Since the first human respiratory syncytial virus (HRSV) genotype classification in 1998, inconsistent conclusions have been drawn regarding the criteria that define HRSV genotypes and their nomenclature, challenging data comparisons between research groups. ‬‬‬‬‬‬‬‬‬In this study, we aim to unify the field of HRSV genotype classification by reviewing the different methods that have been used in the past to define HRSV genotypes and by proposing a new classification procedure, based on well-established phylogenetic methods. All available complete HRSV genomes (>12 000 base pairs) were downloaded from GenBank and divided into the two subgroups: HRSV-A and HRSV-B. From whole genome alignments, the regions that correspond to the open reading frame of the glycoprotein G and the second hypervariable region (HVR2) of the ectodomain were extracted. In the resulting partial alignments, the phylogenetic signal within each fragment was assessed. Maximum likelihood phylogenetic trees were reconstructed using the complete genome alignments. Patristic distances were calculated between all pairs of tips in the phylogenetic tree and summarized as a density plot in order to determine a cut-off value at the lowest point following the major distance peak. Our data shows that neither the HVR2 fragment, nor the G gene contains sufficient phylogenetic signal to perform reliable phylogenetic reconstruction. Therefore, whole genome alignments were used to determine HRSV genotypes. We define a genotype using the following criteria: a bootstrap support of ≥ 70% for the respective clade and a maximum patristic distance between all members of the clade of ≤ 0.018 substitutions per site for HRSV-A or ≤ 0.026 substitutions per site for HRSV-B. By applying this definition, we distinguish 23 genotypes within subtype HRSV-A and six genotypes within subtype HRSV-B. Applying the genotype criteria on subsampled datasets confirmed the robustness of the method.

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Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo. Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans). As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains). Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies.