Recombination in the Evolution of Enterovirus C Species Sub-Group that Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus). However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C) species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A–C). In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5′UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses.     

Genetic relationship between cocirculating Human enteroviruses species C

Recombination events between human enteroviruses (HEV) are known to occur frequently and to participate in the evolution of these viruses. In a previous study, we reported the isolation of a panel of viruses belonging to the Human enterovirus species C (HEV-C) that had been cocirculating in a small geographic area of Madagascar in 2002. This panel included type 2 vaccine-derived polioviruses (PV) that had caused several cases of acute flaccid paralysis in humans. Previous partial sequencing of the genome of these HEV-C isolates revealed considerable genetic diversity, mostly due to recombination. In the work presented herein, we carried out a more detailed characterization of the genomes of viruses from this collection. First, we determined the full VP1 sequence of 41 of these isolates of different types. These sequences were compared with those of HEV-C isolates obtained from other countries or in other contexts. The sequences of the Madagascan isolates of a given type formed specific clusters clearly differentiated from those formed by other strains of the same type isolated elsewhere. Second, we sequenced the entire genome of 10 viruses representing most of the lineages present in this panel. All but one of the genomes appeared to be mosaic assemblies of different genomic fragments generated by intra- and intertypic recombination. The location of the breakpoints suggested potential preferred genomic regions for recombination. Our results also suggest that recombination between type HEV-99 and other HEV-C may be quite rare. This first exhaustive genomic analysis of a panel of non-PV HEV-C cocirculating in a small human population highlights the high frequency of inter and intra-typic genetic recombination, constituting a widespread mechanism of genetic plasticity and continually shifting the HEV-C biodiversity.     

Complete genomic sequencing shows that polioviruses and members of human enterovirus species C are closely related in the noncapsid coding region

The 65 human enterovirus serotypes are currently classified into five species: Poliovirus (3 serotypes), Human enterovirus A (HEV-A) (12 serotypes), HEV-B (37 serotypes), HEV-C (11 serotypes), and HEV-D (2 serotypes). Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 constitute HEV-C. We have determined the complete genome sequences for the remaining nine HEV-C serotypes and compared them with the complete sequences of CAV21, CAV24, and the polioviruses. The viruses were most diverse in the capsid region (4 to 36% amino acid difference). A high degree of capsid sequence conservation (96% amino acid identity) suggests that CAV15 and CAV18 should be classified as strains of CAV11 and CAV13, respectively. In the 3CD region, CAV1, CAV19, and CAV22 differed from one another by only 1.2 to 1.4% and CAV11, CAV13, CAV17, CAV20, CAV21, CAV24, and the polioviruses differed from one another by only 1.2 to 3.6%. The two groups, however, differed from one another by 14.6 to 16.2%. The polioviruses as a group were monophyletic only in the capsid region. Only one group of serotypes (CAV1, CAV19, and CAV22) was consistently monophyletic in multiple genome regions. Incongruities among phylogenetic trees based on different genome regions strongly suggest that recombination has occurred between the polioviruses, CAV11, CAV13, CAV17, and CAV20. The close relationship among the polioviruses and CAV11, CAV13, CAV17, CAV20, CAV21, and CAV24 and the uniqueness of CAV1, CAV19, and CAV22 suggest that revisions should be made to the classification of these viruses.     

Phylogenetic analysis of the first complete hepatitis E virus (HEV) genome from Africa

Hepatitis E virus (HEV) is globally distributed, transmitted enterically and between humans and animals. Phylogenetic analysis has identified five distinct HEV genotypes. The first full-length sequence of an African strain (Chad) is presented and compared to 31 complete HEV genomes available, including the fulminant hepatitis strain from India, swine strains and a strain from Morocco. The two African strains are more closely related to genotype 1 than to any other genotypes and together they possibly form a sub-genotype or sixth genotype. The first evidence for recombination between divergent HEV strains is presented.     

Phylogeny of protostome worms derived from 18S rRNA sequences

The phylogenetic relationships of protostome worms were studied by comparing new complete 18S rRNA sequences of Vestimentifera, Pogonophora, Sipuncula, Echiura, Nemertea, and Annelida with existing 18S rRNA sequences of Mollusca, Arthropoda, Chordata, and Platyhelminthes. Phylogenetic trees were inferred via neighbor-joining and maximum parsimony analyses. These suggest that (1) Sipuncula and Echiura are not sister groups; (2) Nemertea are protostomes; (3) Vestimentifera and Pogonophora are protostomes that have a common ancestor with Echiura; and (4) Vestimentifera and Pogonophora are a monophyletic clade.      

Evidence of recombination between divergent hepatitis E viruses

Phylogenetic and recombination analysis was performed on 32 complete hepatitis E virus (HEV) genomes from infected humans and pigs. For the first time, evidence for recombination between divergent HEV strains was obtained, with at least two strains being found to have discordant phylogenetic relationships consistent with the occurrence of intragenotype recombination. This finding confirms that humans can be dually infected with divergent HEV strains and has implications for the emergence and evolution of new HEV epidemics.     

High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

Background

Hepatitis E virus (HEV) infects a range of species, including humans, pigs, wild boars and deer. Zoonotic transmission may contribute to the high HEV seroprevalence in the human population of many countries. A novel divergent HEV from moose (Alces alces) in Sweden was recently identified by partial genome sequencing. Since only one strain was found, its classification within the HEV family, prevalence in moose and zoonotic potential was unclear. We therefore investigated samples from 231 moose in seven Swedish counties for HEV, and sequenced a near complete moose HEV genome. Phylogenetic analysis to classify this virus within the family Hepeviridae and to explore potential host specific determinants was performed.

