Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts

Abstract

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Fig 1. CPE (Cytopathic effect) results between the six cell lines.

CPE graded 0 through 4 for A549, HEP2, HLF, MA104, RD and Vero cell lines for samples 1 through 6.

Fig 2. Enterovirus typing from preliminary NGS of 6 cell lines.

The A549, HEP2, HLF, MA104, RD and Vero cell lines identified polioviruses (PV3, PV1), echoviruses (E1, E3, etc.), Coxsackieviruses (CV) B5, B2 and CVA13. Color coding shows genus: red—Enterovirus A, green—Enterovirus B, blue—Enterovirus C.

Fig 3. Phylogenetic relationship of the Viral Protein 1 (VP1) typing region of 21 enterovirus sequences from Guatemala and 15 representative genomes of each type using a PAUP* neighbor joining tree building algorithm.

Tree created using MAFFT global alignment (default parameters) and PAUP* phylogenetic analysis software. The PAUP* parameters were as follows: tree building method = neighbor joining, genetic distance model = GTR, rates variation = gamma distribution, outgroup = none, and root method = midpoint.

Fig 4. Detection patterns of enterovirus C (poliovirus, orange circles), echovirus (green), Coxsackievirus (blue) in Guatemala by collection site (ACB, CBM, CQU-San Juan Sacatepéquez.

RMG, PLA, CVP-Villa Nueva) and by month of collection (April 2020 -September 2021), 63 samples were tested using the HLF cell line and 58 samples contained at least one enterovirus through sequence comparison.