- nce of temporal signal in the complete and subdivided datasets as determined by performing a root-to-tip genetic divergence nd sampling date correlation analysis using maximum-likelihood rees generated as described above and implemented in TempEst 1.5.3 ( Rambaut et al., 2016 ). For each dataset, the strict molecu- ar clock model and the coalescence constant tree prior were im- lemented (as convergence was not achieved for many parameters sing the relaxed clock model, even after more than 100 million enerations). The analysis was computed for at least 30 million CMC steps for each dataset, sampling trees at every 10 0 0 genera- ions. Parameter convergence was inspected in TRACER v1.7.1 ( http: /tree.bio.ed.ac.uk/software/tracer/ ) to achieve an effective sample ize (ESS > 200). A maximum clade credibility (MCC) phylogeny as computed from the posterior distributions of the complete ataset, excluding 10% as burn-in using TreeAnnotator (via BEAST). ree visualization and annotation was done with FigTree v1.4.4 http://tree.bio.ed.ac.uk/software/figtree/ ). To visualize how the sequenced strains fit into the African HIKV landscape, a maximum likelihood phylogenetic tree was reated with all available CHIKV partial E1 sequences of frican origin from NCBI, using a partial E1 sequence from an ’nyong’nyong virus (NCBI accession AF079456.1 ) as an exter- al outgroup. Akaike Information Criterion (AIC)-based SMS (smart odel selection) ( Lefort and Longueville, 2017 ) was used to de- ermine the model of best fit. Initial tree(s) for the heuristic earch were obtained automatically by applying Neighbor-Joining nd BioNJ algorithms to a matrix of pairwise distances estimated sing the maximum composite likelihood (MCL) approach. A max- mum likelihood tree using a general time reversible model was hen created in Mega X with a discrete gamma distribution and nvariable sites (GTR + G + I). Tree visualization and annotation was one with FigTree v1.4.4. 67 .7. Mosquito prevalence and insecticide resistance To gain a better perspective on the vector environment where hese sequenced strains were collected, CHIKV vector prevalence or Ae. aegypti and Ae. albopictus alongside insecticide resistance ata were mined from Tedjou et al. ( Tedjou et al., 2020; Ted- ou and Armel, 2019 ) and Kamgang et al. ( Kamgang et al., 2011 ; amgang et al., 2017 ), respectively. These data were presented longside population density data from the 2005 Cameroon census https://microdata.worldbank.org/ ) and visualized on region and istrict-specific maps created in ArcGIS (v10.7.1). . Results Three complete CHIKV genomes were obtained after sequenc- ng. All three strains belong to the ECSA genotype ( Table 1 ). etails of genome structure and organization, as well molecu- ar fingerprints in non-structural proteins (NSPs) are presented in upplementary Material File 1. The opal stop codon between nsP3 nd nsP4 was intact for all three genomes. .1. Structural protein molecular fingerprints In the structural polyproteins, the E1 envelope protein segment 439 amino acids (aa)) demonstrated 10 aa substitutions that were hared among all of the three strains from this study. One sub- titution, K157R, was present in only CHIKV/CAM/2016/Yaoundé. mong the substitutions was the highly characterized A226V, hich has been shown to enhance Ae. albopictus competence or CHIKV and result in increased viral transmission potential Tsetsarkin et al., 2007 ). Also present were the less character- zed mutations M269V, I317V, and V322A, which have been re- orted previously in India ( Harsha et al., 2020 ; Singh et al., 2012 ). ive novel aa substitutions were also present. The E2 protein seg- ent (423 aa) had 21 aa substitutions, and 20 of these mutations ere shared by all three strains, while the Q282K mutation was resent in only the CHIKV/CAM/2018/Mfou strain. Notably, aa sub- titutions