Molecular Epidemiology Of Measles In India Biology Essay

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Measles is a childhood disease of great morbidity and mortality in India and worldwide. Since measles surveillance in India is in infancy, there is a paucity of countrywide data on circulating measles virus genotypes. In order to prepare the baseline sequence data, this study was carried out in 24 states (out of 35 states and Union Territories) of India by MeaslesNetIndia, a network of 27 centres and sentinel practitioners across India. This network investigated 52 measles outbreaks in geographically representative areas during 2005 to 2010 (June). All outbreaks were serologically confirmed by detection of anti-measles virus IgM antibodies in serum and/or oral fluid samples. RTPCR-Sequencing studies, using WHO recommended protocols yielded 203 N gene, 40 H gene and 4 M gene sequences during this period.

Measles genotypes D4, D7, D8 were found to be circulating in various parts of India during the study period. Four lineages of Indian D8 genotypes (D8a, D8b, D8c and D8d) were detected. Lack of geographical or time clustering of lineages indicated free transmission of measles strains across the country.

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In conclusion, this study generated large countrywide sequence database which would form the baseline for future molecular studies on measles virus transmission pathways in India. This study has created support and capabilities for countrywide measles molecular surveillance which needs to be carried forward.

Introduction:

Measles is a childhood disease of great morbidity and mortality worldwide. Although worldwide, the number of reported measles cases declined by 67%, from 852 937 in 2000 to 278 358 in 2008 and deaths attributed to measles have declined by 78%1, risk for illness and death from measles still exists in countries with variable routine vaccination coverage like in India. Though there are various studies carried out describing the measles epidemiology in India, the country lacks knowledge generated from systemic countrywide measles surveillance. In absence of countrywide case based surveillance, globally molecular epidemiology studies have provided an important data for mapping transmission routes, documenting the elimination of endemic virus strains and indicating the risk groups. 2, 3

Measles virus (MeV), an enveloped virus with a single stranded, negative sense RNA genome, is a member of the genus Morbillivirus within the family Paramyxoviridae. MV is highly contagious and causes a disease characterized by high fever, cough, coryza, conjunctivitis and appearance of a maculopapular rash. MV is a monotypic virus, but genetic variability exists among wild type strains.6 The protocols and nomenclature for genetic characterization of wild-type measles viruses have been standardized by the World Health Organization (WHO) which helped in detecting circulation of 23 genotypes of MV.7 In June 2010, new genotype of measles, provisionally assigned as d11, was detected which need to be finally designated by WHO.8 Molecular studies have been instrumental in many countries for tracking national and international transmission pathways of measles (MV)strains. 3,9,10,11,12,13 In Southeast Asia, during 2005-2006, virological surveillance activities expanded throughout the region, and D4, D7, D8, D9, D5 and G3 genotypes have been detected in the countries of the region.14

The National Institute of Virology (NIV), Pune has been instrumental in initiating molecular surveillance in the state of Maharashtra which detected presence of indigenous genotypes D4, D8 until 2005 and circulation of D7 was detected in 2006.4,5 Since data of circulating genotypes was limited from other parts of the country, the study was extended to other geographic areas of India in 2005.15 The baseline sequence databases of measles genotypes circulating in all the geographic areas of India would be the most useful resource in tracing the origin of strain/s and tracking transmission pathways and assessing the impact of control programs when India moves into measles elimination phase. The findings of this study from 2005 to 2010 have been described in this paper.

Materials and Methods

Establishment of MeaslesNetIndia Network:

In order to accomplish the objectives of this study, a voluntary participatory network of 17 centres in India was initiated in August 2005 and named as MeaslesNetIndia.15 In the year 2006, MeaslesNetIndia enrolled private paediatric and general practitioners from various states, first ever large scale public-private partnership for measles related work. Subsequently in 2008, network expanded to include 10 centres from Northeastern region of India. Network centres included ICMR centres, medical colleges and other research centres giving geographical representation in the country. The participating sentinel site collaborators were trained at three workshops on various aspects of this study including outbreak investigation strategies, specimen collection, lab diagnostics and role of each partner. These centres carried out outbreak investigations using local resources including funding. Financial assistance was made available through WHO for training of participants, logistics and testing at NIV, Pune.

