Mucosal bivalent live attenuated vaccine protects against human metapneumovirus and respiratory syncytial virus in mice.

Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.

Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d strain: genome sequencing, in vivo virus replication kinetics, and viral dose effect.

BACKGROUND: Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21). RESULTS: We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10- 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality. CONCLUSIONS: These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.

Recombination at the emergence of the pathogenic rabbit haemorrhagic disease virus Lagovirus europaeus/GI.2.

Rabbit haemorrhagic disease is a viral disease that emerged in the 1980s and causes high mortality and morbidity in the European rabbit (Oryctolagus cuniculus). In 2010, a new genotype of the rabbit haemorrhagic disease virus emerged and replaced the former circulating Lagovirus europaeus/GI.1 strains. Several recombination events have been reported for the new genotype Lagovirus europaeus/GI.2, with pathogenic (variants GI.1a and GI.1b) and benign (genotype GI.4) strains that served as donors for the non-structural part while GI.2 composed the structural part; another recombination event has also been described at the p16/p23 junction involving GI.4 strains. In this study, we analysed new complete coding sequences of four benign GI.3 strains and four GI.2 strains. Phylogenetic and recombination detection analyses revealed that the first GI.2 strains, considered as non-recombinant, resulted from a recombination event between GI.3 and GI.2, with GI.3 as the major donor for the non-structural part and GI.2 for the structural part. Our results indicate that recombination contributed to the emergence, persistence and dissemination of GI.2 as a pathogenic form and that all described GI.2 strains so far are the product of recombination. This highlights the need to study full-genomic sequences of lagoviruses to understand their emergence and evolution.

Retrospective Analysis Shows That Most RHDV GI.1 Strains Circulating Since the Late 1990s in France and Sweden Were Recombinant GI.3P-GI.1d Strains.

Recombination is one of the major sources of genetic variation in viruses. RNA viruses, such as rabbit hemorrhagic disease virus (RHDV), are among the viruses with the highest recombination rates. Several recombination events have been described for RHDV, mostly as a consequence of their genomic architecture. Here, we undertook phylogenetic and recombination analyses of French and Swedish RHDV strains from 1994 to 2016 and uncovered a new intergenotypic recombination event. This event occurred in the late 1990s/early 2000s and involved nonpathogenic GI.3 strains as donors for the nonstructural part of the genome of these recombinants, while pathogenic GI.1d strains contributed to the structural part. These GI.3P-GI.1d recombinant strains did not entirely replace GI.1d (nonrecombinant) strains, but became the dominant strains in France and Sweden, likely due to a fitness advantage associated with this genomic architecture. GI.3P-GI.1d (P stands for polymerase) strains persisted until 2013 and 2016 in Sweden and France, respectively, and cocirculated with the new genotype GI.2 in France. Since strains from the first GI.2 outbreaks were GI.3P-GI.2, we hypothesize that GI.3P-GI.1d could be the parental strain. Our results confirm the outstanding recombination ability of RHDV and its importance in the evolution of lagoviruses, which was only revealed by studying complete genomic sequences.

Genetic diversity and evolution of Hare Calicivirus (HaCV), a recently identified lagovirus from Lepus europaeus.

First recognized as highly pathogenic viruses, hare lagoviruses belonging to genotype GII.1 (EBHSV) infect various Lepus species. Genetically distinct benign lagoviruses (Hare Calicivirus, HaCV) have recently been identified but few data have been available so far on these strains. The analysis of 199 samples from hunted hares collected throughout France allowed the detection of 20 HaCV and showed that they were widely distributed in this country. Ten HaCV capsid protein gene sequences were characterized. A first HaCV capsid protein structural model was proposed, revealing a global structure similar to that of a pathogenic GII.1 strain. The HaCV sequences showed an even higher genetic diversity than previously appreciated, with the characterization of two genotypes (GII.2, GII.3) and several additional putative genotypes. The most recent common ancestor for HaCV VP60 gene was estimated to be much older than that for GII.1 pathogenic strains. These results give new insights into the phylogenetic relationships of HaCV within the Lagovirus genus.

First Complete Genome Sequence of a Hare Calicivirus Strain Isolated from Lepus europaeus.

The first full-genome sequence of a hare calicivirus (HaCV), recently characterized as a novel member of the Caliciviridae, is described. This presumed nonpathogenic lagovirus is 7,433 nucleotides long, shows the same genomic organization as that of other lagoviruses, and has the highest nucleotide identity (79%) with pathogenic European brown hare syndrome viruses.