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Chikungunya outbreak in Delhi, India, 2016: report on coinfection status and comorbid conditions in patients
Accepted Manuscript Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and co-morbid conditions in patients Navjot Kaur, Jaspreet Jain, Ankit Kumar, Moninder Narang, Mohammad Khalid Zakaria, Alessandro Marcello, Dilip Kumar, Rajni Gaind, Sujatha Sunil PII:
S2052-2975(17)30063-X
DOI:
10.1016/j.nmni.2017.07.007
Reference:
NMNI 351
To appear in:
New Microbes and New Infections
Received Date: 11 May 2017 Revised Date:
2052-2975 2052-2975
Accepted Date: 23 July 2017
Please cite this article as: Kaur N, Jain J, Kumar A, Narang M, Zakaria MK, Marcello A, Kumar D, Gaind R, Sunil S, Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and co-morbid conditions in patients, New Microbes and New Infections (2017), doi: 10.1016/ j.nmni.2017.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and comorbid conditions in patients
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Navjot Kaur1, 2, Jaspreet Jain2, Ankit Kumar2, Moninder Narang3, Mohammad Khalid Zakaria4, Alessandro Marcello4, Dilip Kumar3, Rajni Gaind1*, Sujatha Sunil2*
Affiliations
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1. Department of Microbiology, VMMC & Safdarjang Hospital, New Delhi, India.
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2. Vector Borne Disease Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India 3. Department of Medicine, VMMC & Safdarjang Hospital, New Delhi, India
*Corresponding Authors
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4. Laboratory of Molecular Virology, International Center for Genetic Engineering and Biotechnology, Trieste, Italy
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Rajni Gaind: Department of Microbiology, VMMC & Safdarjang Hospital, New Delhi, India.
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Sujatha Sunil: Vector Borne Disease Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India
ACCEPTED MANUSCRIPT Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and co-morbid conditions in patients Navjot Kaur1, 2, Jaspreet Jain2, Ankit Kumar2, Moninder Narang3, Mohammad Khalid Zakaria4, Alessandro Marcello4, Dilip Kumar3, Rajni Gaind1*, Sujatha Sunil2*
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Abstract:
Objective: Chikungunya fever is a major public health issue in India affecting billions. Post 2010, the infection was in a decline until in 2016, when a massive
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outbreak affected the country. In this report, we present serological and molecular investigations of 600 patient samples for chikungunya and dengue viruses along with
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clinical and co-morbidity features.
Methods: We recruited 600 patients during this outbreak and evaluated them for CHIKV and DENV antibodies and viral RNA through IgM, NS1 antigen and RTPCR. We further evaluated Zika virus RNA by qRT-PCR. Additionally, we documented all clinical and co-morbid features that were observed during the
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outbreak in the hospital.
Results: We report a total incidence rate of 58% of chikungunya during the outbreak in our hospital. Within the recruited patients, 70% of the patients were positive for CHIKV IgM whereas 24.17% were positive by RT-PCR. None of the samples were
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positive for ZIKV viral RNA. Additionally, co-infection of dengue and chikungunya was seen in 25.33% of patients. Analysis of clinical features revealed that 97% of patients had restricted movements of the joints with other features like swelling,
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itching and rashes observed with varying severity. Twelve patients presented with comorbid conditions and two fatalities reported among these co-morbid patients. Conclusion: High incidence of co-infection in the current outbreak warrants implementation of routine testing of both CHIK and DEN in suspected patients for better patient management. Post-acute phase complications reported in the hospitals require in-depth studies to understand the actual impact of the current outbreak.
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Keywords: Chikungunya, outbreak, 2016, India
ACCEPTED MANUSCRIPT Introduction India has been affected by Chikungunya fever (CHIKF) every two to three decades since the 1960s [1]. The latest pandemic that occurred in several parts of the globe in 2005 resulted in huge outbreaks in India ensuing in huge loss of disability-adjusted life years (DALY) and a major public health concern in the country [2,3,4]. In 2010,
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Delhi witnessed an outbreak [5] and since then CHIKF has been in a decline with only sporadic cases been reported. However, in August 2016, another massive outbreak broke in New Delhi and National Capital Region (NCR) inflicting thousands of households bedridden [6]. The present report is the status of the CHIKF outbreak in
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New Delhi during 2016, with emphasis on the status of co-infections and co-morbid
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conditions of recruited patients.
Methods
Between August and December 2016, 5,180 patients reported in the Department of Microbiology and outpatient wards of Safdarjung Hospital for suspected CHIKF. Of these, 600 patients were enrolled in this study that was part of an ongoing project that
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was cleared by the institutional ethical board (IEC/VMMC/SJH/Project/February2016/575, ICGEB/IEC/2016/01, and Version-3). Blood samples were collected and clinical information such as age, gender, symptoms, joint movement, and onset of
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fever were documented. Sera was separated and stored in -80°C until further use. Admission criteria also included patients with complications and associated comorbid conditions. Cerebrospinal fluid (CSF) was collected from two such patients
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and stored at -80°C. All sera samples were subjected to IgM serology for the presence of dengue virus (DENV) and chikungunya virus (CHIKV) using government recommended kits from National Institute of Virology, Pune, NS1 antigen for DENV was detected using Qualpro diagnostics, RT-PCR for the presence of CHIKV RNA was performed using previously published primers [7] and Zika viral RNA was tested using qPCR kits from Altona Diagnostics. CSF was tested directly and was also inoculated in C6/36 cells to evaluate the presence of CHIKV through RT-PCR. RTPCR positive bands were eluted from gel, sequenced and phylogenetic analysis performed for the sequences (Accession Nos; Awaited)
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Results A total of 2982 patients were tested positive for CHIKF out of the total 5180, resulting in the incidence rate of 58% during the collection period. Of these,
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demographic and laboratory data were collected for a total of 600 patients enrolled for this study (Table 1). The median age of patients was 35 years (11 - 68 years range). Male-to-female ratio was 1.1:1.0. At the time of patient recruitment, onset of fever ranged from 1-30 days with a median at day 4.
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Serological investigations of the samples revealed that 420 samples (70%) were positive for CHIKV IgM, 145 samples (24.17%) were positive by RT-PCR and 40
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samples (6.67%) were positive by both methods. With respective to dengue, DENV IgM tested positive in 168 samples (28%) and only 3 samples tested positive for the NS1 antigen. Based on the investigations above, it was observed that 152 samples (25.33%) tested positive for both CHIKV and DENV; as measured either by RT PCR and/or IgM. None of the samples tested positive for ZIKV viral RNA by quantitative RT-PCR.
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Analysis of the clinical features in 150 patients revealed that 97% (n=146) of patients had restricted movements of the joints with 41 patients displaying extreme restriction and required help even in lifting their limbs (Table 1). Swelling in joints was observed
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in 39% of the patients with severe swelling seen in two patients. Itching and rashes occurred in 23.3% (n=35) and 30.6% (n=46) of patients respectively with variable levels of severity (Table 1). Details of clinical features, muscoskeletal involvement
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and co-morbid conditions are documented in table 1. Complications in CHIKF during this outbreak were seen in twelve patients during the time of sample collection (Table 1). Of these, hypotension was seen in three patients with additional complication like abdominal tenderness, co-infection with dengue and hematemesis. Encephalopathy/encephalitis was seen in two patients. Other co-morbid conditions are listed in table 1. Cerebrospinal fluid that was collected from two patients with co-morbid conditions was cultured and revealed positivity for CHIKV through RT-PCR.
