An Outbreak of Acute Hemorrhagic Conjunctivitis Caused by Coxsackievirus A24 in Eastern Uttar Pradesh, India 2023

Acute hemorrhagic conjunctivitis (AHC) is an epidemic form of highly infectious conjunctivitis characterized by swollen red eyes, photophobia, excessive lacrimation, and conjunctival hemorrhage. Viral conjunctivitis accounts for 80% of infectious conjunctivitis followed by bacterial infection [1]. Non-infectious conjunctivitis (allergic or toxin-induced) is a less common cause of conjunctivitis that is often reported [2]. Enteroviruses and adenoviruses are the main etiological agents of highly contagious AHC [3, 4]. Human adenovirus serotypes (3, 4, 7, 8, 9, 11, 19a, 21, 35, and 37) and among the human enterovirus group, enterovirus-70 (EV-70), and CV-A24 variants are reported as major causes of AHC [4, 5]. CV-A24 and EV-70 are the members of enterovirus C and D species, respectively. Widespread epidemics of AHC have occurred in many parts of the world by an antigenic variant of the CV-A24, which was first isolated in Singapore from an outbreak in 1970 [6, 7]. Following this, several outbreaks of AHC caused by CV-A24 virus have also been reported from India and neighboring countries like China, Nepal, and Pakistan [8‒13]. In India outbreaks of AHC have been reported from different parts, like Vellore (1979), Delhi (1988), Uttar Pradesh (1994 and 2010), Chennai (1999), Gujarat and Maharashtra (2003), and Mumbai (2007) [8‒10, 14‒17].

The CV-A24 genomic RNA is translated into a single polyprotein, which is catalytically processed by the viral protease into four structural proteins (VP1-VP4) and seven nonstructural proteins (2A-2C, 3A-3D) [18]. The capsid proteins (VP1–VP4) assemble to form an icosahedral virion. The 3C region with high recombination capacity and VP1 capsid protein is under constant evolutionary pressure to induce changes in epitope to evade the host immune response [13, 19, 20]. Therefore, the 3C and VP1 regions were selected for the genetic analysis and epidemiological relationship among CV-A24 strains responsible for AHC outbreaks. In this study, we have conducted a molecular investigation of the AHC outbreak that occurred in Eastern Uttar Pradesh, India, during July-August 2023.

The study was conducted among patients attending the outpatient department of the Ophthalmology of Baba Raghav Das Medical College (BRDMC) and Netaji Subhash Chandra Bose District Hospital (NSCBDH) of Gorakhpur. Both hospitals are tertiary care hospitals located in Gorakhpur. Patients presenting with symptoms of sudden onset of foreign body sensation in the eye, watery discharge, red eyes, itching, or burning sensation in the eye were recruited for the study. Written informed consent was taken from patients or their parents or guardians. Conjunctival swabs were collected from the infected eye by the ophthalmologist. Two drops of proparacaine hydrochloride ophthalmic solution were added to the affected eye of each patient and, after 1 min, a sterile swab was swiped over the conjunctiva and stored in a tube containing viral transport media (VTM). Further, the VTM tubes were transferred in the cold chain to the Viral Research Diagnostic Laboratory of ICMR-RMRC, Gorakhpur, for investigation.

Two hundred microliter of VTM containing the conjunctiva swab was used for the nucleic acid (DNA and RNA) extraction using PureLink^™ Mini Kit, Invitrogen, USA. The extraction procedure was conducted as per the manufacturer’s instructions. The extracted nucleic acid was stored at −20°C for further use.

Human adenovirus was detected from DNA isolated from a conjunctiva swab using nested PCR. First-round PCR was carried by ADV_F1 and ADV_R1 primers and second-round PCR using ADV_F2 and ADV_R2 primers (Table-1) [21]. PCR conditions were 94°C for 3 min, followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1.45 min, with a final extension at 72°C for 10 min.

