Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction

Recombination in Enteroviruses, a Multi-Step Modular Evolutionary Process

International audienceRNA recombination is a major driving force in the evolution and genetic architecture shaping of enteroviruses. In particular, intertypic recombination is implicated in the emergence of most pathogenic circulating vaccine-derived polioviruses, which have caused numerous outbreaks of paralytic poliomyelitis worldwide. Recent experimental studies that relied on recombination cellular systems mimicking natural genetic exchanges between enteroviruses provided new insights into the molecular mechanisms of enterovirus recombination and enabled to define a new model of genetic plasticity for enteroviruses. Homologous intertypic recombinant enteroviruses that were observed in nature would be the final products of a multi-step process, during which precursor nonhomologous recombinant genomes are generated through an initial inter-genomic RNA recombination event and can then evolve into a diversity of fitter homologous recombinant genomes over subsequent intra-genomic rearrangements. Moreover, these experimental studies demonstrated that the enterovirus genome could be defined as a combination of genomic modules that can be preferentially exchanged through recombination, and enabled defining the boundaries of these recombination modules. These results provided the first experimental evidence supporting the theoretical model of enterovirus modular evolution previously elaborated from phylogenetic studies of circulating enterovirus strains. This review summarizes our current knowledge regarding the mechanisms of recombination in enteroviruses and presents a new evolutionary process that may apply to other RNA viruse

Evolution and Emergence of Enteroviruses through Intra- and Inter-species Recombination: Plasticity and Phenotypic Impact of Modular Genetic Exchanges in the 5’ Untranslated Region

<div><p>Genetic recombination shapes the diversity of RNA viruses, including enteroviruses (EVs), which frequently have mosaic genomes. Pathogenic circulating vaccine-derived poliovirus (cVDPV) genomes consist of mutated vaccine poliovirus (PV) sequences encoding capsid proteins, and sequences encoding nonstructural proteins derived from other species’ C EVs, including certain coxsackieviruses A (CV-A) in particular. Many cVDPV genomes also have an exogenous 5’ untranslated region (5’ UTR). This region is involved in virulence and includes the cloverleaf (CL) and the internal ribosomal entry site, which play major roles in replication and the initiation of translation, respectively. We investigated the plasticity of the PV genome in terms of recombination in the 5’ UTR, by developing an experimental model involving the rescue of a bipartite PV/CV-A cVDPV genome rendered defective by mutations in the CL, following the co-transfection of cells with 5’ UTR RNAs from each of the four human EV species (EV-A to -D). The defective cVDPV was rescued by recombination with 5’ UTR sequences from the four EV species. Homologous and nonhomologous recombinants with large deletions or insertions in three hotspots were isolated, revealing a striking plasticity of the 5’ UTR. By contrast to the recombination of the cVDPV with the 5’ UTR of group II (EV-A and -B), which can decrease viral replication and virulence, recombination with the 5’ UTRs of group I (EV-C and -D) appeared to be evolutionarily neutral or associated with a gain in fitness. This study illustrates how the genomes of positive-strand RNA viruses can evolve into mosaic recombinant genomes through intra- or inter-species modular genetic exchanges, favoring the emergence of new recombinant lineages.</p></div

Phylogenetic relationships between the 5’ UTR sequences of the eight 5’ partners.

<p>This neighbor-joining tree was constructed with MEGA, version 6.06 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.ref065" target="_blank">65</a>] using aligned 5’ UTR nt sequences. The reliability of tree topology was estimated using 1000 bootstrap replicates. The nt sequence of rhinovirus A16 (RV-A16) was used as an outgroup. The name of each isolate includes the type of the isolate followed by the laboratory number or name for the prototype strains. For each 5’ partner, the percentage similarity with MAD4 is shown in brackets. The enterovirus species (EV-A to -D) and the 5’ UTR group I and II are indicated.</p

Recombination efficiency following the co-transfection of L20B and HEp-2c cells with the MAD4 3’ RNA partner and each 5’ RNA partner.

<p>^<i>a</i> The 5’ UTR group of the 5’ partner is indicated.</p><p>^<i>b</i> The period following co-transfection was optimized before staining and counting PFUs, depending on the RNA partner pairs.</p><p>^<i>c</i> The average numbers of plaques ± the standard deviation of two experiments is given. The same amount (in μg) of 5’ and 3’ partner was co-transfected.</p><p>Recombination efficiency following the co-transfection of L20B and HEp-2c cells with the MAD4 3’ RNA partner and each 5’ RNA partner.</p

Competition assays comparing the fitness of MAD4 and selected CV-A17/MAD4 or EV-70/MAD4 homologous recombinants.