L210Q and I211T, which have been shown to enhance HIKV infectivity in Ae. albopictus , were present ( Sahu et al., 2013 ; setsarkin et al., 2009 ). The remaining 19 mutations in the E2 pro- ein have not been reported previously and could be novel. The 64 a long E3 protein showed a single substitution, I23T, which was resent in all strains. The 6K protein (61 aa) showed 2 aa sub- titutions, I54V and S60N, which were shared between all of the trains in the study. In the capsid protein (261 aa), 2 aa substi- utions (K63R and K122R) were observed and were present in all hree stains. A summary of all of the aa substitutions is presented n Table 2 , and those that are unique to the three Cameroon strains re detailed in Table 3 . .2. Phylogenetic relationships The strains recovered from this study belong to the ECSA enotype and formed a monophyletic taxon, the new Central frican Clade (nCAC), with strains collected in Congo, Gabon, and ameroon ( Fig. 1 ). The time scaled phylogeny indicates that the ommon recent ancestor of the Cameroon CHIKV strains existed round 2012, with a 95% highest posterior density (HPD) inter- al of 2010 to 2013. However, the nCAC was estimated to have riginated around January 1998, with a 95% HPD interval of April 995 to March 2000. The nCAC was evolving at 3.0 × 10−4 nu- leotide substitutions per site per year, with a 95% HPD interval of .94 × 10−4 to 4.1 × 10−4 substitutions/site/year. The substitution ates of the datasets and the root-to-tip genetic divergence results re presented in Table 4 . Partial CHIKV E1 phylogenetic analyses B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 Table 1 CHIKV sequencing and assembly results Strain Number of reads G + C content (%) Mean coverage (X) Assembled sequence length GenBank accession number CHIKV/CAM/2018/Yaoundé 734 581 50.5 8526 11 561 MT666071 CHIKV/CAM/2016/Yaoundé 1 889 829 50.4 20 932 11 562 MT666072 CHIKV/CAM/2018/Mfou 2 727 185 50.4 31 695 11 562 MT666073 Table 2 Amino acid substitutions in ORFs. AA (amino acid), n (number of strains); substitutions in bold are those that have been reported previously Non-structural polyprotein Structural polyprotein nsP1 nsP2 nsP3 nsP4 E1 E2 E3 6K Capsid AA AA AA AA AA AA AA AA AA change n change n change n change n change n change n change n change n change n 29PS 3 R34C 3 T122I 3 Q46H 3 N9S 3 G57K 3 I23T 3 I54V 3 K63R 3 D75E 3 H374Y 3 V175I 3 T101I 3 T37I 3 I74T 3 S60N 3 K122R 3 V156I 3 A604V 3 A196V 3 E119K 2 K157R 1 G79E 3 L172V 3 C642Y 3 P326S 3 K230N 3 A226V 3 V85A 3 K224N 1 Q328P 3 Q235R 1 S250P 3 S118G 3 E234K 3 V331A 3 V497A 3 M269V 3 K149R 3 M383L 3 T337A 3 Q500L 3 I317V 3 N160T 3 I384L 3 T338M 3 I514T 3 V322A 3 A164T 3 T481I 3 Q344R 3 V604I 3 K324R 3 L181M 3 P482L 1 K352E 3 G348E 3 S194G 3 A487T 3 I376T 3 V399I 3 L210Q 3 L507R 3 T378M 3 I211T 3 A382T 3 V222I 3 V399I 3 A227V 3 M449T 3 A246D 3 S462N 3 M267R 3 N483S 3 Q282K 1 S299N 3 A344T 3 I415L 3 ORF, open reading frame. Table 3 Unique amino acid substitutions in strains of the new Central African clade (nCAC) Strain Non-structural polyprotein Structural polyprotein nsP1 nsP2 nsP3 nsP4 E1 E2 E3 6K Capsid CHIKV/CAM/2018/Yaoundéa K224N R34C, T122I, A196V, Q46H, T101I, K157R, S250P, W64R, V85A, - - - A604V Q328P, S462N E119K, Q235R, I317V, K324R, L210Q, A227V, V497A G348E, V339I A246D CHIKV/CAM/2016/Yaoundéa P482L R34C, T122I, A196V, Q46H, T101I, S250P, I317V, V85A, L210Q, - - - A604V Q328P, S462N E119K, V497A K324R, G348E, A227V, A246D V339I CHIKV/CAM/2018/Mfoua - R34C, T122I, A196V, Q46H, T101V, S250P, I317V, V85A, L210Q, - - - A604V Q328P, S462N V497A K324R, G348E, A227V, A246D, V339I Q282K - - Q328P, V349A, - - A246D - CHIKV/ H.sapiens /CMR/667/2006a S462I H60R Q37R GABOPY1b - I457S T122I, S153T, T101I, E119K S250P - - - S462N R13G BRAZZA_MRS1c E508V A604V, T122I, A196V, T101I, E119K, S250P, K324R V85A, L210Q, - - - T17I, Q328L, S462N