ACCEPTED MANUSCRIPT PCR products corresponding to complete gene of the structural envelope protein E1, from 21 samples including the cerebrospinal fluid samples were purified and sequenced. Sequence analysis revealed that all samples belonged to ECSA genotype at the CHIKV E1 region (Figure 1). Nucleotide sequence analysis performed on the whole gene of E1 revealed variations at 40 nucleotide positions when compared with
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the 1953 Tanzanian strain (Accession no: 045811.1). Of these, 29 variations resulted in synonymous and 11 variations in non-synonymous mutations. At the amino acid level, 11 amino acid substitutions, namely, R123K, T145S, V179M, K211E, P232Q, I261D, M269V, D284E, I317V/M, V322A, N389K were observed. Of these
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mutations, K211E, M269V, D284E have been reported previously in Delhi samples (5). With respect to K211E, the mutation has been seen in all CHIKV samples post 2010 episode since its initial report in 2006 in a single sample in South India (8). This
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mutation has been observed until 2014 in all reported samples (9). However, for the first time, in the present study, two samples from the 2016 outbreak revealed reversion of wild type lysine from the mutant glutamic acid. Considering this amino acid site showed positive selection for the mutation in our previous study (5), warranting in-depth analyses to understand the implication of this reversion in
Conclusions
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evolution of CHIKV and its impact in disease prevalence.
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Since 2010, CHIKF had been declining in the country [10] until the current outbreak in 2016. However, chikungunya has been reported as co-infection in almost 10% of
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dengue cases in recent studies [11]. The present outbreak reveals that the percentage is much higher with 25% of CHIKF cases to be positive for DENV RNA/antibodies thereby making it imperative to check all suspected dengue cases for possible chikungunya infection as well. The current outbreak reports of neurological complications in CHIKF patients as evidenced in a recent study on samples collected during the same outbreak in another hospital [12]. The chikungunya outbreak in 2016 has left long lasting effects on the affected population. Post-acute phase complications are still being reported in the hospitals and detailed analyses of the aftermath of the outbreak will be important to understand the actual impact of the current outbreak.
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Disclosure The authors have no conflicts of interest to disclose. Acknowledgements
participate
in
this
study.
Funding
from
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We would like to thank all the patients enrolled in this study for their agreement to research
grants
to RG and SS were utilized for this work.
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BT/PR14725/AGR/36/672/2010 granted to SS and DST/INT/JST/P-25/2014 granted
Work in the AM laboratory is supported by grants PAR FSC 2007-2013 Flavipoc and LR 17/2014 Sevare from the Region FVG. MKV benefits from an Arturo Falaschi
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postdoctoral Fellowship from the ICGEB:
ACCEPTED MANUSCRIPT References: 1.
Shah KV, Gibbs Jr CJ, Banerjee G. Virological Investigation of the Epidemic
of Haemorrhagic Fever in Calcutta: Isolation of Three Strains of Chikungunya Virus. The Indian journal of medical research. 1964 Jul; 52:676-83. Mavalankar D, Shastri P, Raman P. Chikungunya epidemic in India: a major
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2.
public-health disaster. The Lancet infectious diseases. 2007 May 31; 7(5):306-7. 3.
Krishnamoorthy K, Harichandrakumar KT, Kumari AK, Das LK. Burden of
chikungunya in India: estimates of disability adjusted life years (DALY) lost in 2006
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epidemic. Journal of vector borne diseases. 2009 Mar 1;46(1):26. 4.
Jain J, Nayak K, Tanwar N, Gaind R, Gupta B, Shastri JS, Bhatnagar RK,
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Kaja MK, Chandele A, Sunil S. Clinical, Serological and Virological analysis of 572 chikungunya patients during the years 2010-2013 from India. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2017 Apr 1. 5.
Shrinet J, Jain S, Sharma A, Singh SS, Mathur K, Rana V, Bhatnagar RK,
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Gupta B, Gaind R, Deb M, Sunil S. Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates. Virology journal. 2012 May 25;9(1):100.
NVBDCP 2016.http://nvbdcp.gov.in/chik-cd.html Status as of Dec 2016.
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6.
Accessed on Feb 16th, 2017.
Niyas KP, Abraham R, Unnikrishnan RN, Mathew T, Nair S, Manakkadan A,
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7.
Issac A, Sreekumar E. Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedesalbopictus mosquitoes emerged from larvae from Kerala, South India. Virology journal. 2010 Aug 13; 7(1):189. 8.
Krishnamoorthy N, Kamarajs T, Joseph R, Jambulingam P. Genotyping of
virus involved in the 2006 Chikungunya outbreak in South India (Kerala and Puducherry). Current Science. 2007 Nov 25;93(10). 9.
Singh P, Sharma P, Kumar S, Chhabra Mala, Rizvi MA, MittalV,
BhattacharyaD, VenkateshS, Rai A. Continued persistence of ECSA genotype with
ACCEPTED MANUSCRIPT replacement of K211E in E1 gene of Chikungunya virus in Delhi from 2010 to 2014. Asian Pacific Journal of Tropical Disease. 2016 Jul 31;6(7):564-6. 10.
Muniaraj M. Fading chikungunya fever from India: beginning of the end of
another episode?. The Indian journal of medical research. 2014 Mar; 139(3):468. Londhey V, Agrawal S, Vaidya N, Kini S, Shastri JS, Sunil S. Dengue and
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11.
Chikungunya Virus Co-infections: The Inside Story. Journal of The Association of Physicians of India. 2016 Mar;64:36. 12.
Agarwal A,Vibha D, Srivastava AK, Shukla G, Prasad K. Guillain-Barre
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syndrome complicating chikungunya virus infection.Journal of NeuroVirology. 2017
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February.1538; 2443.
Figure detail:
Fig.-1: Genome sequence analysis of CHIKV E1 region of all samples confirms that
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all samples belonged to ECSA genotype.
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Table 1 General Information (n=600) Patient Information n (%)
Age, mean (years)
35
Range (years)
11-68
Male : Female
1.1:1
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Days of onset of fever (median)
4 days
Clinical Features (n=150) Symptoms
% of patients suffering
Fever with chills
9 (6%)
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Vomiting
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Diarrhoea
35 (23.3%) Severe 2 (1.3%)
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Itching
Rashes
1-30 days
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Days of onset of fever (range)
Nausea
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General Observations
12 (8%) 46 (30.6%), Severe 11 (7.3%) Moderate 21 (14.0%) 2 (1.3%) 5 (3.3%)
Ulcers
2 (1.3%)
Headache
7 (5%)
Muscoskeletal Swelling
59 (39%), Severe 2 (1.3%)
Restricted Movement of the Joints
146 (97%), Extreme 41 (27%)
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3 (2%)
Morning stiffness
32 (21.3)
Body ache
3 (2%)
Pedal edema
22 (14.6%)
Partial paraparesis
4 (2.6%)
