Haider Al-Hello

An enterovirus strain isolated from diabetic child belongs to a genetic subcluster of echovirus 11, but is also neutralised with monotypic antisera to coxsackievirus A9

An enterovirus strain (designated D207) isolated from a Slovakian diabetic child and originally serotyped as coxsackievirus A9 (CAV-9) was found to cause rapid cytolysis coinciding with severe functional damage of the surviving cells in primary cultures of human pancreatic islets. This finding prompted us to clone the isolate for full-length genome sequencing and molecular characterization as the prototype strain of CAV-9 is known to cause only minimal damage to insulin-producing beta-cells. Based on capsid-coding sequence comparisons, the isolate turned out to be echovirus 11 (E-11). Phylogenetic analyses demonstrated that E-11/D207 was closely related to a specific subgroup B of E-11 strains known to cause uveitis. To study further antigenic properties of isolate E-11/D207 and uveitis-causing E-11 strains, neutralization experiments were carried out with CAV-9- and E-11-specific antisera. Unlike the prototype strains, the isolate E-11/D207 and uveitis-causing E-11 strains were well neutralized with both CAV-9- and E-11-specific antisera. Attempts to identify recombination of the capsid coding sequences as a reason for double-reactivity using the Simplot analysis failed to reveal major transferred motifs. However, peptide scanning technique was able to identify antigenic regions of capsid proteins of E-11/D207 as well as regions cross-reacting with an antiserum raised to CAV-9. Thus, double specificity of E-11/D207 seems to be a real characteristic shared by the phylogenetically closely related virus strains in the genetic subgroup B of E-11.

Amino acids of coxsackie B5 virus are critical for infection of the murine insulinoma cell line, MIN-6

It was shown recently that 15 successive passages of a laboratory strain of the Coxsackie B virus 5 in a mouse pancreas (CBV‐5‐MPP) resulted in apparent changes in the virus phenotype, which led to the capacity to induce a diabetes‐like syndrome in mice. For further characterization of islet cell interactions with a passaged virus strain, a murine insulinoma cell line, MIN‐6, was selected as an experimental model. The CBV‐5‐MPP virus strain was not able to replicate in MIN‐6 cells in vitro but required adaptation over a few days for progeny production and the generation of cytopathic effects. In order to determine the genetic characteristics required for virus growth in MIN‐6 cells, the whole genome of the MIN‐6‐adapted virus variant was sequenced, and critical amino acids were identified by comparing the sequence with that of a virus strain passaged repeatedly in the mouse pancreas. The results of site‐directed mutagenesis demonstrated that only one residue, amino acid 94 of VP1, is a major determinant for virus adaptation to MIN‐6 cells. J. Med. Virol. 81:296–304, 2009.

Highly Divergent Type 2 and 3 Vaccine-Derived Polioviruses Isolated from Sewage in Tallinn, Estonia

ABSTRACT Highly divergent vaccine-derived polioviruses (VDPVs) have been isolated from sewage in Tallinn, Estonia, since 2002. Sequence analysis of VDPVs of serotypes 2 and 3 showed that they shared common noncapsid region recombination sites, indicating origination from a single trivalent oral polio vaccine dose, estimated to have been given between 1986 and 1998. The sewage isolates closely resemble VDPVs chronically excreted by persons with common variable immunodeficiency, but no chronic excretors have yet been identified in Estonia.

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.

Characteristics of an Environmentally Monitored Prolonged Type 2 Vaccine Derived Poliovirus Shedding Episode that Stopped without Intervention