V497A A246D, T265I M557T a Cameroonian partial E1 sequences ( KJ508821.1 , KJ508819.1 (Maurice et al., 2015) and EF051584 ( Peyrefitte et al., 2007 )) were derived from sporadic CHIKV surveil- lance, stemming from febrile dengue-like symptom outbreaks. b GABOPY1 ( KP003812.1 ) from a 2007 CHIKV outbreak in Gabon. c BRAZZA_MRS1 ( KP003813.2 ) from a 2011 CHIKV outbreak in Congo. Table 4 Nucleotide substitution rates of datasets used in the molecular clock analysis and the root-to-tip genetic divergence results Substitution rate (BEAST analysis) Root-to-tip analysis (TempEst) Dataset Mean ( × 10−4 ) −4 95% HPD ( × 10 ) Correlation coefficient R2 Complete 2.15 1.98–2.32 0.57 0.33 ECSA 1.65 1.46–1.84 0.81 0.67 Old ECSA 1.20 0.99–1.41 0.90 0.81 IOL 4.49 3.54–5.50 0.70 0.48 Asian 4.20 3.66–4.74 0.99 0.99 West African 2.02 1.64–2.40 0.93 0.87 nCAC 3.00 1.94–4.10 0.99 0.99 ECSA, East/Central/South African; HPD, highest posterior density; IOL, Indian Ocean Lineage; nCAC, new Central African Clade. 68 B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 Figure 1. Maximum clade credibility (MCC) phylogeny based on the complete ORFs of 70 CHIKV strains used for molecular clock analysis. Black asterisks indicate sequences containing the A226V mutation. The MCC tree was based on the strict molecular clock model and the coalescence constant tree prior. Taxon labels include accession number/country and year of isolation for sequences extracted from GenBank, while new sequences are labeled with strain names. Posterior probabilities are present on all clades. The inset shows a summary of the maximum number of unique amino acid substitutions between each strain in each gene for the three new CHIKV strains from Cameroon described in Table 3 . Figure 2. Molecular phylogenetic analysis of 60 CHIKV partial E1 sequences. Sequences obtained in this study are denoted in bright red with three red asterisks. Taxa are represented by NCBI accession number, county(-city), and year. Each clade is separated by color and bracketed by strain (WA = West African, ECSA = East/Central/South African, IOL = Indian Ocean Lineage). Black asterisks indicate sequences containing the A226V mutation. Evolutionary history was inferred by the maximum likelihood method based on the general time reversible model with discrete gamma distribution rates among invariant sites (GTR + G + I). Bootstrap values are present on major clades. The tree is drawn to scale, with branch lengths measured by the number of substitutions per site. The accompanying map of Africa is colored by clade. ( p o r t 3 i b A e Fig. 2 ) further support this finding and suggest that there may be ther smaller clades akin to the nCAC within the ECSA group. .3. Mosquito prevalence and insecticide resistance Merged mosquito prevalence data from Tedjou et al. (Tedjou t al., 2019; Tedjou et al., 2020) show that Ae. albopictus vectors w 69 redominate in downtown and suburban areas in Yaoundé, while ural areas seem to have roughly equal Ae. aegypti and Ae. albopic- us populations. Although no published data exist for Mfou, a sim- lar prevalence trend towards Ae. albopictus can be seen in the ur- an and suburban surrounding areas of Yaoundé, Mbalmayo, and konolinga ( Fig. 3 ). Within Yaoundé, the 2018 sequenced sample as collected from Bastos in Yaoundé II, while the 2016 sample B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 Figure 3. Geographic distribution of Aedes albopictus and Aedes aegypti in Centre, Cameroon. Black borders denote administrative districts within Centre, Cameroon and are shaded based on population density gathered from the 2005 census. ‘ + ’ denotes cities with mosquito insecticide resistance data; a purple cross indicates susceptible populations, while a red cross indicates deltamethrin-, bendiocarb-, and DDT-resistant populations that were susceptible to