Methods and Findings

The HEV prevalence of moose was determined by PCR (marker for active infection) and serological assays (marker of past infection) of sera and 51 fecal samples from 231 Swedish moose. Markers of active and past infection were found in 67 (29%) animals, while 34 (15%) were positive for HEV RNA, 43 (19%) were seropositive for anti-HEV antibodies, and 10 (4%) had both markers. The number of young individuals positive for HEV RNA was larger than for older individuals, and the number of anti-HEV antibody positive individuals increased with age. The high throughput sequenced moose HEV genome was 35-60% identical to existing HEVs. Partial ORF1 sequences from 13 moose strains showed high similarity among them, forming a distinct monophyletic clade with a common ancestor to HEV genotype 1-6 group, which includes members known for zoonotic transmission.

Conclusions

This study demonstrates a high frequency of HEV in moose in Sweden, with markers of current and past infection demonstrated in 30% of the animals. Moose is thus an important animal reservoir of HEV. The phylogenetic relationship demonstrated that the moose HEV belonged to the genotype 1-6 group, which includes strains that also infect humans, and therefore may signify a potential for zoonotic transmission of this HEV.     

DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)

Partial (～ 780 bp) mitochondrial cytochrome c oxidase subunit I (COI) and near complete nuclear 18S rDNA (～ 1,780 bp) sequences were directly compared to assess their relative usefulness as markers for species identification and phylogenetic analysis of coccidian parasites (phylum Apicomplexa). Fifteen new COI partial sequences were obtained using two pairs of new primers from rigorously characterised (sensu Reid and Long, 1979) laboratory strains of seven Eimeria spp. infecting chickens as well as three additional sequences from cloned laboratory strains of Toxoplasma gondii (ME49 and GT1) and Neospora caninum (NC1) that were used as outgroup taxa for phylogenetic analyses. Phylogenetic analyses based on COI sequences yielded robust support for the monophyly of individual Eimeria spp. infecting poultry except for the Eimeria mitis/mivati clade; however, the lack of a phenotypically characterised strain of E. mivati precludes drawing any firm conclusions regarding this observation. Unlike in the 18S rDNA-based phylogenetic reconstructions, Eimerianecatrix and Eimeria tenella formed monophyletic clades based on partial COI sequences. A species delimitation test was performed to determine the probability of making a correct identification of an unknown specimen (sequence) based on either complete 18S rDNA or partial COI sequences; in almost all cases, the partial COI sequences were more reliable as species-specific markers than complete 18S rDNA sequences. These observations demonstrate that partial COI sequences provide more synapomorphic characters at the species level than complete 18S rDNA sequences from the same taxa. We conclude that COI performs well as a marker for the identification of coccidian taxa (Eimeriorina) and will make an excellent DNA 'barcode' target for coccidia. The COI locus, in combination with an 18S rDNA sequence as an 'anchor', has sufficient phylogenetic signal to assist in the resolution of apparent paraphylies within the coccidia and likely more broadly within the Apicomplexa.     

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.     

Whole Genomic Sequence and Replication Kinetics of a New Enterovirus C96 Isolated from Guangdong,China with a Different Cell Tropism

Enterovirus 96 (EV-C96) is a newly described serotype within the enterovirus C (EV-C) species, and its biological and pathological characters are largely unknown. In this study, we sequenced the whole genome of a novel EV-C96 strain that was isolated in 2011 from a patient with acute flaccid paralysis (AFP) in Guangdong province, China and characterized the properties of its infection. Sequence analysis revealed the close relationship between the EV-C96 strains isolated from the Guangdong and Shandong provinces of China, and suggested that recombination events occurred both between these EV-C96 strains and with other EV-C viruses. Moreover, the virus replication kinetics showed EV-C96 Guangdong strain replicated at a high rate in RD cells and presented a different cell tropism to other strains isolated from Shandong recently. These findings gave further insight into the evolutionary processes and extensive biodiversity of EV-C96.     

Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.     

Genetic and Experimental Evidence for Cross-Species Infection by Swine Hepatitis E Virus

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared ≥97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.     

Complete genome sequence of the genotype 4 hepatitis E virus strain prevalent in swine in Jiangsu Province, China, reveals a close relationship with that from the human population in this area

Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human viruses. In a previous study, we sequenced the complete genome of a human genotype 4 HEV strain (HM439284) that is prevalent in Jiangsu Province, China. Here we report the complete genome of one genotype 4 HEV strain which is prevalent in swine herds in Jiangsu Province. Phylogenetic analysis indicated that the swine HEV strain in the present study has high sequence homology (>92%) with the genotype 4 HEV strains prevalent in the human population of Jiangsu Province. These results suggested that the genotype 4 HEV strain in the present study is involved in cross-species transmission between swine and humans in this area.     

Molecular characterisation of Neoparamoeba strains isolated from gills of Scophthalmus maximus

Small subunit ribosomal RNA gene sequences were determined for 5 amoeba strains of the genus Neoparamoeba Page, 1987 that were isolated from gills of Scophthalmus maximus (Linnaeus, 1758). Phylogenetic analyses revealed that 2 of 5 morphologically indistinguishable strains clustered with 6 strains identified previously as N. pemaquidensis (Page, 1970). Three strains branched as a clade separated from N. pemaquidenis and N. aestuarina (Page, 1970) clades. Our analyses suggest that these 3 strains could be representatives of an independent species. In a more comprehensive eukaryotic tree, strains belonging to Neoparamoeba spp. formed a monophyletic group with a sister-group relationship to Vannella anglica Page, 1980. They did not cluster with Gymnamoebae of the families Hartmannellidae, Flabellulidae, Leptomyxidae or Amoebidae presently available in GenBank.