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Back ache
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Co-morbid Conditions (n=12) Hypotension with additional complications
3 (2%)
2 (1.3%)
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Hypertonia Decreased sensation of pain, touch & temperature
2 (1.3%)
Lipoma in head
2 (1.3%)
Chest Pain
2 (1.3%)
Dermatomyositis Respiratory presentation
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Severe thrombocytopenia
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Encephalopathy and encephalitis
2 (1.3%) 1 (0.5%) 1 (0.5%) 1 (0.5%) 1 (0.5%)
Intra uterine death
1 (0.5%)
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Demyelinating disorder presenting as traverse myelitis
Mortality rate
2 (1.3%)
Diagnostics (n=600)
CHIKV IgM positive
420 (70%)
CHIKV RT-PCR positive
145 (24.17%)
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40 (6.67%)
Only CHIKV IgM positive (CHIKV RT PCR negative)
244 (40.67%)
Only CHIKV RT-PCR positive (CHIKV IgM negative)
103 (17.17%)
DENV IgM positive
168 (28%)
DENV NS1 positive
3 (0.5%)
ZIKV qRT-PCR
0
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CHIKV IgM and RT-PCR positive
Co-infection status (n=600)
CHIKV RTPCR/DENV IgM
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CHIKV/DENV
40 (6.67%)
CHIKV RTPCR/DENV NS1 CHIKV IgM/DENV IgM
0 (0%)
112 (18.67%) 1 (0.5%)
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CHIKV IgM/DENV NS1
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Overall co-infection (CHIKV IgM/RT-PCR/DENV IgM/NS1
152 (25.33%)
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e e om me m n A no no ge e RN ge m ge ete c i e e no pl om let et ge n pl com mp e te g m o ple 065 co 108 8 c m / 7 0 co -0 H20 217 /08 me e 8 Y E 0 no m 4 M D C e 5 o ) S 22 g n e DR (EH ICH 1 lat e ge ete l o D e e p t s t la e LK EH ICH e i om enom ple c o G m m t H 3 g o o is 23 s sola te S GE gen 10 c ete K l u e a i S p r C l vi us iso te lete 5/20 08 nom om me e c a o a r 0 l g p i 0 n y v s o e -2 08 te un ya viru s is com n GD iland H10 ete g ple g m l i u s n n u ya ir ru p co )C ra me ha iku ung un ya v a vi s st in T K(PB com 4108 eno g h w g u te e C ik n un ny ir tra e L bT HS42 ple nom 1| Ch hiku ung ngu ya v rus s olat 0810 m C e . o I g c n vi 6 | H is k u te 0 me 0079.1.2| C Chi hik ungu nya irus strain SGE hik1 omple 2 eno C C v e | t c g k 7 s u 1 | 6 i a a U e 2 2 l u g t 3 5 le iso U3 13 43 2. 5. Ch un uny vir te C 0/1 me omp c |EFJ5 445 550548 5.1| Chik ung unya virus isola e NL1 eno 2 g b 5 ge k t | e 4 s J 9 i R t g 0 a | g a le | gb |F J4 N2 87 .1 Ch kun uny lete -K iru isol p p v D 0 m m 9 | b N o i c 1 7 g |F |F 08 4 1| h ya us eI ng 8 co m 87397 95|6|gbmb |JX7968628. .1| C hiku ungun ya vir isolat 810aTw S42280 9 geno n s C b e 9 0 e 3 6 H k t u u J | i 7 | 1 e r 9 7 C n g l 1 i g i I 2 h | stra comp 69 0 7 27 03 1 b|K J5 78 3.2 | C kun ya v GEH lete |1|268288557244008874|g|gb|F|FJ804544780.11| Chi ungun a virus olate S -Chik009 i comp s 0 ggi i|2 8 93 2 82 47 gb FJ4 01 29. y i k 8 U i n s K C h | | 0 3 3 C viru isolate |2 2 9 ngu 008 C comp 0 ggi i|4i|395167399554|gbb|GUC862 69.1| Chiku gunya s and-2 l u i r i a 00 g g |65 20 10 70 4|g b|K 000 | n v h K11/53 1 T u C a . k 9 n y i i 4 8 6 0 2 i n a 0 h 0 000 g i|2 62 57 27702|g |FJ 0789 .2| C nd-2 ngu s str mpl 8894839773|gbb|FJ8 45433 .1| Chiku gunya viru strain Thaila 01 ggi|i2 K539 co 2 C | 9 0 5 9 0 9 0 n s 4 g 2 0 ggi|2 52310221909521||gb|FJU301778.1| Chiku ngunya viru train Thailand00 gi|2 mplete g | i Chiku virus s H6708 co 624 270 |gb|G 9684 g 00 )C H (E K 1 gii||2288547827712|gb|KJ7796850.1| hikungunya L C ete s isolate 9 g i|299 6828 |gb|KJ 1 6 6852.1| H17708 compl gunya viru )C H (E 14 g LK e at ggi|i|6655551668822890|gb|KJJ75913654.1| Chikun unya virus isol ggi|i|6256521073965|gbb||F 673.1| Chikung ya virus isolate LK(EH)CH4408 complete 13 J5 F |g 71 4 622 |2620739 13645.1| Chikungun 063 gi )CH3008 complete gi|262073962|gb|FJ5 36 gi|262073953|gb|FJ513632.1| Chikungunya virus isolate LK(PB gi|288572683|gb|FJ445426.2| Chikungunya virus isolate LKEHCH13908 complet e gi| 2073974|gb|FJ51367 gi|226 5.1| Chikungunya virus iso 80983626|gb|G 57 g i| lat 4 e LK U 1 (EH 19 0 )CH18608 complete 067116|em 9351.1| Chiku 41 gi|4100 ngunya virus st 67119|e b|FR717336 2 ggi|i|25901 ra in .1 m S D | 08 b C 4 |F P h 1 an 7 ik R71733 complete geno 34|g gi 894 7.1| Ch ungunya virus comp m 4 gi |1567 18704 b|FJ95 lete genom 135 gi||111162951972||ggb|EU569103.1| Ch ikungunya v e is gi|1 604 5203 b|EU 4334 ir o ik u la s comple te IMTSS u te genom 549 |gb|EF 03796 .1| Chik ngunya virus g |g1i|2224297 | u 5 2 g e s 113 gi|i3 n 0 1 t . is 5 r 2 g b 1 a 1 o 2 u | la in 7 nya te IMTS BNI-CH 1 gi|3 34429529059|gb|DQ44 2359.1 Chikun S IKV 89 111 ggi|3 34 145 58 8|g |EF0 354 | Ch guny virus strain i 9 3 1 i 2 a 4 | 6 k c b 5 2 TM25 omplet ungu virus 6 233441 457765 |gb| |FJ 713 .2| C 4 e gen comp Wu 14457 67| |gb| EF0 0000 5.1| hikun nya vi lete g 58 rus s erzburg HQ 271 64. Chik gun 6 5 g g e 9 b 7 n g i g o 1 tra me comp gi i|2 i|3|29 71| |gb |HQ 456 36. 1| C ungu ya vir 2556 us s in D570 |HQ 45 25 1| C hiku lete g nya 93 ggi|2|29 96 804612gb|H t / 1 e r 6 n 0 h a n 6 co ome in i|1 96612124 67 450 Q 456 252 .1| C ikun guny virus mple stra LR200 24 12 45 58 95 4| 456 253 .1| hik gun a vir t e 6 i g n 0 u C g enom O 29 44 91 8|g |g b| 254 .1| hik ng ya v us is I e olat ND-06- PY1 co 55 77 |g b| b|J HM .1| Ch ung unya irus m e K b H i Q stra plet A15 91 |g |H M 0 045 Ch kun uny vir IND e u i b 6 n com gen ss |g |H M0 04 76 79 iku gun a v IND -GJ5 ome plet t b| M 4 58 2 3. ng i e ge EF 0 58 2 4.1 1| un ya v rus s rain C -06-M 1 com 2 i t 4 p C y n r rai om lete ome H2 02 57 23 .1 | C hi a v us com gen 71 84 .1| | C hik kun iru stra n CO 25 c o o h s p M in me m 39 .1 Ch ik un gu lete s .1 | C iku ung gun nya train Com J com plete gen | C hi n u gen 1 y om ple v K e om te hi kun gun nya a vir irus PA1 25 c g o ku g y e u s e 5 m v t n s a i r co pl om ng un ru a s e un ya viru s s trai in C mpl ete g n e t A s e r ya vir t a n R e C s om ge vir us tra in H HI2 256 e no 01 us str in A B7 co m e st ain ng 8 c 0 co mpl ra ete om m in Da ola ple ple ge M IN kA t D- r B 202 te g e ge nom e en 00 2 n 1 co om om -M 68 e m e H4 78 pl co com ete ge m pl plet no et e m e ge e ge no no m e m e
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42 Chikungunya virus strain RGCB729/09 complete genome 195 gi|655168276|gb|KJ796845.1| gi|6551 78|gb|KJ796846.1| Chikungunya viru 0 gi|65516682 s strain RGCB755/09 complete gen 0 gi|6 82 |gb|KJ796844.1| 0 gi| 5516828874 ome Chikungunya viru |g 6 b 0 g |K 5 5 J 7 1 9 6 s 6 strain RGCB730/0 851.1| Chik 0 g i|2985 8280|gb|K ungunya vir 9 complete geno 0 gii|| 2885778082|gb|GJ796847.1| us str me
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S2052-2975(17)30063-X
DOI:
10.1016/j.nmni.2017.07.007
Reference:
NMNI 351
To appear in:
New Microbes and New Infections
Received Date: 11 May 2017 Revised Date:
2052-2975 2052-2975
Accepted Date: 23 July 2017
Please cite this article as: Kaur N, Jain J, Kumar A, Narang M, Zakaria MK, Marcello A, Kumar D, Gaind R, Sunil S, Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and co-morbid conditions in patients, New Microbes and New Infections (2017), doi: 10.1016/ j.nmni.2017.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and comorbid conditions in patients
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Navjot Kaur1, 2, Jaspreet Jain2, Ankit Kumar2, Moninder Narang3, Mohammad Khalid Zakaria4, Alessandro Marcello4, Dilip Kumar3, Rajni Gaind1*, Sujatha Sunil2*
Affiliations
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1. Department of Microbiology, VMMC & Safdarjang Hospital, New Delhi, India.
