Lymphocytic Choriomeningitis Virus Infections and Seroprevalence, Southern Iraq.

Acute febrile neurological infection cases in southern Iraq (N = 212) were screened for lymphocytic choriomeningitis virus (LCMV). Two LCMV IgM-positive serum samples and 2 cerebrospinal fluid samples with phylogenetically distinct LCMV strains were found. The overall LCMV seroprevalence was 8.8%. LCMV infections are common and associated with acute neurological disease in Iraq.

Global Distribution of Human Protoparvoviruses.

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.

Increase in outbreaks of gastroenteritis linked to bathing water in Finland in 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.

[Recurrent epidemics of gastroenteritis caused by norovirus GI.3 in a small hotel]. / Norovirus GI.3 aiheutti toistuvia vatsatautiepidemioita pienessä hotellissa kesälä 2013.

BACKGROUND: Recurrent cases of gastroenteritis occurred in a small hotel. The causative agent of disease could not be detected. MATERIAL AND METHODS: The cause and the source of the disease were established through epidemiological investigations and laboratory diagnosis. RESULTS: The causative agent of the disease was norovirus GI.3. Norovirus GI was detected in the water from the well and on surfaces at the hotel. CONCLUSIONS: Both epidemiological investigations and laboratory diagnostics are needed in resolving epidemics. Continuous development of laboratory methods is important.

Highly divergent type 2 and 3 vaccine-derived polioviruses isolated from sewage in Tallinn, Estonia.

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.

Norovirus GII.17 Caused Five Outbreaks Linked to Frozen Domestic Bilberries in Finland, 2019.

In March 2019, the Finnish Institute for Health and Welfare and Finnish Food Authority started an outbreak investigation after a notification of food business operators' recall of frozen bilberries due to a norovirus finding. A retrospective search was conducted in the food and waterborne outbreak notification system to identify the notifications linked to norovirus and consumption of bilberries in January-March 2019. Five outbreaks were found in which norovirus GII or GII.17 had been detected in patient samples. A pooled retrospective cohort study was performed for those four in which a questionnaire study had been done. A case was defined as a person with diarrhoea or vomiting within 2 days after consuming a meal studied at one of the outbreak locations. Of 79 participants, 45 (57%) cases were identified. Persons that had consumed foods containing unheated bilberries were three times more likely to get ill than those who had not consumed them (RR 3.1, CI 95% 1.2-8.1, p = 0.02). Norovirus GII.17 was found in 16/17 patient samples sent for further typing. Identical norovirus GII.17 was detected in frozen Finnish bilberries and patient samples. At the berry packaging premises, signs of norovirus GII contamination were found in packaging lines. A new procedure for extracting viral nucleic acid from food and environmental samples was used during the outbreak investigation. Consumption of industrially packed frozen berries as heated would be one of the means to prevent norovirus infections.

Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland.

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area. Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered. Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland.

Wastewater-based surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used to monitor the population-level prevalence of the COVID-19 disease. In many cases, due to lockdowns or analytical delays, the analysis of wastewater samples might only be possible after prolonged storage. In this study, the effect of storage conditions on the RNA copy numbers of the SARS-CoV-2 virus in wastewater influent was studied and compared to the persistence of norovirus over time at 4 °C, -20 °C, and -75 °C using the reverse-transcription quantitative PCR (RT-qPCR) assays E-Sarbeco, N2, and norovirus GII. For the first time in Finland, the presence of SARS-CoV-2 RNA was tested in 24 h composite influent wastewater samples collected from Viikinmäki wastewater treatment plant, Helsinki, Finland. The detected and quantified SARS-CoV-2 RNA copy numbers of the wastewater sample aliquots taken during 19-20 April 2020 and stored for 29, 64, and 84 days remained surprisingly stable. In the stored samples, the SARS betacoronavirus and SARS-CoV-2 copy numbers, but not the norovirus GII copy numbers, seemed slightly higher when analyzed from the pre-centrifuged pellet-that is, the particulate matter of the influent-as compared with the supernatant (i.e., water fraction) used for ultrafiltration, although the difference was not statistically significant. Furthermore, when wastewater was spiked with SARS-CoV-2, linear decay at 4 °C was observed on the first 28 days, while no decay was visible within 58 days at -20 °C or -75 °C. In conclusion, freezing temperatures should be used for storage when immediate SARS-CoV-2 RNA analysis from the wastewater influent is not possible. Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency.

Continuing rotavirus circulation in children and adults despite high coverage rotavirus vaccination in Finland.

OBJECTIVES: To determine occurrence of residual rotavirus (RV) disease in different age groups in Finland after five to nine years of high coverage (≥90%) mass-vaccination with RotaTeqⓇ vaccine, and to examine the vaccine effect on circulating genotypes. METHODS: Since 2013 all clinical laboratories in the country were obliged to send RV positive stool samples for typing. RVs were genotyped by RT-PCR for VP7 and VP4 proteins, sequenced and compared to reference strains. RESULTS: RV continued to circulate throughout the study period at low level with a small increase in 2017-2018. There were three age-related clusters: young children representing primary or secondary vaccine failures, school-age children who may not have been vaccinated, and the elderly. Genotype distribution differed from the pre-vaccination period with a steady decline of G1P[8], emergence of G9P[8] and especially more recently G12P[8]. In the elderly, G2P[4] was predominant but was also replaced by G12P[8] in 2017-18. CONCLUSIONS: RV vaccination with a high coverage keeps RV disease at low level but does not prevent RV circulation. New RV genotypes have emerged replacing largely the previously predominant G1P[8]. Increase of overall RV activity with emergence of G12P[8] in the latest follow-up season 2017-18 might be a potential alarm sign.