Vaccine derived poliovirus (VDPV) type 2 strains strongly divergent from the corresponding vaccine strain, Sabin 2, were repeatedly isolated from sewage in Slovakia over a period of 22 months in 2003–2005. Cell cultures of stool specimens from known immune deficient patients and from an identified putative source population of 500 people failed to identify the potential excretor(s) of the virus. The occurrence of VDPV in sewage stopped without any intervention. No paralytic cases were reported in Slovakia during the episode. According to a GenBank search and similarity plotting-analysis, the closest known relative of the first isolate PV2/03/SVK/E783 through all main sections of the genome was the type 2 poliovirus Sabin strain, with nucleotide identities in 5′UTR, P1, P2, P3, and 3′UTR parts of the genome of 88.6, 85.9, 87.3, 88.5, and 94.0 percent, respectively. Phenotypic properties of selected Slovakian aVDPV strains resembled those of VDPV strains isolated from immune deficient individuals with prolonged PV infection (iVDPV), including antigenic changes and moderate neurovirulence in the transgenic mouse model. One hundred and two unique VP1 coding sequences were determined from VDPV strains isolated from 34 sewage specimens. Nucleotide differences from Sabin 2 in the VP1 coding region ranged from 12.5 to 15.6 percent, and reached a maximum of 9.6 percent between the VDPV strains under study. Most of the nucleotide substitutions were synonymous but as many as 93 amino acid positions out of 301 in VP1 showed substitutions. We conclude that (1) individuals with prolonged poliovirus infection are not as rare as suggested by the studies on immune deficient patients known to the health care systems and (2) genetic divergence of VDPV strains may remain extensive during years long replication in humans.

Molecular surveillance of norovirus, 2005-16: an epidemiological analysis of data collected from the NoroNet network

BACKGROUND The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. METHODS We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. FINDINGS We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2-3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16-GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17-GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015-16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2-53·9], as did GII.17 [median 6·3%, range 0-44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe-GII.4 or GII.P4-GII.4). INTERPRETATION Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. FUNDING European Unions Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific ‘signature’ amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific ‘signature’ amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

Increase in outbreaks of gastroenteritis linked to bathing water in Finland in summer 2014

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare (THL) and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks.

Enterovirus strain and type-specific differences in growth kinetics and virus-induced cell destruction in human pancreatic duct epithelial HPDE cells

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1 diabetes. In addition, the capability for rapid adaptation to different cell types suggests that, on occasion, enterovirus strains with different pathogenetic properties may arise from less pathogenic ancestors.

Evolution of type 2 vaccine derived poliovirus lineages. Evidence for codon-specific positive selection at three distinct locations on capsid wall.

Partial sequences of 110 type 2 poliovirus strains isolated from sewage in Slovakia in 2003–2005, and most probably originating from a single dose of oral poliovirus vaccine, were subjected to a detailed genetic analysis. Evolutionary patterns of these vaccine derived poliovirus strains (SVK-aVDPV2) were compared to those of type 1 and type 3 wild poliovirus (WPV) lineages considered to have a single seed strain origin, respectively. The 102 unique SVK-aVDPV VP1 sequences were monophyletic differing from that of the most likely parental poliovirus type 2/Sabin (PV2 Sabin) by 12.5–15.6%. Judging from this difference and from the rate of accumulation of synonymous transversions during the 22 month observation period, the relevant oral poliovirus vaccine dose had been administered to an unknown recipient more than 12 years earlier. The patterns of nucleotide substitution during the observation period differed from those found in the studied lineages of WPV1 or 3, including a lower transition/transversion (Ts/Tv) bias and strikingly lower Ts/Tv rate ratios at the 2nd codon position for both purines and pyrimidines. A relatively low preference of transitions at the 2nd codon position was also found in the large set of VP1 sequences of Nigerian circulating (c)VDPV2, as well as in the smaller sets from the Hispaniola cVDPV1 and Egypt cVDPV2 outbreaks, and among aVDPV1and aVDPV2 strains recently isolated from sewage in Finland. Codon-wise analysis of synonymous versus non-synonymous substitution rates in the VP1 sequences suggested that in five codons, those coding for amino acids at sites 24, 144, 147, 221 and 222, there may have been positive selection during the observation period. We conclude that pattern of poliovirus VP1 evolution in prolonged infection may differ from that found in WPV epidemics. Further studies on sufficiently large independent datasets are needed to confirm this suggestion and to reveal its potential significance.