Ethics statement
The specimens were collected as a part of disease outbreak investigation mandated by the Disease Outbreak Investigation and Control Manual V1 2015 and NEWARS guideline (www.rcdc.gov.bt/web/). Hence, no written consent was obtained from patients. To further characterize the specimens, the RCDC provided a written request for public health support to the WRAIR-AFRIMS, Bangkok, Thailand for analysis of de-identified specimens by RT-PCR and Next Generation Sequencing (NGS). The material was reviewed by the Walter Reed Army Institute of Research (WRAIR) and clearance was obtained for its presentation and/or publication (WRAIR No. 3105).
Outbreak notification
Bhutan established the Notifiable Disease Surveillance System (NDSS) in 2010, which was later revised into the NEWARS system. This comprehensive, web-based national surveillance and response system monitors priority diseases and syndromes of public health concern. NEWARS integrates indicator-based surveillance (IBS) for 11 weekly and 15 immediately notifiable diseases and event-based surveillance (EBS) for ad hoc reporting of unusual health events.
During the AHC outbreak in 2023, the EBS component was used to collect outbreak information by reporting clusters of AHC cases. AHC cases are defined as individuals presenting with acute bilateral or unilateral redness of the eye(s), pain or discomfort, and watery discharge, with no other underlying cause identified. A risk assessment tool, outlined in the NEWARS guideline, evaluated epidemiological factors such as case numbers, potential for widespread outbreaks, morbidity, mortality, and contamination risks based on eight pre-set questions. Each question was scored, and the system classified the outbreak risk as low, medium, or high. Based on the risk level, RCDC provided recommendations to reporting healthcare centers. Local reporting sites submitted investigation reports with basic epidemiological data, including the number and characteristics of cases, clinical manifestations, geographical distribution, and control measures implemented. In addition, RCDC increased outreach to other health centers to increase reporting of similar events in their localities and as of this report, outbreaks of AHC continue to be tracked through NEWARS surveillance, particularly from southern bordering districts with India and within central Bhutan. Additionally in hospitals with ophthalmologists, RCDC requested to ship samples for laboratory investigation as part of outbreak response.
Specimen collection, Storage and Shipment
Eighteen paired samples (9 conjunctival and 9 throat/nasal swabs) were collected from 9 patients on 17 August 2023, in Phuntsholing. This selection was part of an exploratory investigation to identify a potential etiological agent during a large-scale outbreak of AHC. The outbreak disproportionately affected vulnerable groups, such as school children, and was characterized by similar clinical symptoms among reported cases. The decision to collect a limited number of samples was due to Bhutan’s constrained diagnostic capacity for identifying the causative agent of AHC. Given these limitations, we focused on a small, representative subset to maximize the likelihood of detecting the pathogen while conserving resources.
Specimens were collected in Universal Transport Medium (UTM), provided by Copan, California, USA, and subsequently shipped to RCDC. The specimens were transported in insulated containers with ice packs to ensure that the temperature remained between 2–8 °C throughout transit. Temperature storage conditions were monitored to prevent degradation of the samples and preserve their integrity for further analysis. Upon receipt, the specimens were stored at −80 °C for further investigation. Specimens were initially tested for influenza virus using the FLU real-time RT-PCR assay following the CDC protocol. Influenza-negative specimens were subsequently shipped on dry ice to WRAIR-AFRIMS for further analysis. At WRAIR-AFRIMS, total nucleic acids were extracted and analyzed using the FTD21 multiplex real-time PCR assay for the preliminary detection of pathogens. Next-generation sequencing (NGS) was performed to confirm the presence of pathogens identified by the FTD21 assay; however, only 17 specimens underwent NGS due to insufficient volume in one specimen.
Nucleic acid extraction
Total nucleic acid (NA) was extracted from 200 µl of UTM from collected 18 specimens, which contained a conjunctival or throat/nasal swab, using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Catalogue No. 06543588001, Roche Life Science, Switzerland), on MagNa Pure 96 instrument (Roche Life Science, Switzerland), following the manufacturer’s instructions. The elution buffer was automatically added to archieve a final NA volume of 50 µl.
Multiplex real-time RT-PCR
A total of 50 µl of extracted total NA was used for multiplex real-time RT-PCR testing with the Fast-Track Respiratory 21 (FTD21) assay (Catalogue No. SMN 10,921,703, Siemens Healthineers, Berkeley, CA) following the manufacturer’s instructions. The assay utilized equine arteritis virus (EAV) as an internal control (IC), which was added to each sample and the negative control during the extraction process. The IC was extracted, processed, and amplified simultaneously with each sample to monitor the extraction process and identify any PCR inhibition. The FTD21 assay uses five PCR reaction tubes, each containing a proprietary mixture of primers and probes for detecting respiratory pathogens. A 10 µl of extracted total NA was added to the reactions in Tube-1 through Tube-5 as a template for pathogens detection. The targets for each tube were as follows: Tube-1 reaction was for detection of influenza A (Flu A), influenza A subtype H1N1 (pandemic H1N1), human rhinovirus (HRV), influenza B (Flu B). Tube-2 reaction was for detection of human coronaviruses NL63 (HCoV-NL63), 229E (HCoV 229E), OC43 (HCoV-OC43), and HKU1 (HCoV HKU1). Tube-3 reaction was for detection of human parainfluenza viruses, 2, 3, and 4 (HPIV- 2, 3 and 4 and internal control. Tube-4 reaction was for detection of human parainfluenza viruses-1, Mycoplasma pneumoniae (M.pneu), human bocavirus (HBoV), human metapneumovirus (HMPV A/B). Tube-5 reaction was for detection of respiratory syncytial virus (RSVA/B), HAdV, EV, human parechovirus (HPeV).The real-time RT-PCR was conducted on an ABI7500 Fast PCR instrument (Life Technologies, USA) following the manufacturer’s instructions, using the following thermal cycler program; 50 °C for 15 min, 94 °C for 1 min, 40 cycles of 94 °C for 8 s, 60 °C for 1 min. The cycle threshold (Ct) value was used to determine the presence of pathogens according to the FTD21 assay manual. A Ct value of 40 was considered negative, while a Ct value of less than 33 with a typical S-shaped amplification curve was considered positive. Samples with Ct values between 33 and 40 required repeat testing for confirmation.
NGS with hybrid capture-based target enrichment
Prior to library preparation, 50 µl of extracted total NA was precipitated by adding 25 µl (0.5 volume) of 7.5 M ammonium acetate and 165 µl (2.2 volume) of ice-cold absolute ethanol. The mixture was centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was carefully removed and discarded, ensuring the pellet remained undisturbed. The pellet was washed twice with ice-cold 70% ethanol to remove impurities. After washing, the pellet was air-dried at room temperature for approximately 10 min, or until no residual liquid was visible. Finally, the pellet was resuspended in 8.5 µl of nuclease-free water for subsequent library preparation. The Viral Surveillance Panel kit (VSP, Catalogue No. 20088154, Illumina, USA) was used following the manufacturer’s instructions for library preparation and hybrid-capture target enrichment for detection of 66 viral genomes (listed in supplementary Table S1)26. Briefly, 8.5 µl of the resuspended NA sample with concentration ranging from 10 to 100 ng was denatured and used for first and second-strand cDNA syntheses followed by tagmentation and cDNA library construction. DNA libraries were cleaned with Agencourt AMPure XP (Beckman Coulter, USA) before normalization in resuspension buffer. For three-plex enrichment, three libraries were each diluted to a concentration of 200 ng in a volume of 2.5 µl and then combined into one pool. Subsequently, 7.5 µl of the pool was used for target enrichment. Target enrichment was performed by bead-based capture of hybridized probes, amplification, and purification. For quality control check, fragment size of enriched libraries was analyzed using QIAxcel Advanced System (QIAGEN, Germany). Concentration of enriched libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fischer Scientific, USA). Enriched libraries were equimolarly pooled at 2 nM and diluted to a loading concentration of 0.8 pM. The pool was then sequenced (15 samples per run) on a MiSeq instrument using a 500-cycle (2 × 250-bp, paired-end) MiSeq v2 reagent kit (Illumina), following the manufacturer’s instructions.
NGS data analysis
The obtained paired-end reads were subjected to quality trimming using BBtools v.37.62 with the following criteria: base quality threshold of > 25, minimum length requirement of 100, and minimum average quality threshold of 2027. The viral genome consensus sequence of each sample was derived by aligning the trimmed reads to the CA24v reference genome sequence (KR478685.1) from the US National Institute of Health (NIH) GenBank nucleotide database. This alignment was performed using BWA-MEM v.0.7.1728 and iVAR v1.3.129 with the following criteria: mapping quality threshold > 30, base quality > 30, and a minimum of depth of coverage of 10. Ambiguous bases were identified and confirmed by genome-guided assembly using Trinity v2.14.030. The obtained consensus sequences were aligned with other sequences in the NCBI database to search for sequence similarity using BLAST31.
Phylogenetic analysis was conducted using maximum-likelihood (ML) trees constructed for the VP1 gene (915 nucleotides) and the complete coding sequences (CDS, 6,645 nucleotides) of CVA24v. The analysis included sequences generated in this study along with additional sequences retrieved from GenBank. From a dataset of 1,620 CVA24v sequences available in GenBank as of November 2023, a random subset of 96 VP1 gene sequences and 83 CDS sequences were selected for ML tree construction. Nucleotide alignment was performed using MAFFT v7.31019, and tree building using IQ-TREE v2.0.332,33. The trees were constructed with 1,000 ultrafast bootstrap replicates and visualized using FigTree v1.4.434. The Treesub program (https://github.com/tamuri/treesub) was utilized to deduce amino acid substitution along the tree. MEGA11 software was used to identify amino acid substitutions35. Molecular CVA24v genotyping based on the complete genome sequences was also performed using online Genome Detective, Enterovirus Genotyping Tool v.2.132136.