Extracted RNA samples were subjected to pan-enterovirus detection using the nested RT-PCR (Promega, USA). The outer pair of primers EV64F and EV578R was used in the RT-PCR. While EV160F and EV547R were used in the nested PCR, the primers targeted the 5′UTR region used in the study (Table 1) [22]. RT-PCR amplification reaction was performed under the following conditions: 45°C for 45 min, 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 68°C for 1 min, with final extension at 68°C for 7 min. The nested PCR was conducted using the same thermal profile except for the reverse transcription step, i.e., 45°C for 45 min.

The pan-enterovirus positive samples were further tested for enterovirus-70 (EV-70) using its specific primers (Table 1) [10]. PCR was carried out using the following thermal profile: 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 55°C for 2 min, and 72°C for 3 min, with final extension at 68°C for 7 min.

The pan-enterovirus positive samples were further targeted using the partial 3C and full-length VP1 region to identify the enterovirus genotype. The PCR primer sets used in amplification are given in Table 1. Amplification reaction using CA24-3C_F and CA24-3C_R6047 primer was performed using the one-step RT-PCR kit (Promega) using the following conditions: 94°C for 3 min, followed by 40 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 1.30 min, with a final extension at 72°C for 10 min. The VP1 region was amplified using the cDNA prepared using the reverse primer (CA24-VP1_R3417) following the manufacturer’s protocol (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific). Further, the cDNA templates were used in the conventional PCR using CA24_VP1_F and CA24-VP1_R3417 primer. The cycling condition used 94°C for 3 min, followed by 40 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 1.30 min, with a final extension at 72°C for 10 min.

The sequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), followed by enumeration on an ABI 3130 Genetic Analyzer (Applied Biosystems). Both the DNA sequences of the 3C and VP1 genes obtained in the study were identified by comparison of derived sequences with available GenBank reference sequences using N-BLAST software. The retrieved nucleotide sequences were submitted to the GenBank database under accession number (GenBank OR887070-OR887088). The multiple sequence alignment of newly generated sequences from clinical samples of this study and reference sequences retrieved from NCBI was performed using the Clustal W algorithm [23]. Finally, the phylogenetic analysis was performed using Mega X software [24]. The evolutionary history was inferred using the Neighbor-Joining method with 1,000 bootstrap replications [25].

We enrolled 128 patients with AHC (91 from BRDMC and 37 from NSCBDH, Gorakhpur). The male-to-female ratio of the patients was found to be 0.59:0.41. The mean age of the patients was found to be 27.9 years. The common symptoms were watering of the eyes (125/128; 97.7%), followed by redness in the eye (123/128; 96.1%). Other ocular symptoms like itching or burning symptoms, foreign body sensation, and swelling of eyelids were reported by 32 (25%), 35 (27.3%), and 121 (94.5%) patients, respectively. Ten patients (7.8%) also had conjunctival hemorrhage. Generalized symptoms like fever and headache were reported by 13 (10.2%) patients.

All 128 samples were negative for adenovirus gene-specific PCR. Of the total number (128) samples 96 (75.5%) samples were positive for the pan-enterovirus PCR. All samples were negative for enterovirus-70 gene-specific PCR. Further, the pan-enterovirus-positive samples were confirmed as CV-A24 virus, by amplifying the partial gene targeting the 3C and VP1 region. We could successfully amplify these two regions of CV-A24, which were further used for genetic analysis. Among the 96 samples positive for CV-A24, the best sequencing reads were obtained from 42 for the 3C region and 19 samples for the VP1 region. Alignment reports of the retrieved nucleotide sequences for both these showed identical with corresponding sequences; therefore, 21 sequences for 3C and 14 sequences for VP1 region were used for the construction of the respective phylogenetic tree. The phylogenetic analysis of both VP1 and 3C isolated sequences, along with global sequences retrieved from the GenBank database, clustered with genotype IV (G IV) of CV-A24 as shown in Figures 1 and 2. In the phylogenetic tree, the studied 3C nucleotide sequences grouped along with isolates reported from Northeast India (GenBank No. PP327391), China (GenBank No. OR361390 and OR361388), and Pakistan (GenBank No. OR633288) with genetic similarity was >99%. These isolated sequences other than our study were also reported from conjunctivitis outbreak patients circulating during the same period. Furthermore, the genetic relatedness with the Indian Mumbai outbreak strain isolated during 2007 (GenBank No. GU477563) was found to be 99.95%, while earlier outbreak strains reported from Uttar Pradesh (GenBank No. JX417157) during 2010 ranged from 99.94 to 99.95%. The overall mean distance was found to be 0.09. The four amino acid substitutions were observed at 3C region are 21 (Serine [S]→asparagine[N]), 30 (Valine[V]→Isoleucine[I]), 66 (Serine[S]→Isoleucine[I]), and 75 (Valine[V] →Isoleucine[I) positions after comparison with earlier Indian outbreak strains (GU477562 and JX417157) reported during 2007 and 2010. The studied VP1 nucleotide sequences also clustered together with sequences reported from Northeast India (GenBank No. PP327391), China (GenBank No. OR361390 and OR361388), and Pakistan (GenBank No. OR633288). The genetic similarity between the studied sequences and the above sequences was found to be identical, ranging from (99.98–100%). Studied VP1 nucleotide sequences showed 99.90–99.91% genetic relatedness with earlier outbreak CV-A24 strains (GenBank No. JX417165 and JX417160) reported in 2010 from Uttar Pradesh, India. Further, we found three amino acid substitutions at 16 (Leucine [L]→ Isoleucine [I]), 21 (Proline [P]→ Serine [S]), and 301 (Asparagine [N]→Aspartic acid [D]) positions, after comparison with 2007 outbreak strain reported from Mumbai, India (GenBank No.GU477576). While in three of our sequences (OR887072, OR887078 and OR887080), the amino acid substitution at 21 (Proline [P]→Serine [S]) position was not observed. The average evolutionary divergence over all sequence pairs was found to be 0.08.

Epidemics of AHC have been reported across the world. In 2023, there was an unprecedented surge of AHC cases affecting most parts of North India, including the Eastern Uttar Pradesh region. Our investigations indicated that the outbreak was due to CV-A24, a member of enterovirus group C.

The first epidemics of AHC by CV-A24 were reported from Singapore in 1970 [7]. From 1970 to 1984, the disease appeared in southwest Asia and India [7, 8, 14, 26]. However, since 1985, AHC outbreaks have been reported from several parts of the world, namely China, Japan, Brazil, Europe, and Africa [13, 27‒31]. Since 1970, India witnessed several sporadic conjunctivitis outbreaks every year throughout the country, but most of the outbreaks or cases go unnoticed due to weak surveillance systems, the use of over-the-counter drugs, and the self-limiting nature of the disease [32]. Compared with the last 3 years of conjunctivitis data, cases that remained undocumented might be due to the SARS-COVID-19 pandemic [33, 34]. Probable reasons for hiking in many conjunctivitis cases in a short period may be due to the immunosuppression caused by COVID-19 or loss of herd immunity to the CV-A24 virus [35, 36].

Due to high recombination rate of 3C region and presence of neutralizing sites in VP1 region, majority of the studies has targeted 3C and VP1 region of CV-A24 for molecular epidemiological investigations [13, 19, 20, 37, 38]. Therefore, we targeted the 3C and VP1 regions of CV-A24 to perform molecular epidemiological study. The phylogenetic analysis using nucleotide sequences of 3C and VP1 obtained in this study were clustered along with global genotype IV CV-A24 strains. Further, these two target sequences showed >99% resemblance with the latest available sequences of CV-A24 circulating in Northeast India (GenBank No. PP327391), China (GenBank No OR361388 and OR361390), and Pakistan (GenBank No OR633288) during the same period [39, 40]. These findings further highlight that the same genotype of CV-A24 may have caused similar AHC outbreaks in Northeast India, China, and Pakistan. Additionally, this study found a variation of four amino acids in the 3C region and three in the VP1 region in comparison with earlier outbreak strains reported from India during 2007 and 2010, which demonstrates continuous change in both the structural and nonstructural protein of CV-A24.