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2. Vector Borne Disease Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India 3. Department of Medicine, VMMC & Safdarjang Hospital, New Delhi, India
*Corresponding Authors
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4. Laboratory of Molecular Virology, International Center for Genetic Engineering and Biotechnology, Trieste, Italy
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Rajni Gaind: Department of Microbiology, VMMC & Safdarjang Hospital, New Delhi, India.
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Sujatha Sunil: Vector Borne Disease Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India
ACCEPTED MANUSCRIPT Chikungunya outbreak in Delhi, India, 2016: A Report on the co-infection status and co-morbid conditions in patients Navjot Kaur1, 2, Jaspreet Jain2, Ankit Kumar2, Moninder Narang3, Mohammad Khalid Zakaria4, Alessandro Marcello4, Dilip Kumar3, Rajni Gaind1*, Sujatha Sunil2*
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Abstract:
Objective: Chikungunya fever is a major public health issue in India affecting billions. Post 2010, the infection was in a decline until in 2016, when a massive
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outbreak affected the country. In this report, we present serological and molecular investigations of 600 patient samples for chikungunya and dengue viruses along with
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clinical and co-morbidity features.
Methods: We recruited 600 patients during this outbreak and evaluated them for CHIKV and DENV antibodies and viral RNA through IgM, NS1 antigen and RTPCR. We further evaluated Zika virus RNA by qRT-PCR. Additionally, we documented all clinical and co-morbid features that were observed during the
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outbreak in the hospital.
Results: We report a total incidence rate of 58% of chikungunya during the outbreak in our hospital. Within the recruited patients, 70% of the patients were positive for CHIKV IgM whereas 24.17% were positive by RT-PCR. None of the samples were
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positive for ZIKV viral RNA. Additionally, co-infection of dengue and chikungunya was seen in 25.33% of patients. Analysis of clinical features revealed that 97% of patients had restricted movements of the joints with other features like swelling,
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itching and rashes observed with varying severity. Twelve patients presented with comorbid conditions and two fatalities reported among these co-morbid patients. Conclusion: High incidence of co-infection in the current outbreak warrants implementation of routine testing of both CHIK and DEN in suspected patients for better patient management. Post-acute phase complications reported in the hospitals require in-depth studies to understand the actual impact of the current outbreak.
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Keywords: Chikungunya, outbreak, 2016, India
ACCEPTED MANUSCRIPT Introduction India has been affected by Chikungunya fever (CHIKF) every two to three decades since the 1960s [1]. The latest pandemic that occurred in several parts of the globe in 2005 resulted in huge outbreaks in India ensuing in huge loss of disability-adjusted life years (DALY) and a major public health concern in the country [2,3,4]. In 2010,
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Delhi witnessed an outbreak [5] and since then CHIKF has been in a decline with only sporadic cases been reported. However, in August 2016, another massive outbreak broke in New Delhi and National Capital Region (NCR) inflicting thousands of households bedridden [6]. The present report is the status of the CHIKF outbreak in
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New Delhi during 2016, with emphasis on the status of co-infections and co-morbid
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conditions of recruited patients.
Methods
Between August and December 2016, 5,180 patients reported in the Department of Microbiology and outpatient wards of Safdarjung Hospital for suspected CHIKF. Of these, 600 patients were enrolled in this study that was part of an ongoing project that
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was cleared by the institutional ethical board (IEC/VMMC/SJH/Project/February2016/575, ICGEB/IEC/2016/01, and Version-3). Blood samples were collected and clinical information such as age, gender, symptoms, joint movement, and onset of
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fever were documented. Sera was separated and stored in -80°C until further use. Admission criteria also included patients with complications and associated comorbid conditions. Cerebrospinal fluid (CSF) was collected from two such patients
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and stored at -80°C. All sera samples were subjected to IgM serology for the presence of dengue virus (DENV) and chikungunya virus (CHIKV) using government recommended kits from National Institute of Virology, Pune, NS1 antigen for DENV was detected using Qualpro diagnostics, RT-PCR for the presence of CHIKV RNA was performed using previously published primers [7] and Zika viral RNA was tested using qPCR kits from Altona Diagnostics. CSF was tested directly and was also inoculated in C6/36 cells to evaluate the presence of CHIKV through RT-PCR. RTPCR positive bands were eluted from gel, sequenced and phylogenetic analysis performed for the sequences (Accession Nos; Awaited)
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Results A total of 2982 patients were tested positive for CHIKF out of the total 5180, resulting in the incidence rate of 58% during the collection period. Of these,
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demographic and laboratory data were collected for a total of 600 patients enrolled for this study (Table 1). The median age of patients was 35 years (11 - 68 years range). Male-to-female ratio was 1.1:1.0. At the time of patient recruitment, onset of fever ranged from 1-30 days with a median at day 4.
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Serological investigations of the samples revealed that 420 samples (70%) were positive for CHIKV IgM, 145 samples (24.17%) were positive by RT-PCR and 40
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samples (6.67%) were positive by both methods. With respective to dengue, DENV IgM tested positive in 168 samples (28%) and only 3 samples tested positive for the NS1 antigen. Based on the investigations above, it was observed that 152 samples (25.33%) tested positive for both CHIKV and DENV; as measured either by RT PCR and/or IgM. None of the samples tested positive for ZIKV viral RNA by quantitative RT-PCR.
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Analysis of the clinical features in 150 patients revealed that 97% (n=146) of patients had restricted movements of the joints with 41 patients displaying extreme restriction and required help even in lifting their limbs (Table 1). Swelling in joints was observed
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in 39% of the patients with severe swelling seen in two patients. Itching and rashes occurred in 23.3% (n=35) and 30.6% (n=46) of patients respectively with variable levels of severity (Table 1). Details of clinical features, muscoskeletal involvement
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and co-morbid conditions are documented in table 1. Complications in CHIKF during this outbreak were seen in twelve patients during the time of sample collection (Table 1). Of these, hypotension was seen in three patients with additional complication like abdominal tenderness, co-infection with dengue and hematemesis. Encephalopathy/encephalitis was seen in two patients. Other co-morbid conditions are listed in table 1. Cerebrospinal fluid that was collected from two patients with co-morbid conditions was cultured and revealed positivity for CHIKV through RT-PCR.
ACCEPTED MANUSCRIPT PCR products corresponding to complete gene of the structural envelope protein E1, from 21 samples including the cerebrospinal fluid samples were purified and sequenced. Sequence analysis revealed that all samples belonged to ECSA genotype at the CHIKV E1 region (Figure 1). Nucleotide sequence analysis performed on the whole gene of E1 revealed variations at 40 nucleotide positions when compared with
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the 1953 Tanzanian strain (Accession no: 045811.1). Of these, 29 variations resulted in synonymous and 11 variations in non-synonymous mutations. At the amino acid level, 11 amino acid substitutions, namely, R123K, T145S, V179M, K211E, P232Q, I261D, M269V, D284E, I317V/M, V322A, N389K were observed. Of these
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mutations, K211E, M269V, D284E have been reported previously in Delhi samples (5). With respect to K211E, the mutation has been seen in all CHIKV samples post 2010 episode since its initial report in 2006 in a single sample in South India (8). This
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mutation has been observed until 2014 in all reported samples (9). However, for the first time, in the present study, two samples from the 2016 outbreak revealed reversion of wild type lysine from the mutant glutamic acid. Considering this amino acid site showed positive selection for the mutation in our previous study (5), warranting in-depth analyses to understand the implication of this reversion in
Conclusions
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evolution of CHIKV and its impact in disease prevalence.
