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Corrigendum: Updated classification of norovirus genogroups and genotypes
Updated classification of norovirus genogroups and genotypes
Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1 respectively. In recent years several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 49 (9 GI 27 GII 3 GIII 2 GIV 2 GV 2 GVI and 1 genotype each for GVII GVIII GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1 GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region noroviruses can be divided into 60 P-types (14 GI 37 GII 2 GIII 1 GIV 2 GV 2 GVI 1 GVII and 1 GX) 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.
Comparison of norovirus genogroup I, II and IV seroprevalence among children in the Netherlands, 1963, 1983 and 2006
Noroviruses are a major cause of acute gastroenteritis worldwide and are a genetically diverse group of viruses. Since 2002 an increasing number of norovirus outbreaks have been reported globally but it is not clear whether this increase has been caused by a higher awareness or reflects the emergence of new genogroup II genotype 4 (GII.4) variants. The hypothesis that norovirus prevalence has increased post-2002 and is related to the emergence of GII.4 is tested in this study. Sera collected from children aged <5 years of three Dutch cross-sectional population based cohorts in 1963 1983 and 2006/2007 (n=143 n=130 and n=376 respectively) were tested for specific serum IgG by protein array using antigens to GII.4 and a range of other antigens representing norovirus GI GII and GIV genotypes. The protein array was validated by paired sera of norovirus infected patients and supernatants of B-cell cultures with single epitope specificity. Evidence for norovirus infection was found to be common among Dutch children in each cohort but the prevalence towards different genotypes changed over time. At the genogroup level GI seroprevalence decreased significantly between 1963 and 2006/2007 while a significant increase of GII and in particular specific antibodies of the genotype GII.4 was detected in the 2006/2007 cohort. There were no children with only GII.4 antibodies in the 1963 cohort. This study shows that the high GII.4 norovirus incidence in very young children is a recent phenomenon. These findings are of importance for vaccine development and trials that are currently focusing mostly on GII.4 viruses.
Influenza A virus evolution and spatio-temporal dynamics in Eurasian wild birds: a phylogenetic and phylogeographical study of whole-genome sequence data
Low pathogenic avian influenza A viruses (IAVs) have a natural host reservoir in wild waterbirds and the potential to spread to other host species. Here we investigated the evolutionary spatial and temporal dynamics of avian IAVs in Eurasian wild birds. We used whole-genome sequences collected as part of an intensive long-term Eurasian wild bird surveillance study and combined this genetic data with temporal and spatial information to explore the virus evolutionary dynamics. Frequent reassortment and co-circulating lineages were observed for all eight genomic RNA segments over time. There was no apparent species-specific effect on the diversity of the avian IAVs. There was a spatial and temporal relationship between the Eurasian sequences and significant viral migration of avian IAVs from West Eurasia towards Central Eurasia. The observed viral migration patterns differed between segments. Furthermore we discuss the challenges faced when analysing these surveillance and sequence data and the caveats to be borne in mind when drawing conclusions from the apparent results of such analyses.
Genetic evolution of the neuraminidase of influenza A (H3N2) viruses from 1968 to 2009 and its correspondence to haemagglutinin evolution
Each year influenza viruses cause epidemics by evading pre-existing humoral immunity through mutations in the major glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). In 2004 the antigenic evolution of HA of human influenza A (H3N2) viruses was mapped (Smith et al. Science 305 371–376 2004) from its introduction in humans in 1968 until 2003. The current study focused on the genetic evolution of NA and compared it with HA using the dataset of Smith and colleagues updated to the epidemic of the 2009/2010 season. Phylogenetic trees and genetic maps were constructed to visualize the genetic evolution of NA and HA. The results revealed multiple reassortment events over the years. Overall rates of evolutionary change were lower for NA than for HA1 at the nucleotide level. Selection pressures were estimated revealing an abundance of negatively selected sites and sparse positively selected sites. The differences found between the evolution of NA and HA1 warrant further analysis of the evolution of NA at the phenotypic level as has been done previously for HA.
Fusion protein is the main determinant of metapneumovirus host tropism
Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds respectively. This study confirmed the difference in host range in turkey poults and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.
Evolutionary dynamics of human and avian metapneumoviruses
Human (HMPV) and avian (AMPV) metapneumoviruses are closely related viruses that cause respiratory tract illnesses in humans and birds respectively. Although HMPV was first discovered in 2001 retrospective studies have shown that HMPV has been circulating in humans for at least 50 years. AMPV was first isolated in the 1970s and can be classified into four subgroups A–D. AMPV subgroup C is more closely related to HMPV than to any other AMPV subgroup suggesting that HMPV has emerged from AMPV-C upon zoonosis. Presently at least four genetic lineages of HMPV circulate in human populations – A1 A2 B1 and B2 – of which lineages A and B are antigenically distinct. We used a Bayesian Markov Chain Monte Carlo (MCMC) framework to determine the evolutionary and epidemiological dynamics of HMPV and AMPV-C. The rates of nucleotide substitution relative genetic diversity and time to the most recent common ancestor (TMRCA) were estimated using large sets of sequences of the nucleoprotein the fusion protein and attachment protein genes. The sampled genetic diversity of HMPV was found to have arisen within the past 119–133 years with consistent results across all three genes while the TMRCA for HMPV and AMPV-C was estimated to have existed around 200 years ago. The relative genetic diversity observed in the four HMPV lineages was low most likely reflecting continual population bottlenecks with only limited evidence for positive selection.
Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters
Human metapneumovirus (HMPV) causes acute respiratory tract illness primarily in young children immunocompromised individuals and the elderly. Vaccines would be desirable to prevent severe illnesses in these risk groups. Here we describe the generation and evaluation of cold-passage (cp) temperature-sensitive (ts) HMPV strains as vaccine candidates. Repeated passage of HMPV at low temperatures in Vero cells resulted in the accumulation of mutations in the viral genome. Introduction of these mutations in a recombinant HMPV by reverse genetics resulted in a ts-phenotype judged on the decreased shut-off temperature for virus replication in vitro. As an alternative approach three previously described cp-respiratory syncytial virus (cp-HRSV) mutations were introduced in a recombinant HMPV which also resulted in a low shut-off temperature in vitro. Replication of these ts-viruses containing either the cp-HMPV or cp-HRSV mutations was reduced in the upper respiratory tract (URT) and undetectable in the lower respiratory tract (LRT) of hamsters. Nevertheless high titres of HMPV-specific antibodies were induced by both ts-viruses. Upon immunization with the ts-viruses the LRT of hamsters were completely protected against challenge infection with a heterologous HMPV strain and URT viral titres were reduced by 10 000-fold. In conclusion we provide proof-of-principle for two candidate live-attenuated HMPV vaccines that induce cross-protective immunity to prevent infection of the LRT in Syrian golden hamsters. Further mapping of the molecular determinants of attenuation of HMPV should be the subject of future studies.
Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses
Human metapneumovirus (HMPV) and avian metapneumovirus (AMPV) have a similar genome organization and protein composition but a different host range. AMPV subgroup C (AMPV-C) is more closely related to HMPV than other AMPVs. To investigate the specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses a minireplicon system was generated for AMPV-C and used in combination with minireplicon systems for HMPV lineages A1 and B1. Viral RNA-like molecules representing HMPV-A1 and -B1 AMPV-A and -C and human respiratory syncytial virus were replicated efficiently by polymerase complexes of HMPV-A1 and -B1 and AMPV-C but not by polymerase complexes of bovine parainfluenza virus 3. Upon exchange of HMPV and AMPV-C polymerase complex components all chimeric polymerase complexes were functional; exchange between HMPVs did not result in altered polymerase activity whereas exchange between HMPVs and AMPV-C did. Recombinant HMPV-B1 viruses in which polymerase genes were exchanged with those of HMPV-A1 replicated with normal kinetics in vitro whilst replacement with AMPV-C genes resulted in moderate differences in virus replication. In hamsters recombinant HMPV-B1 viruses in which individual polymerase genes were exchanged with those of AMPV-C were attenuated irrespective of the results obtained with minireplicon systems or in vitro replication assays. This study provides insight into the specificity and functional interaction of polymerase complex proteins of human and avian metapneumoviruses but neither minireplicon systems nor in vitro replication kinetics were found to be predictive for attenuation in permissive animals.
Immunization of Syrian golden hamsters with F subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains
Human metapneumovirus (hMPV) a newly discovered paramyxovirus is associated with acute respiratory-tract illness primarily in young children individuals with underlying disease and the elderly. Two genetic lineages of hMPV circulate around the world and viruses from these two lineages demonstrate antigenic differences. The clinical impact of hMPV warrants the development of vaccines. Recombinant soluble fusion (F) proteins of prototype viruses of the two main lineages of hMPV that can be produced in high yields have been constructed. In this study the antigenicity immunogenicity and protective efficacy of these soluble F subunit vaccines were evaluated in Syrian golden hamsters (Mesocricetus auratus). Immunization of hamsters with the soluble F proteins adjuvanted with Specol or iscom matrix induced high virus-neutralization titres with higher titres against the homologous than the heterologous virus. The neutralizing antibodies protected from subsequent infection of the lungs with both homologous and heterologous virus. Upon challenge viral titres in the nasal turbinates of immunized animals were reduced significantly compared with those of PBS-immunized animals. In conclusion a soluble F subunit vaccine for hMPV that induces cross-protective immunity for infection of the lower respiratory tract in Syrian golden hamsters has been generated.
Experimental infection of macaques with human metapneumovirus induces transient protective immunity
Human metapneumovirus (hMPV) a member of the family Paramyxoviridae is a causative agent of acute respiratory-tract illness. Two main hMPV lineages circulate worldwide and reinfections occur frequently. It is unclear what level of protection is induced by natural hMPV infection what the durability of this protection is and whether it differs for reinfection with homologous or heterologous viruses. Here protective immunity in cynomolgus macaques at different time points after inoculation with molecularly cloned prototype viruses of the two main lineages of hMPV has been addressed. Animals received a homologous challenge at 4 6 or 12 weeks after the primary infection. In addition animals that had been inoculated three times within 10 weeks were challenged with homologous or heterologous virus 8 months later. Primary infection with 107 TCID50 resulted in virus shedding and induction of virus-neutralizing antibody responses with higher titres against the homologous than the heterologous virus. Infections associated with virus shedding and seroconversion protected completely from homologous reinfection within 6 weeks and partly at 12 weeks after primary infection. Eight months later protection had waned to virtually undetectable levels. This study demonstrates that experimental hMPV infection induces transient protective immunity.