Molecular Epidemiology of Enteroviruses Associated with Hand, Foot and Mouth Disease in South India during 2015 to 2017

doi:10.21203/rs.3.rs-1635954/v1

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Molecular Epidemiology of Enteroviruses Associated with Hand, Foot and Mouth Disease in South India during 2015 to 2017

https://doi.org/10.21203/rs.3.rs-1635954/v1

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Hand, foot and mouth disease (HFMD) is a common childhood infection caused by human enteroviruses and clinically characterised by fever with vesicular rash on hand, foot or mouth. While Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CVA16) were the major aetiologies of HFMD in India earlier, there were sparse data on recently circulating enteroviruses associated with HFMD. Here we describe the molecular epidemiology of enteroviruses associated with HFMD in South India from 2015 to 2017. We used archived enterovirus real-time reverse transcriptase (RT) PCR positive vesicle swab and or throat swab specimens from clinically suspected HFMD cases collected from four secondary care hospitals in South India between July 2015 to December 2017. PCR amplification and sequencing were done based on 5’VP1, 3’VP1, VP2 or 5´NCR regions to identify enterovirus types. Genetic diversity among enteroviruses was inferred by phylogenetic analysis. Of the 107 enterovirus RNA real-time RT-PCR positive HFMD cases, 69 (64%) were typed as CVA6, 16 (15%) were CVA16, and 1 (1%) was CVA10. Whereas 21 (20%) cases were not typable by any of the methods used in the study. The majority of HFMD cases (89, 83%) were children below five years, while 11 (10.3%) were adults. 5’VP1 yielded the maximum number of enteroviruses genotyped and the phylogenetic analysis showed the CVA6 strains belongs to the subclade D3 while the subclades of CVA16 and CVA10 were B1c and D, respectively. The predominant etiology of HFMD in South India during 2015-2017 period was CVA6 followed by CVA16 and CVA10.

Hand

Foot and Mouth Disease virus

HFMD

Coxsackievirus

Enterovirus

maculopapular rashes

vesicular rashes

Hand, Foot and Mouth Disease (HFMD) is an acute febrile infection caused by enteroviruses (EVs), mainly found in children below five years of age and clinically characterised by fever and typical vesicular lesions on hand, foot, mouth or buttocks (1). Enteroviruses are single-stranded RNA viruses belonging to Picornaviridae family and the genus Enterovirus (2). Even though HFMD is a self-limiting infectious disease, this can also lead to severe clinical manifestations such as meningitis, encephalitis and myocarditis in children (3–5). Since 1950s, HFMD was reported in the form of outbreaks and sporadic cases, caused mainly by coxsackievirus A16 (CVA16), enterovirus 71 (EV-A71), coxsackievirus A6 (CVA6), coxsackievirus A10 (CVA10), and also by other EVs in EV-A, EV-B and EV-C groups (6,7). The transmission of HFMD is mainly through direct contact with mucus, saliva, fluid from blisters, or feces of an infected patient and indirectly by fomites (8).

India reported the first outbreak of HFMD with mild symptoms in children caused by EV-71 from Kerala in 2003 (9). In the following years, a significant morbidity and mortality were reported in children with EV-71 infection from Uttar Pradesh during 2004 to 2006 (10). In 2007, an epidemic of HFMD was reported in children with mild symptoms from West Bengal (11). Since then, the enterovirus types such as CVA16, CVA6, EV-A71, CVA10 and E-9 were circulating sporadically in India and caused mild to severe infections in children (12–16). Recent reports from Europe, North America and Asia indicated the emergence of CVA6 associated HFMD outbreaks and sporadic cases, meanwhile, CVA16, EV-A71, and CVA10 were sparsely distributed (17,18). CVA6 is one of the major enterovirus strains identified in both mild and severe cases of HFMD globally in recent years (19,20).

While there were insufficient data on etiology and clinico-epidemiological profile of HFMD cases from south India in the recent years, our retrospective study mainly focused on the molecular epidemiology and clinical features of HFMD in South India from July 2015 to December 2017.

Clinical specimens and epidemiological data collection

The archived pan enterovirus real-time RT-PCR positive vesicle swab and or throat swab specimens of clinically diagnosed HFMD (1) cases collected as a part of the sentinel surveillance in 2015 and those received for virological diagnosis in 2016 and 2017 were used in this study. Any clinically diagnosed HFMD cases with vesicular lesions anywhere in the body with or without fever and from South India during the period of July 2015 to December 2017 were enrolled. The specimens were anonymised using a unique identifier. Clinical and epidemiological data were obtained from the case record forms or laboratory request forms.

Enterovirus detection and typing

The algorithm for enterovirus detection and typing were shown in Figure 1. The total RNA was extracted from vesicle and throat swab specimens using QIAamp Viral RNA Mini Kit (QIAGEN, Germany) as per manufacturer’s protocol. The extracted RNA was subjected to pan enterovirus real-time reverse transcriptase (RT) PCR using respiratory pathogens 21 kit (Fast Track Diagnostics - FTD, Luxembourg).

The multi locus-specific PCR and sequencing enabled the screening of enteroviruses associated with HFMD. Enteroviruses were typed from the positive samples by PCR based on partial 5’VP1, 3’VP1, VP2 and 5’NCR regions, followed by Sanger sequencing. Pan enterovirus nested RT-PCR was performed by initial cDNA synthesis and subsequent nested RT-PCR targeting 5’VP1 (PCR-1: ~992 bp and PCR-2: ~375 bp) (21). Upon PCR failure based on 5’VP1 region, VP2 based semi-nested RT-PCR was performed (PCR-1: ~583 bp and PCR-2: ~367 bp) (22). Subsequently, nested RT-PCR targeting 5’NCR region was carried out (PCR-1: ~529 bp and PCR-2: ~397 bp) when other sequence regions failed to amplify in PCR (23). CVA6 specific semi-nested RT-PCR was carried out for samples collected in 2015 based on 3’VP1 (PCR-1: ~657 bp and PCR-2: ~420 bp) (24). For each PCR, the reaction mix contains forward and reverse primers (Table S1), AgPath-ID One-Step RT-PCR reagents (Applied Biosystems, USA) and the extracted RNA. All the PCR assays were performed according to the previously described methods (21–24). BigDye terminator V3.1 cycle sequencing kit was used to sequence purified PCR products in ABI-3500 Genetic Analyser (Applied Biosystems, USA). The sequence quality was analysed and edited using Sequencher 5.4.6 software (Gene Codes Corporation, USA). BLAST was used to determine the sequence identities with reference strains in GenBank (NCBI). Those could not be typed by previous methods were subjected to CVA16 and EV-A71 specific real-time RT-PCR targeting VP1 locus as described elsewhere (25,26).

Phylogenetic analysis

All the study sequences were aligned with the reference sequences retrieved from GenBank- NCBI by multiple sequence alignment in ClustalW (Table S2). Phylogenetic analysis was performed using the software MEGAX (version 10.2.5) (27). Phylogenetic dendrograms based on 5’VP1, 3’VP1, VP2 and 5’NCR partial sequence regions were constructed using the neighbour-joining method with Kimura 2-parameter (K2P) model. Bootstrap values (>70%) on each node were generated using 1000 replicates, and thereby, the statistical efficiency of the tree was calculated.

Statistical analysis for the demographic and clinical data was done using STATA/SE software (version 14.1).

A total of 172 archived samples from clinically suspected HFMD cases were included in this study. Of which, 148 samples were collected from secondary care hospitals in Kozhikode, followed by 18 from Wayanad, 4 from Malappuram districts of Kerala state, and 2 from Shimoga district of Karnataka state in South India. Enteroviruses were identified in 107 (62%) cases out of 172 clinically suspected cases of HFMD. Among these 172 samples, 107 were pan enterovirus RNA positive by real-time RT-PCR. Of the 107 cases, 89 (83%) were below five years of age, and 18 (17%) were between 5 and 65 years (Table 1). Eleven (10.3%) adults showed HFMD infection in the study. The number of affected males were 68 (64%), while 39 (36%) were females. The mean age of the patients with HFMD was 4.4 years, ranging from 9 months to 65 years. Meanwhile, the mean age of the male and female population was 5.4 and 2.8, respectively. The most common clinical features observed among the enterovirus associated HFMD cases were fever (90, 84%) and vesicular rash (107, 100%). Respiratory symptoms such as cough (18, 16%), coryza (6, 6%), breathlessness (1, 1%) and sore throat (9, 8%) were also observed among patients. Some of the HFMD cases had oral ulcers (3, 3%), headache (4, 4%), vomiting (2, 2%), myalgia (1, 1%), abdominal pain (1, 1%) and lymphadenopathy (1, 1%). Rash in patients with hand, foot and mouth disease was illustrated in Fig. 2. We also noticed other rash types such as papule (25, 23%), maculopapular rash (19, 18%), crusted lesion (11, 10%), macule (7, 5%), erythema (8, 8%), pustule (3, 3%), erosion (3, 3%) and bullae (1, 1%) in patients. Rash was seen in hand (87, 81%), foot (91, 85%), mouth (51, 48%), soles (39, 36%), palms (41, 38%), buttocks (32, 30%), and other parts of the body (< 20%).

Table 1

Demographic and clinical characteristics of enterovirus associated HFMD
Variable	CVA6 (n = 69)	CVA16 (n = 16)	CVA10 (n = 1)	EV types not identified (n = 21)	Total EVs (n = 107)
	n (%)	n (%)	n (%)	n (%)	n (%)
Gender
Male	44 (64)	10 (63)	1 (100)	12 (57)	67 (63)
Female	25 (36)	6 (37)	0	9 (43)	40 (37)
Age
Mean (± SD)	2.56 ± 3.9	12.68 ± 17.3	NA	4.6 ± 5.9	4.4 ± 8.4
Range (in years)	0.8 to 30	1 to 65	NA	0.8 to 22	0.8 to 65
Age group (in years)
≤ 5	63 (91)	9 (56)	1 (100)	16 (76)	89 (83)
> 5	6 (9)	7 (44)	0	5 (24)	18 (17)
Clinical Parameters
Cutaneous rash	69 (100)	16 (100)	1 (100)	21 (100)	107 (100)
Fever	58 (84)	11 (69)	1 (100)	20 (95)	90 (84)
Cough	10 (15)	3 (19)	0	5 (19)	18 (16)
Sore throat	3 (4)	4 (25)	0	2 (10)	9 (8)
Coryza	3 (4)	0	0	3 (14)	6 (6)
Headache	1 (1)	0	0	3 (14)	4 (4)
Oral ulcers	1 (1)	2 (13)	0	0	3 (3)
Vomiting	0	0	0	2 (10)	2 (2)
Breathlessness	1 (1)	0	0	0	1 (1)
Abdominal pain	0	0	0	1 (5)	1 (1)
Lymphadenopathy	1 (1)	0	0	0	1 (1)
Myalgia	0	0	0	1 (1)	1 (1)
Types of Rash
Vesicle	69 (100)	16 (100)	1 (100)	21 (100)	107 (100)
Papule	17 (25)	3 (19)	0	5 (25)	25 (23)
Maculopapular rash	12 (17)	2 (13)	0	5 (26)	19 (18)
Crusted lesion	11 (16)	0	0	0	11 (10)
Erythema	5 (7)	0	1 (100)	2 (5)	8 (8)
Macule	5 (7)	1 (6)	0	1 (10)	7 (5)
Pustule	1 (1)	0	0	2 (10)	3 (3)
Erosion	1 (1)	2 (13)	0	0	3 (3)
Bullae	0	1 (6)	0	0	1 (1)
Rash Distribution
Hand	55 (80)	13 (81)	0	19 (91)	87 (81)
Foot	61 (88)	10 (63)	1 (100)	19 (91)	91 (85)
Mouth	33 (48)	9 (56)	0	9 (43)	51 (48)
Palms	30 (44)	5 (31)	1 (100)	5 (24)	41 (38)
Soles	31 (45)	5 (31)	1 (100)	2 (10)	39 (36)
Buttocks	21 (30)	6 (38)	1 (100)	4 (19)	32 (30)
Trunk	13 (19)	1 (6)	0	5 (24)	19 (17)
Knees	11 (16)	3 (19)	0	2 (10)	16 (15)
Thigh	8 (12)	1 (6)	0	3 (14)	12 (11)
Elbow	8 (12)	1 (6)	0	2 (19)	13 (12)
Face	6 (9)	0	0	2 (10)	8 (8)

Enterovirus RNA was detected in vesicular (97, 91%) and throat swabs (10, 9%) of 107 cases of HFMD. Of the typed enterovirus cases (86, 80%), 69 (64%) were CVA6, 16 (15%) were CVA16 and 1 (1%) was CVA10. However, 21 (20%) cases of enteroviruses were not typable by any of the molecular typing strategies used in this study. In 2015, 24 HFMD cases were caused by CVA6 while one was CVA16. There were 20 HFMD cases due to CVA6 infection, while nine were CVA16 in 2016. In the following year 2017, HFMD was identified in 25 cases by CVA6, six by CVA16 and one by CVA10. HFMD cases were predominantly found in the rainy season from June to November.