WHO-SEAR Measles and Rubella Laboratory Network:

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The WHO SEAR measles laboratory network was established in 2003 and has expanded in a phased manner 19 laboratories in 2009. The laboratories are recruited through a process of consultation with the national governments. In India, WHO has designated NIV at Pune as WHO Regional Reference Laboratory and two measles laboratories based at NIV field unit, Bangalore and King Institute of Preventive Medicine, Chennai as WHO National Measles laboratories. All laboratories have been accredited and contributed isolates and clinical specimens for virus characterization.

Outbreak investigations by MeaslesNetIndia:

During 2005 to 2010, the network investigated 52 suspected measles outbreaks from 24 states. WHO case definition of measles was used. Measles case was defined as any person with fever of 38oC or more, and maculopapular rash (i.e. non-vesicular) and any one of the following, cough, coryza, or conjunctivitis or any person in whom the health professional suspects as measles was used for such investigations.16 Outbreak was defined arbitrarily as occurrence of 5 cases in 15 days period from a block of approximately 5000 population. Appropriate specimens for serology confirmation and virus isolation were collected and transported in appropriate cold chain conditions to NIV for laboratory studies. Sporadic cases were investigated from Imphal, Dimapur, Dabra and Valsad areas where no outbreaks were reported.

Specimen collection:

Throat swabs (TS) and urine specimens were collected for virus isolations and blood samples for serology, from 5-10 representative acute measles cases in each outbreak. Oral fluid proved to be most convenient sample for collection and transport to the laboratory in Indian climatic conditions wherever serum could not be obtained.17 Clinical samples (Throat Swabs, urine, oral fluid and blood) collected were processed and stored appropriately until transportation to NIV at Pune. For molecular studies on fulminant SSPE and MIBE cases (one each), brain, serum/CSF samples have been sourced from Human Brain Tissue Repository (Human Brain Bank) of Dept of Neuropathology, NIMHANS, Bangalore. A total of 658 sera, 283 Throat swabs, 80 oral fluids & 302 urine samples from 845 measles cases were obtained and received during the study period.

Serological studies:

All outbreaks were serologically confirmed as Measles by IgM ELISA using WHO recommended kits (Dade Behring/currently Siemens and/or Microimmune kits) using manufacturer's instructions. Oral fluid samples were also tested by Microimmune kits for detection of IgM antibodies to measles. The measles IgM negative samples were tested for Rubella IgM using Microimmune kits as per manufacturers' instructions.

Virological studies:

Virus isolation and identification of measles strains was done according to the standardized methods at NIV, Pune, NIV Bangalore and KIPM, Chennai using B95a cells or Vero-SLAM cell line (from Dr Yanagi via CDC/WHO for Network Laboratories) according to the standard CDC-WHO protocol.18,19 In brief, processed throat swabs and urine samples were inoculated onto the cell lines and observed for cytopathic effect (CPE). Inoculated cells were blind-passaged up to three times before being discarded as negative for virus isolation. Cells were harvested when the CPE was maximum (3+ to 4+) in the third passage culture flasks.

Molecular studies:

Clinical samples and virus isolates obtained and/or sent by collaborators were used for RT-PCR and sequencing studies. Viral RNA was extracted from infected cell lysates or clinical specimens using TRIzol LS reagent according to the manufacturer's instructions. RNA pellets were dried and resuspended in 50 μl of nuclease free water and stored at -70° C until amplification by RT-PCR. Sequencing studies were performed by Standardized protocols using WHO recommended primers. Primers MV63 (5'CCT CGG CCT CTC GCA CCT AGT 3') and MV60 primers (5'GCT ATG CCA TGG GAG TAG GAG TGG 3') were used to sequence 456 bp fragment of the N gene COOH terminal according to WHO recommendations. (Ref) Primers H2 (GAT CAT CCA CAA TGT CAC CAC), H4R (AGA TGA CAC ATT GTA ACC TCG), H4 (CGA GGT TAC AAT GTG TCA TCT) H6R (CGT ATG AAG GAA TCC TGT TAT) H7 (CCG AGA TTC AAG GTT AGT CCC), H10 (GTA TGC CTG ATG TCT GGG TGA) were used to amplify 1854bp complete coding region of H gene.3, 4, 7

