Piet Maes

Full Genome-Based Classification of Rotaviruses Reveals a Common Origin between Human Wa-Like and Porcine Rotavirus Strains and Human DS-1-Like and Bovine Rotavirus Strains

ABSTRACT Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.

RotaC: A web-based tool for the complete genome classification of group A rotaviruses

BackgroundGroup A rotaviruses are the most common cause of severe diarrhea in infants and children worldwide and continue to have a major global impact on childhood morbidity and mortality. In recent years, considerable research efforts have been devoted to the development of two new live, orally administered vaccines. Although both vaccines have proven to confer a good protection against severe rotavirus gastroenteritis, these vaccines will have to be screened and may have to be updated regularly to reflect temporal and spatial genotype fluctuations. In this matter, the genetic characterization of circulating and new emerging rotavirus strains will need to be compulsory and accurate. An extended classification system for rotaviruses in which all the 11 genomic RNA segments are used, has been proposed recently. The use of this classification system will help to elucidate the role of gene reassortments in the generation of genetic diversity, host range restriction, co-segregation of certain gene segments, and in adaptation to a new host species.ResultsHere we present a web-based tool that can be used for fast rotavirus genotype differentiation of all 11 group A rotavirus gene segments according to the new guidelines proposed by the Rotavirus Classification Working Group (RCWG).ConclusionWith the increasing sequencing efforts that are being conducted around the world to unravel complete rotavirus genomes of human and animal origin, this tool will be of great help to analyze and correctly classify the large amount of new data. The web-based tool is freely available at http://rotac.regatools.be.

Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea

Background Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Methods and Findings Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within <72 h of symptoms compared to others. Favipiravir was well tolerated. Conclusions In the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia. Trial registration ClinicalTrials.gov NCT02329054

A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

BackgroundFour human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43) and 229E (HCoV-229E), SARS associated coronavirus (SARS-CoV) and the recently identified human coronavirus NL63 (HCoV-NL63). In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium.MethodsSamples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay.ResultsSeven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004.ConclusionsOur results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

Evolutionary History of the Closely Related Group 2 Coronaviruses: Porcine Hemagglutinating Encephalomyelitis Virus, Bovine Coronavirus, and Human Coronavirus OC43

ABSTRACT The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

Abstract We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8±1.2μM) was significantly lower than its cytostatic activity; CC50 (261.3±14.5μM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.

Investigating the Origin and Spread of Hepatitis C Virus Genotype 5a

ABSTRACT Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes – Dobrava, Kurkino, Saaremaa, and Sochi – that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.

A unifying hypothesis and a single name for a complex globally emerging infection: hantavirus disease.

In the January 2011 issue of this journal, Swedish authors described three severe cases of predominantly pulmonary infection with the European hantavirus Puumala virus (PUUV), leading to death due to refractory shock in two of these patients [1]. Of note, the kidneys in these two fatal cases had, on autopsy, no prominent inflammatory infiltrates or haemorrhages, in contrast with the lungs which showed extensive interstitial oedema and mononuclear cell infiltrates, mainly CD8 T lymphocytes. As the authors justly point out, even in the title of their communication, it is now, perhaps, time to revise the sacro-saint paradigm existing since 1994 of two “different” infectious diseases caused by the same genus of rodent-borne hantaviruses of the Old and the New World, respectively. The former would target mainly the human kidney, and the latter mainly the human lung, resulting in the so-called “haemorrhagic fever with renal syndrome” (HFRS) [2] or the “hantavirus pulmonary syndrome” (HPS) [3]. When it appeared that the failing heart was, in fact, the most direct cause of death in refractory shock in HPS, yet another name, “hantavirus cardio-pulmonary syndrome” (HCPS), was added to the already bewildering list of over 60 mostly exotic synonyms for a disease described already in 1913 by Russian doctors in Vladivostok. On the other side of the Eurasian landmass and in 1934, Swedish doctors described an epidemic renal affection which they called “nephropathia epidemica” (NE), which was proven in 1979 to be, in fact, a milder variant of HFRS, caused by PUUV [4]. To date, 23 distinct species of hantaviruses have been recognized, each carried by their more or less specific rodent or insectivore reservoir. The most important pathogens are Hantaan (HTNV) and Seoul virus (SEOV) in the Far-East (where >90% of worldwide hantavirus infections occur), PUUV and Dobrava-Belgrade virus (DOBV) in Europe and Russia, and Sin Nombre (SNV) and Andes virus (ANDV) in the Americas [5]. The first HPS report that appeared in 1994 [3] described “a newly recognized disease” which was of paramount importance for a better understanding of an emerging viral infection in the New World, and generated an explosive surge in hantavirus research. However, H(C)PS was, and remains, a rare disease, with only 560 confirmed cases in the USA during the 1993–2010 period [6] (meaning 30 cases/year) and about 2,500 cases in the entire Americas, albeit with a much higher case fatality rate (CFR) of 35% [7]. This is in stark contrast with the current 0.1–0.2% CFR for PUUV infections with high morbidity in the Old World, with now over 50,000 registered NE cases in Europe, and even over 175,000 in Western Russia. In some peak years, Russia witnessed more than 10,000 cases/year (e.g. 11,413 in 1985), whereas registered NE numbers in Western Europe recently took epidemic proportions (e.g. 3,259 in Finland, 2008, and 1,874 in Germany, 2010), probably as a result of global warming [8]. Thus, it should not come as a surprise that Russian or European clinicians had or have now identified clinical characteristics claimed as being specific for “American HPS”. The Centers for Disease J. Clement (*) : P. Maes :K. Lagrou :M. Van Ranst National Reference Laboratory for Hantavirus Infections, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium e-mail: jan.clement@uzleuven.be

Development of One-Step, Real-Time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E

ABSTRACT The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 × 108 RNA copies per reaction mixture (5 μl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 103 to 1010 viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 × 109 per reaction mixture can be detected, which corresponds to 104 to 1011 viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.