Furthermore, in enterovirus, the viral proteins (VP1, VP2, and VP3) are located at the viral capsid’s surface and exposed to immune pressure [31]. All the neutralization epitopes resides in VP1, VP2, and VP3 proteins of the viral capsid of CV-A24. Among these three proteins, VP1 contains the most neutralizing epitopes [30]. Furthermore, the C-terminal region (293–302) of the VP1 protein has been characterized as highly antigenic by peptide scanning technology and in silico approaches [37, 41]. In our study, we detected the three amino acid variations in the VP1 region (“L16I,” “P21S,” and “N301D”). Wu et al. [27] have also observed similar amino acid substitutions of VP1 (N301D) located at the C-terminal region (293–302). In addition to N301D of VP1, changes at D43N and A165T of VP2, and Y182N of VP3 together led to the viral structure modification ultimately leading to an outbreak in China [27]. The present study helped disseminate data on the CV-A24 virus after a long gap following two major AHC outbreaks that occurred in Maharashtra (2007) and Uttar Pradesh (2010) states of India. Therefore, this study recommends continuous genomic surveillance of the CV-A24 virus, to track the viral evolution, epidemiology, and management of infectious disease outbreaks.

In conclusion, our study identifies CV-A24 as the etiological agent responsible for a major outbreak of AHC in Eastern Uttar Pradesh. Phylogenetic analysis of the 3C and VP1 genetic regions revealed that the outbreak was caused by genotype IV of CV-A24. This study also detected variations of amino acids in both 3C and VP1 regions on comparison with earlier Indian outbreak strains isolated during 2007 and 2010, suggesting continuous change of the virus. Furthermore, based on the close genetic resemblance between studied and reported CV-A24 strains from Northeast India, China, and Pakistan circulating during the same period, it was concluded that the same genotype of the virus might be responsible for the AHC outbreak in this region. This study highlights the requirement of continuous molecular surveillance of CV-A24 to track down the genetic changes and management of AHC outbreaks.

We are thankful to health officials (Principal BRD Medical College, CMO, ACMO, DMO, CMS of NSCB District Hospital, Gorakhpur), Government of Uttar Pradesh, for their timely support during the sample collection. The authors are also thankful to Dr. Pooja Bhardwaj for her suggestions in data analysis, Mr. Sonu Gutam for assistance in data entry, and Mr. Satyendra Mourya for clinical sample transportation.

The study was approved by the Institutional Ethical Committee (RMRCGKP/EC/2023/4.5) of ICMR-RMRC, Gorakhpur, adheres to the 2006 ethical principles for biomedical research involving human subjects issued by the Indian Council of Medical Research (ICMR), New Delhi. Written informed consent was obtained from individuals aged ≥18 years, while for individuals below 18 years, written consent was taken from their parents or guardians.

The authors declare that there is no conflict of interest.

The research was supported by the Department of Health Research (DHR) with Grant/Award No. R.15012/39/2021-HR-VRDL.

Sthita Pragnya Behera conceived and designed the experiments, performed investigations wrote the original draft, and approved the final draft. Nalini Mishra performed formal analysis and laboratory supervision, authored or reviewed drafts of the article, and approved the final draft. Ramyash Yadav directed hospital supervision, authored or reviewed drafts of the article, and approved the final draft. Aishwarya Shukla, Moni Kumari, Sonal Rajput, Satish S. Ranawade, Shashikant Tiwari, and Prashansha Srivastava performed experiments, authored or reviewed drafts of the article, and approved the final draft. Imbisat Fatma and Ashutosh Tiwari collected specimens, performed experiments, authored or reviewed drafts of the article, and approved the final draft. Rajeev Singh and Manoj Murhekar reviewed and revised the draft manuscript for intellectual content and approved the final manuscript. Gaurav Raj Dwivedi conceived and designed the experiments, funding acquisition, investigation, and methodology and approved the final draft.

The nucleotide sequences of 3C and VP1 regions derived in this study are available in the GenBank database with GenBank accession numbers OR933607-OR633627 and OR887070-OR887088, respectively. Further inquiries can be directed to the corresponding author.