Sequence analysis

The sequences of the N gene PCR products were derived by automated sequencing with primers MV60 and MV63 and the BigDye terminator 3.1v chemistry using reaction conditions that were recommended by the manufacturer (ABI 373, ABI 3100, Perkin Elmer-Applied Biosystems). Sequence proof reading and editing was conducted with Sequencerâ„¢ (Gene Codes Corporation). Measles N and H gene sequences obtained in this study were confirmed with both strands and subjected to alignment along with WHO standard reference N and H gene sequences14 using software ClustalX version 1.83. A phylogenetic analysis was conducted using MEGA version-4.20 The robustness of the groupings was assessed using bootstrap re-sampling of 1000 replicates and the trees were visualized with Mega programs. NCBI BLAST analysis was used to find Percent Nucleotide Identity (PNI) with the related strains.

Submission to Public genetic databases:

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Sequence cards were prepared for all the sequences including the available clinico-epidemiological information on each strain. All the sequences were deposited to WHO Sequence Database (Featherstone D - personal communication). 243 representative nucleotide sequences (N and H gene) were deposited in GenBank under accession numbers (EU812245-EU812316, FJ387130-FJ387157, FJ765064-FJ765087, FJ979795-FJ979797, FJ968742, FJ719484-FJ719489, FJ223133-FJ223168, FJ19763, GU306169-GU306174, GU827659, GU953671-GU953672, GQ420695-GQ420699, HM567314-HM567315, HM452160-HM452161, HM358867-HM358877, AY873971-AY873981, DQ987230-DQ987235, DQ335129-DQ335131, DQ345392-DQ345393, AY953414- AY953418, AY957557-AY957558, DQ923615, AY841168, HQ148303-HQ148309).

Indian Measles Sequence Database is also maintained at NIV for future use for measles elimination programme. Detailed analysis was done to find out intra-genotypic diversity of measles strains circulating in India.

Results and Discussion:

This paper describes overview of outbreak investigations and molecular studies done during the period 2005 till June 2010 giving comprehensive baseline data for a large country like India. Of the total 28 states and 7 union territories in India, 24 were covered during this investigation covering 85.7 % of India's geographical areas. Total of 52 outbreaks and several sporadic cases (where outbreaks were not reported) were investigated. Key data elements as available are described in Table 1. The details of individual outbreak investigations would be published elsewhere.21-24 Since the aim of the study was to create countrywide baseline molecular data on circulating genotypes of measles virus, representative outbreaks were investigated during this period.

Table 2 describes the age group distribution among 845 clinically suspected measles cases from which samples were collected and confirmed as measles on IgM ELISA. This didn't reflect the extent of the outbreak and include all cases affected. Convenient sampling could not be ruled out. Most affected age group was 1 yr to 6 yr (51%), and notable proportion of cases (30.3%) was recorded in 6-15 years age group as well. Small proportions of cases were less than one year age. Measles in infants especially in orphanages have been documented in India from previous studies in Pune and Chennai.5, 22 Table 1 gives reported age range from each of the outbreaks, described on the basis of line listing maintained by the public health authorities of the area and could be taken as accurate. Variety of age groups involvement in different geographic areas indicate diversity of measles transmission patterns in India and calls for need to investigate the outbreaks in a standardized manner considering all cases and extent of the outbreak but not limiting the age groups. Other studies documented cases in above 5 years age group.21, 23 Table 1 describes the limited documentation of attack rates and mortality data as encountered during the outbreak investigations. This documentation should be improved further in Indian settings.