The PCR and sequencing enabled CVA6 typing in 27 samples based on 5’VP1, followed by VP2 based typing in five samples and 5’NCR based typing in nine samples. Meanwhile, CVA6 specific nested RT-PCR based on 3’VP1 region enabled the typing of CVA6 strains in the other 28 samples, of which sequencing was possible only in 15 samples. Out of 16 (15%) samples positive for CVA16, PCR and sequencing could identify CVA16 in six samples based on 5’VP1, followed by VP2 targeted typing in two samples and 5’NCR based typing in one sample. Meanwhile, CVA16 specific real-time RT-PCR could type CVA16 in the other seven samples. CVA10 strain was identified only in one sample (1%) by 5’VP1 based PCR and sequencing. However, 21 samples were not typed by any of the enterovirus typing methods described in the study. A total of 66 sequences obtained from the study were deposited to GenBank database under the following accession numbers: MG885750, MG885751, MG840455 - MG840478, MG840480 - MG840487, MG869706 - MG869710, MG885752 - MG885768, MH160045 - MH160049, and MH160051 - MH160055.

The phylogenetic dendrogram of CVA6 based on 5’VP1 (398 bp), 3’VP1 (475 bp), VP2 (540 bp) and 5’NCR (301 bp) partial sequences are shown in Fig. 3. Based on partial 5’VP1 (n = 74), 22 CVA6 strains clustered together with other Indian strain (MH539787, 2016) with 96–100% nucleotide identity, followed by five strains showed 97–97.5% similarity with Australian strain (MH111055, 2017) in subclade D3 (Fig. 3A). Phylogeny of 3’VP1 sequences (n = 67) showed 14 CVA6 strains clustered together with Indian strain (MH539787, 2016) in 96.2–98.7% identity and one with 98.1% similarity with Australian strain (MH111055, 2017) in subclade D3 (Fig. 3B). VP2 sequence (n = 39) based phylogeny showed two CVA6 subclade D3 strains in high nucleotide similarity (99.1–99.3%) with Indian strain (MH539787, 2016), while the other three strains formed a cluster with Australian strain (MH111055, 2017) in 96.9–97.78% identity (Fig. 3C). Of the 5’NCR (301 bp) targeted phylogenetic tree of 41 sequences, four CVA6 strains showed high similarity (99.0%) with Indian strain (MH539787, 2016), while the other five strains showed 98–99% identity to Australian strain (MH111055, 2017) in subclade D3 (Fig. 3D). Phylogenetic analysis of CVA16 based on 5’VP1 (413 bp), VP2 (533 bp) and 5’NCR (301 bp) regions depicted in Fig. 4. The phylogenic tree based on 5’VP1 (n = 34) showed five CVA16 strains in high similarity (96.4–97.6%) with Indian strain (KY792583, 2013), followed by one with 98.3% identity to Indian strain (KY792578, 2013) in B1c subclade (Fig. 4A). Meanwhile, two CVA16 strains showed high nucleotide identity (97.2%) with Indian strain (KY792584, 2015) based on VP2 targeted phylogeny (n = 33) (Fig. 4B). One CVA16 strain was 99% identical to Indian strain (KY792577, 2012) based on 5’NCR sequences (n = 42) (Fig. 4C). CVA10 strain from the study showed 95.6% similarity with Indian strain (MH118041, 2017) in subclade D based on 5’VP1 region (410 bp, n = 33), depicted in Fig. 5. Nucleotide divergence among the inter-clades of CVA6 showed 13.6–30.9% for 5’VP1, 13.1–24.0% for 3’VP1, 15.6–19.2% for VP2 and 4.7–11.3% for 5’NCR. Inter-clade nucleotide divergence of CVA16 was 24.9–27.9% for 5’VP1, 21.4–24.8% for VP2 and 5.7–10.0% for 5’NCR, whereas nucleotide divergence of CVA10 inter-clades was 14.4–27.8% based on 5’VP1.

The predominant enterovirus type associated HFMD in South India was CVA6 during July 2015 to December 2017. This is in contrast to the earlier reports in 2003 to 2008, where EV-A71 and CVA16 were associated with HFMD both sporadically and in outbreaks (9,11). Since then, the cocirculation of CVA16, CVA6, EV-A71, CV-10, and E-9 was reported to cause sporadic infections in India (12,13). Here we report the cocirculation of CVA6, CVA16 and CVA10 in South India from 2015 to 2017, where CVA6 was the predominant strain, followed by CVA16 and CVA10. CVA6 has been recently emerged as a globally predominant etiology of HFMD, while other enteroviruses were sparsely distributed in recent years (5,17,18,28,29). It is worth noticing the non-detection of EV-A71 in this study.