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Since 2010, CHIKF had been declining in the country [10] until the current outbreak in 2016. However, chikungunya has been reported as co-infection in almost 10% of
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dengue cases in recent studies [11]. The present outbreak reveals that the percentage is much higher with 25% of CHIKF cases to be positive for DENV RNA/antibodies thereby making it imperative to check all suspected dengue cases for possible chikungunya infection as well. The current outbreak reports of neurological complications in CHIKF patients as evidenced in a recent study on samples collected during the same outbreak in another hospital [12]. The chikungunya outbreak in 2016 has left long lasting effects on the affected population. Post-acute phase complications are still being reported in the hospitals and detailed analyses of the aftermath of the outbreak will be important to understand the actual impact of the current outbreak.
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Disclosure The authors have no conflicts of interest to disclose. Acknowledgements
participate
in
this
study.
Funding
from
RI PT
We would like to thank all the patients enrolled in this study for their agreement to research
grants
to RG and SS were utilized for this work.
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BT/PR14725/AGR/36/672/2010 granted to SS and DST/INT/JST/P-25/2014 granted
Work in the AM laboratory is supported by grants PAR FSC 2007-2013 Flavipoc and LR 17/2014 Sevare from the Region FVG. MKV benefits from an Arturo Falaschi
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postdoctoral Fellowship from the ICGEB:
ACCEPTED MANUSCRIPT References: 1.
Shah KV, Gibbs Jr CJ, Banerjee G. Virological Investigation of the Epidemic
of Haemorrhagic Fever in Calcutta: Isolation of Three Strains of Chikungunya Virus. The Indian journal of medical research. 1964 Jul; 52:676-83. Mavalankar D, Shastri P, Raman P. Chikungunya epidemic in India: a major
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2.
public-health disaster. The Lancet infectious diseases. 2007 May 31; 7(5):306-7. 3.
Krishnamoorthy K, Harichandrakumar KT, Kumari AK, Das LK. Burden of
chikungunya in India: estimates of disability adjusted life years (DALY) lost in 2006
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epidemic. Journal of vector borne diseases. 2009 Mar 1;46(1):26. 4.
Jain J, Nayak K, Tanwar N, Gaind R, Gupta B, Shastri JS, Bhatnagar RK,
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Kaja MK, Chandele A, Sunil S. Clinical, Serological and Virological analysis of 572 chikungunya patients during the years 2010-2013 from India. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2017 Apr 1. 5.
Shrinet J, Jain S, Sharma A, Singh SS, Mathur K, Rana V, Bhatnagar RK,
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Gupta B, Gaind R, Deb M, Sunil S. Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates. Virology journal. 2012 May 25;9(1):100.
NVBDCP 2016.http://nvbdcp.gov.in/chik-cd.html Status as of Dec 2016.
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6.
Accessed on Feb 16th, 2017.
Niyas KP, Abraham R, Unnikrishnan RN, Mathew T, Nair S, Manakkadan A,
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7.
Issac A, Sreekumar E. Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedesalbopictus mosquitoes emerged from larvae from Kerala, South India. Virology journal. 2010 Aug 13; 7(1):189. 8.
Krishnamoorthy N, Kamarajs T, Joseph R, Jambulingam P. Genotyping of
virus involved in the 2006 Chikungunya outbreak in South India (Kerala and Puducherry). Current Science. 2007 Nov 25;93(10). 9.
Singh P, Sharma P, Kumar S, Chhabra Mala, Rizvi MA, MittalV,
BhattacharyaD, VenkateshS, Rai A. Continued persistence of ECSA genotype with
ACCEPTED MANUSCRIPT replacement of K211E in E1 gene of Chikungunya virus in Delhi from 2010 to 2014. Asian Pacific Journal of Tropical Disease. 2016 Jul 31;6(7):564-6. 10.
Muniaraj M. Fading chikungunya fever from India: beginning of the end of
another episode?. The Indian journal of medical research. 2014 Mar; 139(3):468. Londhey V, Agrawal S, Vaidya N, Kini S, Shastri JS, Sunil S. Dengue and
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11.
Chikungunya Virus Co-infections: The Inside Story. Journal of The Association of Physicians of India. 2016 Mar;64:36. 12.
Agarwal A,Vibha D, Srivastava AK, Shukla G, Prasad K. Guillain-Barre
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syndrome complicating chikungunya virus infection.Journal of NeuroVirology. 2017
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February.1538; 2443.
Figure detail:
Fig.-1: Genome sequence analysis of CHIKV E1 region of all samples confirms that
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all samples belonged to ECSA genotype.
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Table 1 General Information (n=600) Patient Information n (%)
Age, mean (years)
35
Range (years)
11-68
Male : Female
1.1:1
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Days of onset of fever (median)
4 days
Clinical Features (n=150) Symptoms
% of patients suffering
Fever with chills
9 (6%)
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Vomiting
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Diarrhoea
35 (23.3%) Severe 2 (1.3%)
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Itching
Rashes
1-30 days
M AN U
Days of onset of fever (range)
Nausea
RI PT
General Observations
12 (8%) 46 (30.6%), Severe 11 (7.3%) Moderate 21 (14.0%) 2 (1.3%) 5 (3.3%)
Ulcers
2 (1.3%)
Headache
7 (5%)
Muscoskeletal Swelling
59 (39%), Severe 2 (1.3%)
Restricted Movement of the Joints
146 (97%), Extreme 41 (27%)