Suspected cases in older age group of more than 15 years were encountered in outbreaks investigated in post-tsunami camps in Andaman, in East Siang in Arunachal Pradesh, Supoul in West Bengal, Kangra in Himachal Pradesh, Kargil in Jammu and Kashmir states. Attempts were made to have serological confirmations by IgM ELISA on serum or oral fluid specimens. Of 64 suspected measles cases in more than 15 years group, 33 were confirmed as measles on IgM ELISA which included three pregnant women. Adolescent outbreaks in Jammu and Kashmere, 21 outbreak in Arakkonum, Vellore district of TamilNadu25 and outbreak in medical students in Manipal, Karnataka were also confirmed as measles on lab studies. These studies indicated the possibility of susceptible adolescent population in some areas probably where they neither had an opportunity for immunization nor exposed to natural measles. The susceptibility of this age group should be investigated and documented further on nation-wide studies.

Table 3 describes the seasonality of laboratory confirmed measles based on available data. Data indicated measles transmission all round the year in India, with variation in pattern in different geographic areas. The seasonal patterns would clearly be detected by countrywide laboratory surveillance. Seasonality in this table is an indication of circulation of measles strains which could be transmitted to other geographic areas.

Measles genotype circulation in India:

Table 1. described the indigenous measles genotypes circulation in India. During the study period D4, D8 and D7 genotypes were circulating in various parts of India. Though circulation of D4 and D8 had been documented since 19964 and continued in the study period also, D7 genotype was detected to have limited circulation in three cities only.5 Genotype A had not been detected in this period. In the current extensive study covering 24 states, we didn't detect additional D7 genotype which pointed out to the possibility of limited circulation of D7. During this period, there was no documentation of measles genotype other than D4, D7 and D8.

Intra-genotype differentiation:

Since D4 and D8 was found to be circulating extensively in India in the last 15 years, it was thought worthwhile to look at the intragenotypic diversity. When the phylogenetic analysis of all the D8 N gene sequences was done (Fig 1), it was found that Indian Measles D8 genotype could be differentiated into four distinct lineages labelled as D8a, D8b, D8c and D8d.

MVi/Tumkur.Ind/04.08 (Genbank Accession number FJ719484) was designated as the reference strain for D8a genotype. D8a was the most diverse genotype circulating from 2005 to 2010, in geographically diverse states as Maharashtra, Karnataka, Andhra Pradesh, Orissa, Gujarat, Sikkim, Kerala, and Manipur. Circulation of D8a was detected mostly in central, western and southern part of India. MVi/Gulberga.Ind/14.07 (Genbank Accession number FJ979796) was designated as the reference strain for D8b genotype which was circulating since 2005 till 2009 in the states of Assam, West Bengal, Uttar Pradesh, Himachal Pradesh, Arunachal Pradesh, Nagaland and Andaman and Nicobar islands. MVs/Papumpare.Ind/51.07/1 (Genbank Accession number EU812248) was designated as the reference sequence for D8c genotype. No isolate as yet has been made from this genotype. Circulation of this genotype seemed to be limited to Andhra Pradesh, Orissa and north-eastern states of Nagaland and Tripura during the period 2006-08. MVi/Pune.Ind/33.05 (Genbank Accession number FJ223159) was designated as the reference strain for D8d genotype. Circulation of this genotype was limited from 2005-07 to Maharashtra, Andhra Pradesh and Uttar Pradesh states. Our study did not document further detection of this genotype.

Various countries like China26, Russian Federation10, Indian Ocean islands27 and Kenya28 have documented sub-clusters of H1, D4, D6 genotypes from various studies. It would be interesting to see how the circulation of these D8 sub-clusters would evolve in time especially under pressure of vaccination campaigns in some areas. Genetic heterogeneity of dominant genotype D8 in India appears to be due to the presence of multiple, co-circulating lineages of this virus indicating unrestricted transmission across geographic areas, somewhat similar to circulation of dominant H1 in china. 26

Kenya study28 had indicated sub-clustering of D4 into four different groups Montreal like, India like, Johannesburg like and Ethiopia like sub-clusters. Phylogenetic analysis of Indian D4 genotype could not be differentiated into distinct lineages. With multiple chains of transmission, it could emerge out with distinct pattern in future which should be studied with countrywide surveillance.

