Genome Sequences of 16 Enterovirus Isolates from Environmental Sewage in Guatemala, 2019 to 2021

ABSTRACT

Enteroviruses can cause human infectious disease. We report 16 near-complete genome sequences of enteroviruses that were isolated through environmental surveillance of wastewater in Guatemala.

ANNOUNCEMENT

The genus Enterovirus contains 15 species and belongs to the Picornaviridae family, a large family of nonenveloped positive-sense, single-stranded RNA viruses. The Enterovirus B (EV-B) species contains 63 serotypes and is the largest EV species (1). The EV-C species contains 23 serotypes, which includes the three polioviruses (2). Ten EV-B (1 coxsackievirus [CV] type B5, 2 echovirus type 1 [E-1], 1 E-3, 1 E-7, 2 E-11, 1 E-25, 1 E-29, and 1 E-33) and six EV-C (3 CV A13, 1 CV A20, 1 CV A24, and 1 EV C99) were identified through isolation and genome sequencing from environmental sewage collected in Villa Nueva (VNA; GPS coordinates 14.5269 to 90.5875) and San Juan Sacatepéquez, Guatemala (SJS; GPS 14.7236, 90.6520) from 2019 to 2021 (Table 1).

TABLE 1.

Sequencing summary and characteristics of 16 enteroviruses from Guatemala, 2019 to 2021

Isolate	Virus	Taxonomy	Collection date (mm/dd/yyyy)	Collection site	GenBank accession no.	Total no. of readsa	Length (bp)	GC content (%)
A549-010	Coxsackievirus B5	Enterovirus B	11/22/2019	VNA	OL955504	9,553	7,302	47.7
HLF-000	Echovirus 3	Enterovirus B	11/20/2019	SJS	OL955506	15,035	7,342	47.4
HLF-006	Coxsackievirus A13	Enterovirus C	11/22/2019	SJS	OL955507	23,120	7,395	44.8
MA104-000	Echovirus 1	Enterovirus B	11/20/2019	SJS	OL955509	6,015	7,132	47.0
MA104-002	Echovirus 7	Enterovirus B	11/20/2019	SJS	OL955511	14,881	7,270	47.6
RD-000	Echovirus 29	Enterovirus B	11/20/2019	SJS	OL955512	4,717	7,314	47.8
169-41CQU3372	Echovirus 25	Enterovirus B	09/16/2020	SJS	ON383153	9,325	7,259	47.6
179-51CBM4841	Echovirus 11	Enterovirus B	07/13/2020	SJS	ON383154	35,931	7,312	47.5
183-55CBM2468	Coxsackievirus A13	Enterovirus C	06/10/2020	SJS	ON383155	17,008	7,355	44.4
183-55CBM2468-1	Coxsackievirus A24	Enterovirus C	06/10/2020	SJS	ON383156	13,212	7,365	44.7
190-62ACB0312	Enterovirus C99	Enterovirus C	05/15/2020	SJS	ON383157	8,644	7,302	44.9
129-1CBM1352	Echovirus 33	Enterovirus B	09/01/2021	SJS	ON383146	33,169	7,240	47.9
145-17ACB0328	Coxsackievirus A20	Enterovirus C	05/18/2021	SJS	ON383147	4,154	7,185	45.8
146-18PLA0330	Coxsackievirus A13	Enterovirus C	05/18/2021	VNA	ON383149	57,563	7,318	45.1
148-20CQU0199	Echovirus 11	Enterovirus B	04/16/2021	SJS	ON383150	7,861	7,182	47.7
157-29CVP8542	Echovirus 1	Enterovirus B	01/25/2021	VNA	ON383152	3,399	7,211	47.3

^aNumber of reads after quality control and deduplication.

Sewage samples were processed using the concentration and filter elution (CaFÉ) method, as described previously (3, 4). Resulting concentrates were inoculated into cells for enterovirus isolation according to the World Health Organization protocol (5). Briefly, concentrates were inoculated into rhabdomyosarcoma (RD) cells and incubated for 5 days at 37°C. On day 5, the cells were observed for cytopathic effect (CPE).

Viral RNA was extracted from CPE-positive cell culture supernatants using the MagMAX pathogen RNA/DNA kit on a KingFisher Flex system (Thermo Fisher Scientific). Viral RNA was amplified using a sequence-independent, single-primer amplification (SISPA) protocol (6–8). Viral RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific) and a 28-base primer with eight random nucleotides on the 3′ end (CCTTGAAGGCGGACTGTGAGNNNNNNNN). A complementary strand was synthesized using the Klenow fragment of DNA polymerase I (New England BioLabs). Illumina libraries were prepared using the Nextera XT library preparation kit on 69 pooled samples. The samples were sequenced on an Illumina MiSeq system using a 500-cycle paired-end run as previously described (9).

A custom in-house bioinformatics pipeline (10) was used to process raw FASTQ data and for de novo assembly of each isolate’s read. Within the pipeline, multiple preprocessing steps were conducted before the FASTQ reads were assembled. First, the host data were removed using default parameters in Bowtie 2 v2.3.3.1 (11–13), followed by primer trimming, adapter trimming, and Phred quality score filtering using Cutadapt v2.3 (parameters for filtering: reads with a quality score of <20, read length of <50 nucleotides, and error rates of >0.15) (14), and finally duplicate reads were removed using the Dedup.py script in Python (15). Deduplicated reads were de novo assembled into contigs using default parameters in SPAdes v3.15.0 (16).

Consensus genome sequences were verified through read mapping, BLAST alignments using MAFFT, and annotations using Geneious vR11.

The 16 near-complete genome sequences ranged from 7,132 to 7,395 bp in length. Their GC content was between 44.4% and 47.9%, and the median read coverage was 11,382 (interquartile range [IQR], 7,399 to 18,536). These genome sequences share 80 to 90% pairwise identity to previously submitted nucleotide sequences and, therefore, are distinct from other enterovirus genomes in GenBank.

Data availability.

The 16 EVs have been submitted to GenBank, and the raw sequencing reads have been deposited in the Sequence Read Archive under BioProject PRJNA835862. All accession numbers are reported in Table 1.