<p>HEp-2c cells were infected in triplicate with a 1:1 mix of a recombinant virus and the parental strain MAD4 at an MOI of 0.01 TCID50/cell for three passages. The proportion of each virus at each passage was determined by real time RT-PCR and expressed as the ratio of the CT values of MAD4 competitor versus recombinant. The mean ± the standard deviation is shown (Student’s t-test, n = 3; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). A CT value ratio above 1 indicates that the recombinant is fitter than MAD4.</p

Location of recombination site in selected recombinants.

<p>^<i>a</i> Recombinants are named according to their type, class and laboratory number as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.t002" target="_blank">Table 2</a>.</p><p>^<i>b</i> The first number refers to the nt sequence of the indicated 5’ partner and the second to that of MAD4.</p><p>^<i>c</i> Homologous recombination sites (H) display neither an insertion nor a deletion according to aligned parental sequences. The insertion (+) or deletion (-) of nt in nonhomologous sites is indicated.</p><p>Location of recombination site in selected recombinants.</p

Competition assays comparing the relative fitness of homologous and nonhomologous recombinants.

<p>Each of the four nonhomologous selected CV-B4/MAD4 recombinants presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.t002" target="_blank">Table 2</a> were competed against the homologous recombinant CV-B4/MAD4 B.38, the recombination site of which is located in the spacer between IRES domains dIII and dIV (CV-B4 nt 240 / MAD4 nt 234) (A to D), and against the parental cVDPV MAD4 (E to H). Viruses were mixed at a 1:1 ratio and HEp-2c cells were inoculated in triplicate at an MOI of 0.01 TCID50/cell for three passages. At each passage, the progeny RNA was amplified by RT-PCR and the relative amount of each competitor was evaluated by measuring the intensity of the bands on agarose gel electrophoresis (ImageJ 1.47 software, NIH) corresponding either to the nonhomologous recombinant or to its homologous competitor. The ratio of band intensity for the nonhomologous recombinant versus the homologous competitor is shown for each well for the three passages (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.s006" target="_blank">S6 Fig</a>). An increase in this ratio indicates that the nonhomologous recombinant is more fit than the homologous one. Error bars indicate the standard deviation (Student’s t-test, n = 3; *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i>).</p

Experimental model of recombination in the 5’ untranslated region of enteroviruses.

<p>(A) Schematic representation of the genome of MAD4 cVDPV. The poly-adenylated single positive-strand RNA genome is covalently linked to the viral protein VPg (also named 3B) at the 5’ terminus. The unique large open-reading frame is flanked by two untranslated regions (5’ and 3’ UTRs). The 5’ UTR (nt 1 to 747) is magnified to indicate the seven stem-loop structures (I to VII) forming two functional units, the cloverleaf (CL: I) and the internal ribosome entry site (IRES: II-VI). Genomic regions encoding viral proteins VP4 to 3Dpol are indicated. The MAD4 genome is a PV/EV-C recombinant between mutated Sabin 2 sequences (light shading) and non-vaccine sequences derived from coxsackieviruses A (CV-A) (dark shading). (B) Substitutions in the CL leading to a noninfectious MAD4 genome. The native CL structure can be subdivided into four domains: stem a and stem-loops b to d. Mutations (lowercase) introduced into the CL of MAD4 disrupt stem a and create new predicted base pairing interactions. Secondary structure predictions were generated with mfold, version 3.6 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.ref063" target="_blank">63</a>]. (C) Rescue of the defective MAD4 genomic RNA (3’ partner) by co-transfection with 5’ UTR sequences from EVs (5’ partners). 5’ partners included the complete 5’ UTR followed by sequences encoding VP4, VP2 and part of VP3 from eight different EV strains belonging to the four human EV species (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.g002" target="_blank">Fig 2</a>). Human HEp-2c and murine L20B cells were co-transfected with each RNA partner pair and then incubated in semisolid medium until plaques appeared, indicating the generation of viable recombinants.</p

Neurovirulence of MAD4 and selected recombinant viruses in PVR-Tg mice.

<p>PVR-Tg21 mice expressing the human PV receptor were inoculated intracerebrally with 10⁵ TCID50 of virus (six mice per virus). Animals were checked daily for 21 days post-inoculation for paralysis or death. The number of healthy mice following inoculation with group I/I recombinants (A) or group II/I recombinants (B) relative to that following inoculation with the parental cVDPV MAD4 is shown. Survival curves are numbered to indicate overlap. No additional mice suffered paralysis or died after day 15 post-inoculation. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 in Log Rank tests comparing each selected recombinant virus with MAD4 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005266#ppat.1005266.s017" target="_blank">S6 File</a>).</p