D7 genotype has been found from only three cases as on date from India but on the analysis of H gene sequences showing the genetic diversity, point out to the possibility of three different lineages of D7 circulating in India and that all the three D7 evolved over the time.5 Measles Genotype D7 has been associated with measles cases in Europe during the late 1990's and early 2000's 29 but has not been detected in Europe in 2005-2006. The link between India and European D7 genotypes is not clear at this point of time. Subsequent data from both the continents and molecular evolution studies might throw some light on this link.

Co-circulation of D4 and D8 has also been documented from various areas in our study. Co-circulation of D8b and D8c has been noted in one outbreak from Arunachal Pradesh. Various countries have also documented co-circulation of various genotypes, probably due to multiple chains of transmissions during an outbreak.30, 31

Though measles surveillance in India is in infancy, during the last 15 years period only Clade D genotypes (D4, D7, D8) have been detected in India whereas surrounding countries have detected D4, D7, D8, D9, D5, H1, d11 and G3 genotypes have been identified.8,14 These external genotypes didn't get imported into India throughout the period of 15 years (1995-2010). Table 4 showed year wise data on viruses detected on sequencing studies. Possibility of missing or importation of other genotypes needs to be further studied and documented with continuous surveillance countrywide molecular surveillance in India.

In the study period, sequencing was carried out on autopsied specimens from a case of Sub acute sclerosing panencephalitis (SSPE) 32 and Measles Inclusion Body encephalitis (MIBE), each (Shankar et al unpublished data). There is a paucity of studies documenting genotypes identified from SSPE cases 33,34 but this could be a good resource in India to find out missing links in measles transmission in past.

Though representative measles outbreaks were investigated in laboratory, the baseline sequence data generated from our study would be useful in future to track transmission pathways of Indian strains worldwide and also to document importation of globally circulating genotypes into India. This database would expand with the expansion of laboratory based measles surveillance in India. Increased international collaborations, real-time outbreak investigations supported by molecular studies and global sequence database would help document the pathways of measles transmission to, from and within India. The current study has demonstrated the effective use of molecular methods to monitor the measles scenario and changing epidemiology of measles in modern India.

Table 1: Key data elements from outbreaks investigated by MeaslesNetIndia.

( .xls table)

Table 2 Age group distribution of investigated cases and IgM positivity

AGE

Cases investigated

(N, %)

Samples Tested

(N, %)

IgM +ve for Measles

(N, %)

IgM +ve for Rubella

0-6 months

22 (2.6)

22

11

6 months- 1 year

63 (7.5)

57

37

1-3 years

146(17.3)

128

86

3-6 years

278(33)

240

155

1

6-12 years

231(27.3)

205

130

3

12-15 years

25 (3)

23

14

>15 years

64(7.6)

62

31

5

Unrecorded

16 (1.9)

16

9

TOTAL

845

753

473

08

Table 3. Seasonality pattern of measles transmission

Fig 1 MeaslesNetIndia- Geographic representation

JAMMU and Kashmir

LUCKNOW

JABALPUR

MANIPAL

AHEMEDABAD

MEGHALAYA

AGARTALA

IMPHAL

NAGALAND

DIBRUGARH

ARUNACHAL PRADESH

MIZORAM

SIKKIM

PORT BLAIR

COONOR

PATNA

KOLKATA

CHENNAI

BANGLORE

GUNTUR

BHUBANESHWAR

MUMBAI

PUNE

NAGPUR

JODHPUR

UTTARANCHAL

CHANDIGARH

D8d

D8c

D8b

D8a

Fig 2. Indian D8 subgroups(N gene)

D4 seq analysis

Fig 3.Indian D4 genotype (N gene)

Fig 4. PHYLOGENETIC ANANLYSIS OF REPRESENTATIVE H GENE SEQUENCES FROM INDIA