High rates of infection with novel enterovirus variants in wild populations of mandrills and other old world monkey species

Abstract

Enteroviruses (EVs) are a genetically and antigenically diverse group of viruses infecting humans. A mostly distinct set of EV variants have additionally been documented to infect wild apes and several, primarily captive, Old World monkey (OWM) species. To investigate the prevalence and genetic characteristics of EVs infecting OWMs in the wild, fecal samples from mandrills (Mandrillus sphinx) and other species collected in remote regions of southern Cameroon were screened for EV RNA. Remarkably high rates of EV positivity were detected in M. sphinx (100 of 102 screened), Cercocebus torquatus (7/7), and Cercopithecus cephus (2/4), with high viral loads indicative of active infection. Genetic characterization in VP4/VP2 and VP1 regions allowed EV variants to be assigned to simian species H (EV-H) and EV-J (including one or more new types), while seven matched simian EV-B variants, SA5 and EV110 (chimpanzee). Sequences from the remaining 70 formed a new genetic group distinct in VP4/2 and VP1 region from all currently recognized human or simian EV species. Complete genome sequences were obtained from three to determine their species assignment. In common with EV-J and the EV-A A13 isolate, new group sequences were chimeric, being most closely related to EV-A in capsid genes and to EV-B in the nonstructural gene region. Further recombination events created different groupings in 5' and 3' untranslated regions. While clearly a distinct EV group, the hybrid nature of new variants prevented their unambiguous classification as either members of a new species or as divergent members of EV-A using current International Committee on Taxonomy of Viruses (ICTV) assignment criteria.

Importance: This study is the first large-scale investigation of the frequency of infection and diversity of enteroviruses (EVs) infecting monkeys (primarily mandrills) in the wild. Our findings demonstrate extremely high frequencies of active infection (95%) among mandrills and other Old World monkey species inhabiting remote regions of Cameroon without human contact. EV variants detected were distinct from those infecting human populations, comprising members of enterovirus species B, J, and H and a large novel group of viruses most closely related to species A in the P1 region. The viral sequences obtained contribute substantially to our growing understanding of the genetic diversity of EVs and the existence of interspecies chimerism that characterizes the novel variants in the current study, as well as in previously characterized species A and J viruses infecting monkeys. The latter findings will contribute to future development of consensus criteria for species assignments in enteroviruses and other picornavirus genera.

FIG 1

Collection sites in Cameroon of samples used in the current study. The collection sites in Cameroon were abbreviated as follows: CP, Campo, locations Oveng, Melen, and Grothes (CPOV, CPLM, and CPGR, respectively); DJ, Djoum; EK, Ekom; LM, Lomie; GR, Gribi. C. A. R., Central African Republic, EQUA.Gui., Equatorial Guinea, D. R. Congo, Democratic Republic of the Congo.

FIG 2

Comparison of C_T values of EV RNA detected by real-time PCR in fecal samples collected from mandrills with those in human samples. Each symbol represents the value for one fecal sample. Bars show mean values. Statistical significance was performed using Kruskal-Wallis nonparametric test (above graph).

FIG 3

Maximum likelihood analysis (GTR plus γ plus I model) of VP4/partial VP2 region (A) and whole VP1 (positions 743 to 1168 and 2477 to 3376, respectively, numbered using the PV3 reference sequence) (B). Sequences amplified from study samples are shown as white and solid black symbols, while those previously assigned within EV species A to D, J, and H are color coded according to the key (human EV-B and EV-C sequences were monophyletic and have been collapsed to clarify presentation of simian sequences). Bootstrap resampling of maximum likelihood (ML) trees was performed to indicate robustness of grouping (values of >70% shown).

FIG 4

(A) Distributions of pairwise amino acid distances in P1 (positions 743 to 3376) and 3CD regions (positions 5429 to 7360) between representative human-derived sequences within EV-A, EV-B, and EV-C. Color coding has been used to identify within- and between-species sequences distances for each. The 40% amino acid distance threshold for species assignments using P1 is shown as vertical gray broken line. (B) Pairwise distances between three representative mandrill sequences with EV species A to D, J, and H.

FIG 5

Amino acid sequence divergence between mandrill sequences and A1 and A2 subgroups in EV-A, human variants in species B to D (A) and simian viruses in species A, B, J, and H (B). The values on the y axes show mean pairwise distances between 300-base windows across the coding region, in increments of 30 bases between fragments. To show gene boundaries, the P2 region has been shaded gray, and a representation of gene positions in the EV polyprotein (drawn to scale) is shown above the graphs.

FIG 6

Phylogenetic analysis of parts of 5′UTR (A), 3CD (B), and 3′UTR (C). (A) 5′UTR, positions 1 to 742; (B) 3CD, positions 5429 to 7366; (C) 3′UTR, positions 7377 to 7431. Bootstrapped ML trees were constructed as described for in the legend to Fig. 3 with the exception of the 3′UTR where its short sequence length (55 bases) precluded meaningful model fitting. The tree is therefore shown as a phenogram by neighbor joining of uncorrected nucleotide p-distances.