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3 (2%)
Morning stiffness
32 (21.3)
Body ache
3 (2%)
Pedal edema
22 (14.6%)
Partial paraparesis
4 (2.6%)
RI PT
Back ache
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Co-morbid Conditions (n=12) Hypotension with additional complications
3 (2%)
2 (1.3%)
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Hypertonia Decreased sensation of pain, touch & temperature
2 (1.3%)
Lipoma in head
2 (1.3%)
Chest Pain
2 (1.3%)
Dermatomyositis Respiratory presentation
EP
Severe thrombocytopenia
TE D
Encephalopathy and encephalitis
2 (1.3%) 1 (0.5%) 1 (0.5%) 1 (0.5%) 1 (0.5%)
Intra uterine death
1 (0.5%)
AC C
Demyelinating disorder presenting as traverse myelitis
Mortality rate
2 (1.3%)
Diagnostics (n=600)
CHIKV IgM positive
420 (70%)
CHIKV RT-PCR positive
145 (24.17%)
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40 (6.67%)
Only CHIKV IgM positive (CHIKV RT PCR negative)
244 (40.67%)
Only CHIKV RT-PCR positive (CHIKV IgM negative)
103 (17.17%)
DENV IgM positive
168 (28%)
DENV NS1 positive
3 (0.5%)
ZIKV qRT-PCR
0
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CHIKV IgM and RT-PCR positive
Co-infection status (n=600)
CHIKV RTPCR/DENV IgM
M AN U
CHIKV/DENV
40 (6.67%)
CHIKV RTPCR/DENV NS1 CHIKV IgM/DENV IgM
0 (0%)
112 (18.67%) 1 (0.5%)
TE D
CHIKV IgM/DENV NS1
AC C
EP
Overall co-infection (CHIKV IgM/RT-PCR/DENV IgM/NS1
152 (25.33%)
|H
gi|6
599
022
78|g
b|K
J67
957
8.1|
SC
D D E DEEL L-2 Chi DE L-2-20 01 kun gi|26 D 1 6 g uny 2093 DEEL L-200166-C-CH a vi D 230| 2 r us i gb|G DEEL- L-200116-CHHIKIK5 sola Q428 2 C 6 gi|221 L D 1 te C 211. EL -20016 6- -CHHIIK5534 2290 1 H C K2 7 | D 1 2 0 C I C K | gb|FJ hikun gi|371 VS De EL-20166-CHHIH 00 IKIK35 0000 98583 guny T K l 2 M 0 C 00 68.1| 2 0|gb|J 1 I a 1 W K 0 H v 0 2 6 iru 16 - IK 1 G02 N5588 C gi|2885 0 34.1| C hikungun s strain 72686|g com -CHCI HIK101 10 ya vir RGC hikung b|FJ445 K 1 p 3 9 us E le B0 un 427.2| C D D 27|dbj|AB 725 L-2L0-201te0 gen hikungu ya virus iso isolate IND5/KL06 E 455494.1 1 o c la n ya virus | Chikung te SVU -KA51 omple0-CHCHIKme 8 unya viru CTR-0 comp te ge IK10 2 409 4|dbj|AB455 s genomic isolate LKMTC 9 com lete g nom 8 4 493.1| Chiku H RNA com 3 ngunya virus te geennomee plete ge2n707 compple genomic RN le o gi|326902023|gb|JF m te 2 o e m g 9 A e e co str in: SL nome 615 m lete geno 274082.1| Chikung m straain unya virus isolatepIN : SL1110153711 7 8 D-06-Gujeco gi|334145773|gb|HQ456255.1| Chikungu m ple te genome 41 nya virus strain Lamu33 complete genome genome 0 complete RGCB03/KL06 strain virus 62093227|gb|GQ428210.1| Chikungunya 0
EP
e m e no ge nom e et ge pl e m et e e pl co m m 5 om no no e 15 8 c ge ge m g4 L0 ete te no ple gA /K ge pl 6 U m m te me in B35 co 8 co ple eno e ra 9 st GC 64 P-0 com te g nom 5 s e l R 9 e 1 p 0 g KD ru e vi rain SL VU DP- com lete om K 3 ya st ain S en mp e un rus str late VU -AP co ete g om ng vi us iso te S -06 RJ1 e pl en m m a g D l o ku a ir s en hi uny a v iru iso n IN D-06 06 co plete e eg t m N m | C g ny a v us ai e eno .1 un gu ny vir str in I RDE 1 co ompl e 3 D te g c 12 hik un gu ya irus stra m e K l o I 58 | C hik un un a v us train CH GJ52 comp gen e s in 04 .1 C hik ng ny vir ete D3 om M 5 1| C ku u a rus stra te IN -GJ5 compl gen H 21 1. | hi ng ny vi s e 1 t a D l 1 e l N 5 o b| 8 06 5. C ku gu ya iru e p I |g 42 89 83 .1| Chi kun gun ya v rus is late -KR 1 com nom n i 5 e ge IND so 58 Q 1 8 36 | hi n v 5 e u t u i 1 H 8 G . e C U m o 45 b| |G N5 58 34 .1| hik ung nya irus isolate IND-M compl gen me w e s lete 12 |g b |J 5 71 7 | C hik gu a v t 3 o 2 N y b g n T 2 p u n . a 2 a r l e m i 1 g o o v 1 c s e 96 4 7| |g |J 0 71 57 1| C iku gun i t |2 32 61 33 |gb EF 02 01 5. Ch un nya virus train 06 080008 nom mple gi 9 1 8 6 b| EF 21 33 .1| hik gu 7 co te ge s a 5 n 0 3 D 4 e 0 1 g | l C 2 y 5 F s u p | F 1 m n i H o c a C 6203 98 58 6 gb |E 56 06 .1| hik ungun ya viru 8 om s str |2 7 1 8 57 5| gb EU 00 65 | C KRG 7710 2 e gen ggi i|2i|377129955598967| |gbb| |FJ00000066.11| Chikkungun nya viru solate L ICHS mplet g i|3 4 29 9 0 |g FJ 00 7. hi GEH gu us i 08 co
0 3 gi|1224646187052 |gb| |FJ0 0006 6.1| C hikun nya vir D135 ate S geno 5 g i|1 8 4 9 61 b J0 s isol |C mplete gu 89 EHICH 0 u g o 1 9 n 2 r | c G 7 i 1 . F 0 S B 6040 ggi|i|1821222990647|gb||FJ80 99350 | Chiku gunya v olate -Calif A ome virus is ain DHS4263 00 ggi|i|22221122290646|gbb|GU1 5428.2 | Chikun lete gen 4 unya p tr s m o s c u 2 00 ggi||2221224109623|ggb|FJ4 45510.2 | Chikung ir i 3 | IND-KA5 unya v 0 ggi|268098 2689 gb|FJ4 5511.2 hikung genome s isolate | 4 u te C 7 le 4 2 9 0 p 794.1| gunya vir 0 gi| 885 271 gb|FJ L-CK1 com 0 ggii||2288855772722||gb|HM045063.1| Chikun us strain S genome 0 8 507 ungunya vir J000 6-TN1 complete 0 gi|i|2 2961249055|gb|F 45801.1| Chik rus strain IND-0 0 ggi|2926112224526|gb|HM0 27138.1| Chikungunya vi i|2 24295588|gb|EF0 SL-CR 3 complete genome 0 ggi |1 .1| Chikungunya virus strain 1 gi|296124520|gb|HM045799 1
AC C
0.02
0
TE D
M AN U
237