Children below five years are mainly affected by HFMD with male gender preponderance, as described in previous reports (30,31). Typical HFMD presentation with upper respiratory tract symptoms and atypical rashes such as crusted lesion, macule, erythema, pustule, erosion and bullae were observed in the present study.

The multi-locus PCR and sequencing approach enabled the typing of 80% enteroviruses in clinical specimens. By targeting 5’VP1, 3’VP1, VP2 and 5’NCR regions, the enteroviruses were typed as CVA6, CVA16 and CVA10. The highest success rate of enterovirus typing was achieved by PCR and sequencing based on structural protein-coding genes such as 5’VP1, followed by 3’VP1 and VP2 regions (21,22,24). Sequencing based on 5’VP1 yielded the maximum number of enterovirus typing out of four strategies used in the study. Enteroviruses that could not be typed by previous methods were sequenced using PCR products based on 5’NCR. Hence 5’NCR can also be a target for the genotyping of enteroviruses with certain limits of accuracy. Targeting 5’NCR region for PCR and sequencing will be helpful in genotyping of enteroviruses when amplification of other genomic regions by PCR fails. (23). The reason for unsuccessful genetic identification of enteroviruses in clinical specimens may be due to insufficient viral load, RNA degradation, frequent genetic mutations or recombination (32). The nucleotide variations among enteroviruses in the subclades of CVA6, CVA16 and CVA10 were high based on 5’VP1, 3’VP1 and VP2, while 5’NCR showed less genetic divergence. So that 5’VP1, 3’VP1 and VP2 could be efficiently used for finding the evolutionary relationships among enterovirus strains, unlike 5’NCR region. Based on phylogenetic analysis with multiple sequence regions, most of the CVA6 strains were genetically clustered together with other Indian strains, while some CVA6 grouped with viruses sampled from Australia. Our findings indicate the presence of genetically similar CVA6 strains in India and Australia. Phylogenetic evidence shows that it might have emerged from the large outbreaks in mainland China between 2012 to 2013. Earlier studies also corroborated our findings and reported the predominant circulation of CVA6 strains of Asian origin in Asia-Pacific regions in recent years (29,33–35). These finding increases the concern over the emergence and spread of global strains of CVA6. The CVA16 and CVA10 strains detected during the study period were genetically aligned with virus strains reported from others parts of India.

The present investigation might have missed mild or subclinical cases that occurred during this period as this was a hospital-based HFMD surveillance. Further, the retrospective study using archived samples might have introduced bias in sampling and may not fully represent the distribution of the cases in the study period. Enterovirus typing was not possible for 20% of clinical specimens by any of the molecular methods used in the study. Further investigation should be focused on the typing and characterisation of unidentified enteroviruses by next-generation sequencing (NGS).

There is insufficient laboratory surveillance data on the etiology of HFMD cases from India. The present study adds new knowledge and contributes to the understanding of circulating enterovirus types associated with the HFMD cases in India and their molecular epidemiology. The data from this study may contribute to develop evidence-based outbreak preparedness plan and selection of strains for vaccine development. Changing epidemiology of HFMD, including the emergence of new enterovirus types, emphasises the need for systematic laboratory-based HFMD surveillance in disease-endemic regions like India. In conclusion, over the years, CVA6 has emerged as the predominant etiology of HFMD in South India.

Acknowledgements

We acknowledge the Indian Council of Medical Research (ICMR), Government of India for the grant (VIR/NP/66/2013-ECD-1). We are grateful to the District and Taluk hospitals in Kerala and Karnataka for their assistance in clinical and epidemiological data collection.

Conflict of Interests

The authors declare that they have no conflict of interest.

Ethics approval

Permission to conduct this study was granted by the Institutional Ethics Committee, Kasturba Medical College and Kasturba Hospital (ECR/191/Inst/KL/2013 and ECR/146/Inst/KA/2013/RR-16). All experiments were performed based on the approved guidelines and regulations in the study.

Author contributions

Conceptualisation and design of the study by Sanjay ER and Arunkumar G. Sanjay ER wrote the first draft of the manuscript and contributed to the data acquisition, analysis, validation and interpretation. Arunkumar G supervised the work and acquired the funding. Josmi J contributed to data analysis. Sanjay ER, Arunkumar G, Josmi J and Sabeena S reviewed and edited the manuscript. Sarita S and Michael CJ have done the clinical diagnosis and collection of medical data. Aswathyraj S, Shahin S, Kavitha K, Shilpa C, Prasada V S and Anup J contributed to improve the manuscript.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Molecular Epidemiology of Enteroviruses Associated with Hand, Foot and Mouth Disease in South India during 2015 to 2017

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