permethrin and malathion. Data were merged from Tedjou et al., 2019 and 2020, and Kamgang et al., 2017 and 2011. Figure 4. Geographical distribution of Aedes albopictus and Aedes aegypti in Yaoundé (Centre, Cameroon). Black borders denote municipalities within Yaoundé (I through VII) and are shaded based on population density gathered from the 2005 census. ‘ + ’ denotes municipalities with mosquito insecticide resistance; a red cross indicates deltamethrin-, bendiocarb-, and DDT-resistant populations that were susceptible to permethrin and malathion. Data were merged from Tedjou et al., 2020 and Kamgang et al., 2017 . w 4 m p i a b i t e s P K as from Nkolbisson in Yaoundé VII. Vector prevalence in these unicipalities is largely municipality-specific, with an equal pro- ortion of both vectors in urban Yaoundé II and rural Yaoundé VII; n both areas, suburban environments primarily comprise Ae. al- opictus ( Fig. 4 ). Ae. aegypti and Ae. albopictus from Yaoundé appear o be deltamethrin-, bendiocarb-, and DDT-resistant, while still usceptible to permethrin and malathion ( Kamgang et al., 2011 ; amgang et al., 2017 ). 70 . Discussion The persistent re-emergence of CHIKV highlights the evolution- ry fitness and adaptability of the virus. The strains recovered n this study share the same basic genomic architecture as oth- rs isolated in Congo, Gabon, and Cameroon ( Moyen et al., 2014 ; eyrefitte et al., 2007 ). B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 t p s i c a A p s f s s e a a a g ( m t g a E o n a d u t a t i t ( A s a i b t p c g C C 2 t t t i g u h t e 3 4 t a p p D N s m i t a t j t c t t c ( t E e m s p t r n s n ( m C o g i i p G f m d o t D S s m a t D p e o A s t D s G h t a t 2 1Phylogenetic analyses were based on the complete ORFs and artial E1 segments. The former has proven to be more accurate n comparison to the latter ( Volk et al., 2010 ), but sequence avail- bility is largely skewed towards the latter, and therefore both ap- roaches were incorporated. The analyses revealed that the strains rom the current study are of the ECSA genotype. Molecular clock analysis estimates that the new sequences have xisted since around 2012. However, their mother clade (nCAC) had common ancestor around 1998, the same period as the emer- ence of the IOL outbreak strains. Although the nCAC and IOL ight have emerged around the same period, the phylogeny sug- ests that nCAC is an emerging sub-lineage of the Central African CSA harboring the E1-A226V and E2-I211T substitutions, the sig- ature genomic fingerprints of the IOL. Although, the nCAC and IOL iverged long ago, the selection mechanism resulting in the evolu- ionary convergence of these same independent mutations is likely o be the same since the vector that manifests this selective advan- age has been identified in some of the Central African outbreaks Chen et al., 2016 ). The endemicity of the nCAC to Central Africa uggests that it constitutes a conserved genomic pool with a lim- ted geographical distribution. With 99.0% nucleotide identity be- ween the strains recovered from this study and their sister taxa, it ould be suggested that this sub-clade might have originated from HIKVs that caused epidemics in Central Africa during the mid- 0 0 0s ( Zeller et al., 2016 ). The silent circulation of the nCAC con- aining the Ae. albopictus adaptive mutation for nearly two decades ndicates the need for constant surveillance in these regions and nderscores the idea ( Chen et al., 2016 ) that Central Africa con- ains rich, deeply divergent, co-circulating strains. The occurrence of 42 aa substitutions in virus NSPs, of which 0 were shared among the three strains ( Table 2 ), suggests these re stable adaptive mutations essential for viral survival and athogenicity. Unfortunately, the lack of information on CHIKV SPs means no meaningful inferences can be made