e e om me m n A no no ge e RN ge m ge ete c i e e no pl om let et ge n pl com mp e te g m o ple 065 co 108 8 c m / 7 0 co -0 H20 217 /08 me e 8 Y E 0 no m 4 M D C e 5 o ) S 22 g n e DR (EH ICH 1 lat e ge ete l o D e e p t s t la e LK EH ICH e i om enom ple c o G m m t H 3 g o o is 23 s sola te S GE gen 10 c ete K l u e a i S p r C l vi us iso te lete 5/20 08 nom om me e c a o a r 0 l g p i 0 n y v s o e -2 08 te un ya viru s is com n GD iland H10 ete g ple g m l i u s n n u ya ir ru p co )C ra me ha iku ung un ya v a vi s st in T K(PB com 4108 eno g h w g u te e C ik n un ny ir tra e L bT HS42 ple nom 1| Ch hiku ung ngu ya v rus s olat 0810 m C e . o I g c n vi 6 | H is k u te 0 me 0079.1.2| C Chi hik ungu nya irus strain SGE hik1 omple 2 eno C C v e | t c g k 7 s u 1 | 6 i a a U e 2 2 l u g t 3 5 le iso U3 13 43 2. 5. Ch un uny vir te C 0/1 me omp c |EFJ5 445 550548 5.1| Chik ung unya virus isola e NL1 eno 2 g b 5 ge k t | e 4 s J 9 i R t g 0 a | g a le | gb |F J4 N2 87 .1 Ch kun uny lete -K iru isol p p v D 0 m m 9 | b N o i c 1 7 g |F |F 08 4 1| h ya us eI ng 8 co m 87397 95|6|gbmb |JX7968628. .1| C hiku ungun ya vir isolat 810aTw S42280 9 geno n s C b e 9 0 e 3 6 H k t u u J | i 7 | 1 e r 9 7 C n g l 1 i g i I 2 h | stra comp 69 0 7 27 03 1 b|K J5 78 3.2 | C kun ya v GEH lete |1|268288557244008874|g|gb|F|FJ804544780.11| Chi ungun a virus olate S -Chik009 i comp s 0 ggi i|2 8 93 2 82 47 gb FJ4 01 29. y i k 8 U i n s K C h | | 0 3 3 C viru isolate |2 2 9 ngu 008 C comp 0 ggi i|4i|395167399554|gbb|GUC862 69.1| Chiku gunya s and-2 l u i r i a 00 g g |65 20 10 70 4|g b|K 000 | n v h K11/53 1 T u C a . k 9 n y i i 4 8 6 0 2 i n a 0 h 0 000 g i|2 62 57 27702|g |FJ 0789 .2| C nd-2 ngu s str mpl 8894839773|gbb|FJ8 45433 .1| Chiku gunya viru strain Thaila 01 ggi|i2 K539 co 2 C | 9 0 5 9 0 9 0 n s 4 g 2 0 ggi|2 52310221909521||gb|FJU301778.1| Chiku ngunya viru train Thailand00 gi|2 mplete g | i Chiku virus s H6708 co 624 270 |gb|G 9684 g 00 )C H (E K 1 gii||2288547827712|gb|KJ7796850.1| hikungunya L C ete s isolate 9 g i|299 6828 |gb|KJ 1 6 6852.1| H17708 compl gunya viru )C H (E 14 g LK e at ggi|i|6655551668822890|gb|KJJ75913654.1| Chikun unya virus isol ggi|i|6256521073965|gbb||F 673.1| Chikung ya virus isolate LK(EH)CH4408 complete 13 J5 F |g 71 4 622 |2620739 13645.1| Chikungun 063 gi )CH3008 complete gi|262073962|gb|FJ5 36 gi|262073953|gb|FJ513632.1| Chikungunya virus isolate LK(PB gi|288572683|gb|FJ445426.2| Chikungunya virus isolate LKEHCH13908 complet e gi| 2073974|gb|FJ51367 gi|226 5.1| Chikungunya virus iso 80983626|gb|G 57 g i| lat 4 e LK U 1 (EH 19 0 )CH18608 complete 067116|em 9351.1| Chiku 41 gi|4100 ngunya virus st 67119|e b|FR717336 2 ggi|i|25901 ra in .1 m S D | 08 b C 4 |F P h 1 an 7 ik R71733 complete geno 34|g gi 894 7.1| Ch ungunya virus comp m 4 gi |1567 18704 b|FJ95 lete genom 135 gi||111162951972||ggb|EU569103.1| Ch ikungunya v e is gi|1 604 5203 b|EU 4334 ir o ik u la s comple te IMTSS u te genom 549 |gb|EF 03796 .1| Chik ngunya virus g |g1i|2224297 | u 5 2 g e s 113 gi|i3 n 0 1 t . is 5 r 2 g b 1 a 1 o 2 u | la in 7 nya te IMTS BNI-CH 1 gi|3 34429529059|gb|DQ44 2359.1 Chikun S IKV 89 111 ggi|3 34 145 58 8|g |EF0 354 | Ch guny virus strain i 9 3 1 i 2 a 4 | 6 k c b 5 2 TM25 omplet ungu virus 6 233441 457765 |gb| |FJ 713 .2| C 4 e gen comp Wu 14457 67| |gb| EF0 0000 5.1| hikun nya vi lete g 58 rus s erzburg HQ 271 64. Chik gun 6 5 g g e 9 b 7 n g i g o 1 tra me comp gi i|2 i|3|29 71| |gb |HQ 456 36. 1| C ungu ya vir 2556 us s in D570 |HQ 45 25 1| C hiku lete g nya 93 ggi|2|29 96 804612gb|H t / 1 e r 6 n 0 h a n 6 co ome in i|1 96612124 67 450 Q 456 252 .1| C ikun guny virus mple stra LR200 24 12 45 58 95 4| 456 253 .1| hik gun a vir t e 6 i g n 0 u C g enom O 29 44 91 8|g |g b| 254 .1| hik ng ya v us is I e olat ND-06- PY1 co 55 77 |g b| b|J HM .1| Ch ung unya irus m e K b H i Q stra plet A15 91 |g |H M 0 045 Ch kun uny vir IND e u i b 6 n com gen ss |g |H M0 04 76 79 iku gun a v IND -GJ5 ome plet t b| M 4 58 2 3. ng i e ge EF 0 58 2 4.1 1| un ya v rus s rain C -06-M 1 com 2 i t 4 p C y n r rai om lete ome H2 02 57 23 .1 | C hi a v us com gen 71 84 .1| | C hik kun iru stra n CO 25 c o o h s p M in me m 39 .1 Ch ik un gu lete s .1 | C iku ung gun nya train Com J com plete gen | C hi n u gen 1 y om ple v K e om te hi kun gun nya a vir irus PA1 25 c g o ku g y e u s e 5 m v t n s a i r co pl om ng un ru a s e un ya viru s s trai in C mpl ete g n e t A s e r ya vir t a n R e C s om ge vir us tra in H HI2 256 e no 01 us str in A B7 co m e st ain ng 8 c 0 co mpl ra ete om m in Da ola ple ple ge M IN kA t D- r B 202 te g e ge nom e en 00 2 n 1 co om om -M 68 e m e H4 78 pl co com ete ge m pl plet no et e m e ge e ge no no m e m e
RI PT
|g
98
gb
5|
44
ain Thailand Chikungu gi|126209 2698|gb Q9058 -2009 CK25 nya virus 0 ggii|2672191839233|gb |FJ44543 63.1| Chiku 7 complete s tr a in | | n G 0 T 2 2 1 0 g h genome . u a 7 9 g 2 Q 8 il n | a ya virus nd-2009 Chiku 0 gi|28 857 3959 |gb|E 42821 0 C n 2 U 8 s i K | 2 g tr | g 6 u . a 5 1 gi|426 572 692 b|F 2448 1 0 in n | c y o C C a mplete g U-Chik enome ggi|2 29209 713|g|gb|FJJ5136323.2| C hikungun virus isolat 661 co 00 gi|i|286203233239 m y 4 e h 7 b a p i S 4 1 k le . | G 0 v 1 u te 5 F 9 i 2 E rus s ngun g H 10 g gi| 8809 739 98 |gb J44 430 | Chi e n I C 0 o me HS42 train ya v kun .2| 0 g i|2 26 57836 50 |em |GQ 54 2308 RGC c ggi i|3i|26994207 27029| |gb|Fb|FN 428284.2| CChikun gunya v irus stra B o m 8 0 p g /KL07 lete ge in IT irus gi i|2|2800 20 82 396 7|ggb|G J5 295 14.1 hiku guny nome A0 comp is |2 8 8550 93 77 8| b| U 136 48 | C ng a lete g 6209 7 95 23 7| gb FJ 19 29 7.2 hik uny virus olate LK 7-RA1 co e i nome mple (P 0783 27 36 6|ggb| |FJ 445 935 .1| | Ch ungu a viru solat te ge e SG B)CH5 s is 39 63 10 |gb b| GU 513 445 2.1 Chik ikun nya n 8 o o 0 E G | | me 8 co late HIC 56 2|g gb |G Q 301 65 .2| Ch ung gun viru mple HD9 |g b| |FJ U9 42 78 7.1 Ch ikun uny ya v s stra SGE t e 3 i HIC 508 b| G 4 08 82 1. | C ku gu a v irus geno in R FJ U1 45 2 13 1| hik ng ny iru HT0 com me c GC p 7 51 99 46 23 .1| Ch un uny a v s iso ompl B l 7 e 8 3 t e i . 0 i 5 ete lat gen 8 co 36 35 3.2 1| Ch kun gun a v rus ome gen 5/KL0 mp 35 3. | C Ch iku gu ya irus stra e LK 8 l o e ( com te g me PB .1 1| hi iku ng nya viru iso in F eno )C ple | C Ch ku ng un iso la D s te g me late hi iku ngu un ya virus isol te SG 0801 H160 e MY nom ate ku n n ya vi 8 7 i E 8 s c /08 H om e ng gu ya vi rus ola c L /06 ple un ny vir rus str te K(E ICHS ompl 8 t e ete gen 4 ya a v us iso ain CU H)C ge om ge no H7 2520 vi iru iso lat RG -Ch ic R m n 8 o ru s 7 e e i l m k 08 NA co 68 s str ate CU CB e m c 1 is ai om ple ol n SG -Ch 20/ 3 co te ple at F mp KL ge D EH ik e te no LK 08 IC OB 07 c lete g e me no ge om (P 02 HD F c m n 96 om B) 31 p e om 8 C ple lete e H com 08 g te 53 c ge eno 08 plet om me no p e co le m te m gen e g pl et ome eno e m ge e no m e
2 61
gb
3|
49
|2
12
gi |2 9 gi 61 961 |2 2 2 96 gi| 12 452 449 29 9 2 45 61 12 |gb |gb 24 | |H H 4 |g gi| 86|g b|H M0 M0 gi| 4 2 M b| 4 12 HM 04 580 57 42 961 gi| 8 2 5 9 2 04 12 45 79 55 .1 9.1 42 5 1 9 | 6 7 5 7|g C | 95 . gi|6 8 59 gi| 295 b|E |gb|H 7.1 1| C hik Chi 29 gi| 4 102 h |C |gb F0 M 29 iku ung kun 61 02| 0 h 2 61 | 2 dbj| E 4 7 i 45 24 F0 57 ku ng uny gun 14 AB8 32 u 56 2 1 gi|1 603 71 |gb .1| 97.1 ngu ny a v ya 1|g 33 01. 4 n | b|H HM 0.1 Ch | C ya a v iru vir 77 3| C s 97 M 0 hiku 04 45 | Ch ikun hiku vir irus st us s 30 gi|2 ngu 5 u | 8 g i g n 81 k 03 s s ra t b| 961 gi|2 nya .1| ung unya gun str trai in K rain 961 245 EF45 3.1| viru Ch Ch uny 244 s ge v y a n 0 6 52| 24 iku iku a v irus a vir in S CO 14 44 93 89| gb| nes . n V 3 6 gb| n for HM HM04 1| Ch gun gun irus stra us s 04 92 -95 1-8 non y s y i t 0 i 5 k r n a s a 4 t 8 u rai IN ain 44-9-95 co 8 co truc gi|6 578 10. ng vir v i 157 tura r u 8.1 1| C nya us s us n IN D-7 RS 5 c commp mp 945 l pr |C gi|26 h tra str D- 3 U om p le le 0 o i h v k 7|gb 241 t i gi|42 094 |KJ4 ein str kungu ungu irus s in G ain I 63-W-MH 1 co pleletete g te 8670 9|gb u nya nya tra ibb -63 B 5 c m te g en ge 516 855| |FJ8 24.1 ctural viru viru in A s 63 402 1 c om ple ge en omno emb 078 p | F r s |HE8 s o o C o 9 t 7 h e .1| C ikun in c strai strai 1556-263 9 co mp plette g nommee me 0646 gi|61 n h g mp let e gen e 21 o n 1.1| 1 c i u k m P 5794 n u o T ngu plet O7 H3 com m le e g e om ya v Chik 504|g 2 n p 9 3 n t e i u 5 y 3 r ngun 1 u a vir p le e e o 7 7 b|KJ4 gi|615 ya v us s s strai cds iso460 ccomplete te ggennommee 7 50 5162 79450 i n r t o r u l 3 le ge en om e a a 0 9 s m . 1| Ch |gb|KJ i t gi|5419 ikung complet n 0706a9659 ce: CH pletete genomomee 45162 87890|g e n e I o u 2.1| C 1 T K b|KF31 gi|18940 hikung nya virus genome w commpleteV-13genoomme 11318 8729.1 9700|gb 00 p s u 1 s g t l n e r e t |E | 2 1 y a r e C a U t a in e 703759.1 hikung n 2 gi|4293239 unya v virus strain 3462 coin NC/20genoomme A64139 | Chikun 95|emb|FN iru m le 11-5 e 53 gunya vir 3 295484.2| te 13 6 Chikungun us strain s isolate chik 807 compple y a M gi|429324006|e sy com te ggeennom8e 415 virus comp Y002IMR mb|FN295483.3 p le o le te /0 m te geno e 961 genome is | Chikungunya vi olate MY/06/BP complete m rus complete ge 6 /3 7350 geno genomee nome isolate M gi|189409706|gb|EU703761 m ic Y /0 R 6/3734 genom NA 54 .1| Chikungunya virus strain ic RN MY019IMR/06/BP com8ple 70 te genomeA 20 gi|189409709|gb|EU703762.1| Chikungunya virus strain MY021IMR/06/BP complete genome 22 35 genome 62 32 nome 597 virus strain MY003IMR/06/BP complete te ge ple m co 1 G0 e W gi|189409703|gb|EU703760.1| Chikungunya m M o ST n ge enome 7 39 CHIKV pletete g 6 ngunya virus isolate strain AR 18211 cocm iku le Ch p 1| 7. m 57 o 79 J6 e 0 2 virus -psg plete gengoemneom.2 w ya n lo u gi|659902275|gb|K g s n s u 6 ik o h 2 R m 05.1| C mplete 0416 us strain type co 8|gb|HM0458 gunya vir -African protoya virus 1c|oref|NC-C0HIKe4n8ome321 n u ik gi|29612453 h C | n .1 ungu ain S27 5475 2016 te gK88 21 M045811 virus str 4162.2| Chik gi|277 DEL-com1p6le-CHTI11 0 555|gb|H 4 a 2 y 1 n 6 u 9 2 g i| 30 g 00 FS L-20 6-PAK6 2 C hikun 6004 S C N | | L f .2 e T r 4 | n I 1 E 2 0 i 1 0 4 0 5 A 9 a D H 6 7 r 00 -2 -C -P IK 3 54 st |AF3 gi|277 DE-L2016-20166-C-HPATT19 4686|gb 00 virus a y A gi|2773 n L K 1 L 0 E DE -20 0166-P HI IK87 39 ngu D u k i 2 L 1 h C K 7 H C I 0 E D DELL-2 0166-CCHIK6598 9.1| 4580 0 DE L-2-20116--CHHIKIK557 M |H b g | DEEL -2016 6-CCHHIK 49 5 4 D EL-20 01 6- -C 2 61 D L -2 01 16 gi|29 DEEL L-2 20 D E LD E D
gi
42 Chikungunya virus strain RGCB729/09 complete genome 195 gi|655168276|gb|KJ796845.1| gi|6551 78|gb|KJ796846.1| Chikungunya viru 0 gi|65516682 s strain RGCB755/09 complete gen 0 gi|6 82 |gb|KJ796844.1| 0 gi| 5516828874 ome Chikungunya viru |g 6 b 0 g |K 5 5 J 7 1 9 6 s 6 strain RGCB730/0 851.1| Chik 0 g i|2985 8280|gb|K ungunya vir 9 complete geno 0 gii|| 2885778082|gb|GJ796847.1| us str me
29 gi|
29
96
24
|2 gi
61
52
24
9 |2
gi
61
gi|
29
gi|
e 486 nomme 401 e ge no e 368100 plet e ge nome com plet e genomme e 013 com plet ge no om e e5117 e e 7 t m g H3 0 en omom e 9 21 in S H 28 51 coomplepletete g genenm e S 9 stra le e g irus IPD/A n PM2 237 c comomp pletlete enonommee e86 ya v n 30 48 c m p te g ge no om m rai o t 5 m 0 gun trai c kun irus s virus s in ArDrA 308076 57 5 co ple ete ge en eno l e g g Chi 6 v a 6.1| gunya unya s str rain AHD 1 rA 2 IbH3 commpplet te te 581 ng viru s st ain in A in -75 co m pleple ikun M04 iku a 8 o b|H | Ch .1| Ch gunya a viru s str stra s str 455 2-7 1 c comom 4.1 70|g y viru rus iru in 1 41 /00 3 4 c 580 245 785 kun i un i 4 5 0 4 961 Ch kung nya ya v ya v stra in 3 /NR 548 57 M0 |HM gi|2 u i n .1| b H n ra 5 1 3 | s h g g |gb 815 .1| C ikun ungu ungu viru s st u/8 hH KT2 35| 245 h 480 0 u H P J 045 k k a i 4 r i 2 2 961 HM 58 7.1| C | Ch | Ch uny a vi ain rain ain 961 gi|2 04 gb| t tr g y tr 1 1 .1 gi|2 67| |HM 458 818 786. ikun gun s s us s s s 245 0 5 |gb n h ru ir u 5 i 4 82 961 |HM M0 M04 1| C hiku a v a v vir 45 gi|2 y a y |gb |H 12 C 4. H 3 96 |gb |gb| 581 .1| gun gun uny 57 8 gi|2 76 3 n 24 n g 4 0 8 u 61 45 iku ikun M0 58 hik 44 29 12 2 h 4 H 6 | 1 gi| C 0 29 | C Ch M 96 |gb 1| 1 2 . | 4 gi| H . gi| 56 |gb| 800 790 1.1 24 9 5 6 5 4 61 04 04 457 45 M M 0 |H HM b|
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