Abstract
Human enteroviruses (EV) consist of more than 100 serotypes classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses, which have been implicated in a variety of human illnesses. Being able to simultaneously amplify the whole genome and identify enteroviruses in samples is important for studying the viral diversity in different geographical regions and populations. It also provides knowledge about the evolution of these viruses. Therefore, we developed a rapid, sensitive method to detect and genetically classify all human enteroviruses in mixtures. Strains of EV-A (15), EV-B (40), EV-C (20), and EV-D (2) viruses were used in addition to 20 supernatants from RD cells infected with stool extracts or sewage concentrates. Two overlapping fragments were produced using a newly designed degenerated primer targeting the conserved CRE region for enteroviruses A-D and one degenerated primer set designed to specifically target the conserved region for each enterovirus species (EV-A to -D). This method was capable of sequencing the full genome for all viruses except two, for which nearly 90% of the genome was sequenced. This method also demonstrated the ability to discriminate, in both spiked and unspiked mixtures, the different enterovirus types present.
Highlights
The common human viruses, human enteroviruses (EV), consist of more than 100 serotypes most classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses
Human enteroviruses have been implicated in a variety of human diseases, including the common cold, hand foot and mouth disease, acute hemorrhagic conjunctivitis, myocarditis, encephalitis, and poliomyelitis
The combination of the two primer sets (Table 2), allowed for the amplification of the 5 and 3 parts of the genomes, which led to the synthesis of two overlapping DNA fragments per virus
Summary
The common human viruses, human enteroviruses (EV), consist of more than 100 serotypes most classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses. Human enteroviruses have been implicated in a variety of human diseases, including the common cold, hand foot and mouth disease, acute hemorrhagic conjunctivitis, myocarditis, encephalitis, and poliomyelitis. Enteroviruses are non-enveloped viruses, approximately 7500 nucleotides (nt) in length with a positive, single-stranded RNA genome. There are two untranslated regions (5 and 3 -UTR) flanking a large open reading frame encoding a polyprotein that is cleaved to give three precursors. These precursors are subsequently cleaved to give functional proteins: (1) P1 giving rise to four structural capsid proteins (VP1–VP4) and (2) P2 and P3, the non-structural proteins involved in the virus life cycle
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