Comparative Genomic and Phylogenetic Analysis of the First Usutu Virus Isolate from a Human Patient Presenting with Neurological Symptoms

<div><p>Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5′ and 3′ untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.</p></div

Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

<div><p>Background</p><p>Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.</p> <p>Methodology/Principal Findings</p><p>An rRT-PCR assay targeting the 5′ untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log₁₀ cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1–4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.</p> <p>Conclusions/Significance</p><p>This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.</p> </div

Probe signals for each DENV serotype.

<p>Any positive signal above the threshold is shown as (+). DENV-1 has the strongest and earliest signal in green of any serotype as (+++). Other positive signals are designated (++).</p

Comparison of patients from Sri Lanka that tested positive only by the multiplex rRT-PCR and those that tested positive by both the multiplex rRT-PCR and reference assays.

*<p>Viral Load expressed as log₁₀ cDNA equivalents/mL of patient plasma.</p><p>ND, not determined; SD, standard deviation.</p

Probe sequences for the DENV multiplex rRT-PCR.

<p>Probes are listed by the channel in which signal is detected on the Rotor-Gene Q instrument. Underlined probe segments designate sequences complementary to the DENV consensus; segments on the 5′ and 3′ ends of the probe comprise the beacon stem.</p

Total number of specimens, PCR pools and PCR-positive pools collected for every mosquito species in 2010 survey.

*<p><i>Ochlerotatus</i> taxon was considered an <i>Aedes</i> subgenus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038058#pone.0038058-Savage1" target="_blank">[60]</a>.</p

Molecular Phylogenetic analysis by Maximum Likelihood method based on the Kimura two-parameter model of the WNV sequences of amplified fragments and homologous fragment obtained in GenBank library.

<p>The tree with the highest log likelihood (−720.3421) is shown. Initial tree for the heuristic search were obtained automatically by Neighbor-joining method with Maximum Composite Likelihood distance matrix. The analysis involved 22 nucleotide sequences, there were a total of 409 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038058#pone.0038058-Tang1" target="_blank">[65]</a>, the tree was rooted in RO97 50. Percentages of bootstrap test (1000 replicates) are shown next to the branches.</p

Seasonal distribution of positive USUV mosquito pools (bi-weekly collection) per kind of trap.

<p>Seasonal distribution of positive USUV mosquito pools (bi-weekly collection) per kind of trap.</p

USUV “hot spots” obtained by kernel density estimation of positive mosquito pools (a); overlaying of 95% contour perimeter and quartile maps of temperature range (b); and of hydro-climatic balance (c); from July to September 2010.

<p>USUV “hot spots” obtained by kernel density estimation of positive mosquito pools (a); overlaying of 95% contour perimeter and quartile maps of temperature range (b); and of hydro-climatic balance (c); from July to September 2010.</p

Bi-weekly maximum likelihood estimates (95% CI) of USUV infection rates in <i>Culex pipiens</i> estimated at province level.

<p>Province abbreviations: BO, Bologna; FC, Forlì-Cesena; FE, Ferrara; MO, Modena; PC, Piacenza; PR, Parma; RA, Ravenna; RE, Reggio Emilia; RN, Rimini.</p