about these utations. Mutations in CHIKV E1 and E2 envelope structural pro- eins have been noted to play key roles in viral evolution and host daptation ( Tsetsarkin et al., 2007 ; Tsetsarkin et al., 2009 ). A ma- or shift in the dynamics of CHIKV transmission has been the in- rease in epidemic potential and transmissibility by Ae. albopic- us , as conferred adaptive mutations enhance vector competence Schuffenecker et al., 2006 ; Tsetsarkin et al., 2007 ). Analysis of the 1 protein segment revealed that the positively selected E1-A226V utation, which has increased CHIKV fitness in Ae. albopictus , was resent in all strains recovered from the study. This finding cor- oborates the findings of earlier studies, i.e., Ae. albopictus inva- ion is a rising problem of public health concern in Cameroon Simard et al., 2005 ), which was shortly followed by the first HIKV isolation in Cameroon ( Peyrefitte et al., 2007 ). Although the enomic data of the virus were not available at the time, sequenc- ng was done later (strain: CHIKV/ Homo sapiens /CMR/6 67/200 6, enBank accession number KX262996 ). Analysis of the E1 seg- ent of CHIKV/ Homo sapiens /CMR/6 67/200 6 shows the presence f the E1-A226V mutation, suggesting that Ae. albopictus CHIKV ransmission might have happened earlier than previously thought. ubsequently, the analysis of partial E1 protein segments of CHIKV trains collected from Cameroon in 2013 identified the E1-A226V utation ( Demanou et al., 2015 ). In addition to the E1-A226V mu- ation, three other aa substitutions, which had previously been re- orted in isolates of Indian origin, were present in all of the strains f the current study. Seven remaining substitutions were unique to trains in this study. The E2 protein segment had the highest proportion of aa sub- titutions across the genomic segments. Many of these mutations ave not yet been reported, but the aa substitutions E2-L210Q nd E2-I211T have been characterized previously ( Tsetsarkin et al., 009 ; Tsetsarkin and Weaver, 2011 ). Tsetsarkin et al., 2007 showed 71 hat a synergistic effect of E1-A226V with E2-I211T is needed to ee a significant increase in Ae. albopictus midgut infectivity. This hange, however, did not have any effect on the primary vector e. aegypti ( Tsetsarkin et al., 2009 ). All strains recovered from this tudy harbored the E2-I211T substitution, suggesting that these trains would be transmitted well by Ae. albopictus . The E2-L210Q ubstitution has also been shown to increase CHIKV fitness in Ae. lbopictus ; the presence of E2-L210Q enhanced infectivity of Ae. lbopictus midgut cells but not Ae. aegypti or human cells lines Tsetsarkin and Weaver, 2011 ). Nineteen other uncharacterized mu- ations were observed. The abundance of polymorphic sites on the ntigenic E2 protein presents a great challenge to vaccine devel- pment due to the likelihood of high antigenic variation. The E3 nd 6K protein segments were somewhat conserved, showing few ncharacterized substitutions. The capsid protein, however, showed high number of substitutions, many of which were uncharacter- zed. Previous concerns regarding the rapid geographic expansion of e. albopictus in Central Africa have clearly manifested, as there is n overwhelming presence of Ae. albopictus in urban and subur- an Yaoundé and surrounding areas, displacing native Ae. aegypti opulations ( Kamgang et al., 2011 ; Kamgang et al., 2017 ). This sug- ests that there is an increased epidemic potential for CHIKV in ameroon, as Cameroonian strains (historically and herein) con- ain the E1-A226V Ae. albopictus -adaptive substitution. Amplifying his risk are previous findings that there is widespread Ae. ae- ypti and Ae. albopictus insecticide resistance in Yaoundé, which as appeared despite no known vector control efforts in these ar- as ( Kamgang et al., 2017 ). The evolutionary rate of the nCAC of .0 × 10−4 nucleotide substitutions per site per year, which is wo times that of the main ECSA, presents the nCAC with the op- ortunity for increased infectivity and pathogenicity ( Sanjuán and omingo-Calap, 2016 ); previous literature ( Caron, 2012 ) has also uggested that an increase in CHIKV co-infections could also result n increases in viral fitness. In conclusion, CHIKV strains recovered from this study belong o an emerging sub-lineage of the East/Central/South African geno- ype and had a common recent ancestor around 2012. The iden- ification of E1-A226V, E2-L210Q, and E2-I211T mutations, which onfer an adaptive advantage in the mosquito vector Ae. albopic- us , means there is likely a selective shift towards this vector for ffective dissemination of the virus in both urban and peri-urban ettings. The high evolutionary rate of the emerging clade from he ancestral taxa highlights the possibility of increased CHIKV fit- ess and pathogenicity. Furthermore, the identification of many ovel amino acid substitutions is indicative that there is an im- inent need to characterize these mutations to assess their effects n the evolutionary fitness of the pathogen. These functional stud- es combined with known mosquito prevalence data shed light on athogen adaptation and evolution and highlight the concern for urther CHIKV outbreaks in Cameroon, framing future public health ecisions. eclarations The views expressed in this article are those of the authors nd do not necessarily reflect the official policy or position of the epartment of the Navy, Department of Defense, or the US Gov- rnment. This work was supported by a grant provided by the rmed Forces Health Surveillance Division, Global Emerging Infec- ions Surveillance Branch (GEIS), ProMIS ID P0142_19_N3. NA, SNP, W, and AGL are Military Service member or employees of the US overnment. This work was prepared as part of their official du- ies. Title 17, USC, §105 provides that copyright protection under his title is not available for any work of the US Government. Title 7, USC, §101 defines a US Government work as a work prepared B. Agbodzi, F.B.S. Yousseu, F.B.N. Simo et al. International Journal of Infectious Diseases 113 (2021) 65–73 b F a G p B G e G A H S H C T H K S M K A m K S K f K R A L M B M B N B P C P C P C R R C S D S D S D S E S F Sy a Military Service member or employee of the US Government s part of that person’s official duties. Ethics statement: The study protocol NAMRU3.PJT.19.01 was ap- roved by the Naval Medical Research Center Institutional Review oard in compliance with all applicable Federal regulations gov- rning the protection of human subjects. uthor contributions Conceptualization: MRW, MD, AGL. Writing – original draft: BA. ampling: FBSY, FBNS, MD. Molecular assays: BA, FBSY, FBNS, SK, Y, M-TM, REB. Whole genome sequencing: BA, FBSY, SK, CY, M- M, REB, KP. Bioinformatics analysis and data interpretation: BA, GC, RRD, MRW. Writing – review and editing: BA, FBSY, FBNS, K, CY, M-TM, REB, SMC, NA, SN-P, ATF, JHKB, HGC, RRD, MRW, D, AGL. Coordination: NA, SN-P, ATF, JHKB, WA, DW, MD, AGL. ll authors have read and agreed to the published version of the anuscript. upplementary materials Supplementary material associated with this article can be ound, in the online version, at doi:10.1016/j.ijid.2021.09.058 . EFERENCES lvarez MF, Bolívar-Mejía A, Rodriguez-Morales AJ, Ramirez-Vallejo E. Car- diovascular involvement and manifestations of systemic Chikungunya virus infection: A systematic review. F10 0 0Research 2017;6(May):1–22. doi: 10.12688/f10 0 0research.11078.1. lackley DJ, Wiley MR, Ladner JT, Fallah M, Lo T, Gilbert ML, Gregory C, D’ambrozio J, Coulter S, Mate S, Balogun Z, Kugelman J, Nwachukwu W, Prieto K, Yeiah A, Amegashie F, Kearney B, Wisniewski M, Saindon J, … Palacios G. 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