Molecular epidemiology and dual serotype specificity detection of echovirus 11 strains in Finland.

Echovirus 11 (E-11) has been one of the most frequently discovered human enterovirus (HEV) in Finland during the past few years. We have studied molecular epidemiological patterns of E-11 from 1993 to 2007 exploiting the 257-nucleotide region in the 5'-part of the VP1 used for genetic typing of HEV. Designated genogroup D strains had a striking prevalence among the Finnish strains, a finding in accordance with the recent data from other geographical regions. The subgroup D4, harboring the oldest strains, had become extinct in the beginning of the millennium and D5 strains had taken over. Similarly, a new subgroup of D5 had started to diverge from the main D5 in 2006. However, in addition to endemic D strains, few single strains clustered also to genogroups A and C suggesting importation from more distant locations. The relatively large amino acid sequence variability between and within the genogroups favored the idea of antigenic differences. Neutralization assays confirmed that antigenic differences existed, although all studied E-11 strains were neutralized with antisera against the prototype strain Gregory. Five of the six studied strains belonging to genogroup D were, unexpectedly, also neutralized with antisera against coxsackievirus A9 Griggs.

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.

Two Drinking Water Outbreaks Caused by Wastewater Intrusion Including Sapovirus in Finland.

Drinking water outbreaks occur worldwide and may be caused by several factors, including raw water contamination, treatment deficiencies, and distribution network failure. This study describes two drinking water outbreaks in Finland in 2016 (outbreak I) and 2018 (outbreak II). Both outbreaks caused approximately 450 illness cases and were due to drinking water pipe breakage and subsequent wastewater intrusion into the distribution system. In both outbreaks, the sapovirus was found in patient samples as the main causative agent. In addition, adenoviruses and Dientamoeba fragilis (outbreak I), and noroviruses, astroviruses, enterotoxigenic and enterohemorragic Escherichia coli (ETEC and EHEC, respectively) and Plesiomonas shigelloides (outbreak II) were detected in patient samples. Water samples were analyzed for the selected pathogens largely based on the results of patient samples. In addition, traditional fecal indicator bacteria and host-specific microbial source tracking (MST) markers (GenBac3 and HF183) were analyzed from water. In drinking water, sapovirus and enteropathogenic E. coli (EPEC) were found in outbreak II. The MST markers proved useful in the detection of contamination and to ensure the success of contaminant removal from the water distribution system. As mitigation actions, boil water advisory, alternative drinking water sources and chlorination were organized to restrict the outbreaks and to clean the contaminated distribution network. This study highlights the emerging role of sapoviruses as a waterborne pathogen and warrants the need for testing of multiple viruses during outbreak investigation.

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 Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

Rotavirus epidemiology 5-6 years after universal rotavirus vaccination: persistent rotavirus activity in older children and elderly.

BACKGROUND: Rotavirus (RV) vaccination using RotaTeq® vaccine exclusively was introduced into Finnish National Immunization Program (NIP) in 2009, and soon reached high (≥90%) coverage. Since mid-2013, all stool samples from laboratory diagnosed cases of RV gastroenteritis in the entire country have been typed. METHODS: 364 RV positive stool samples collected from clinical laboratories over a 2-year period were G- and P-typed using RT-PCR, and the results were confirmed by sequencing. In addition, the genome segment encoding for VP6 was sequenced to distinguish between wild-type and vaccine origin (bovine) RVs. RESULTS: RV winter epidemic seasons 2013-2014 and 2014-2015 lasted until July each. The age distribution of RV cases showed two unusual clusters: one in children 6-16 years of age, too old to have been vaccinated in NIP, and the other in elderly over 70 years of age. In children, diverse genotypes were observed without any obvious predominance. The most common ones were G1P[8] (30.0%), G2P[4] (22.4%), G9P[8] (15.8%), G3P[8] (12.2%) and G4P[8] (11.2%). The genotype distribution was not different among vaccinated and unvaccinated children. Most cases in the elderly were associated with G2P[4]. CONCLUSIONS: Even at high vaccine coverage and high effectiveness of RV vaccine, RV activity continues to persist, particularly in unvaccinated older children. RV genotypes show greater diversity than before RV vaccinations. We conclude that RV disease can be controlled but not eliminated by vaccinations. Herd-protection in long-term follow-up may be less than at the start of RV vaccinations.

An Outbreak of Norovirus Infections Among Lunch Customers at a Restaurant, Tampere, Finland, 2015.

On January 29, 2015, the city of Tampere environmental health officers were informed of a possible foodborne outbreak among customers who had eaten lunch in restaurant X. Employees of electric companies A and B had a sudden onset of gastrointestinal symptoms. We conducted a retrospective cohort study to identify the vehicle, source, and causative agent of the outbreak. A case was defined as an employee of companies A or B with diarrhea and/or vomiting who ate lunch at Restaurant X on January 26, 2015. All employees of the companies attending the implicated lunch were invited to participate in the cohort study. Environmental investigation was conducted. Twenty-one responders were included in statistical analysis, of which 11 met with the case definition. Of the 15 food items consumed by participants, four food items were associated with gastroenteritis. Of four kitchen staff, three tested positive for norovirus GIP7, the strain was found earlier in the community. No patient samples were obtained. Level of hygiene in the kitchen was inadequate. Infected kitchen staff probably transmitted norovirus by inadequate hygiene practices. No new cases associated with Restaurant X were reported after the hygiene practices were improved.

The Presence and Seroprevalence of Arthropod-Borne Viruses in Nasiriyah Governorate, Southern Iraq: A Cross-Sectional Study.

The knowledge on the presence and seroprevalence of arboviruses in Iraq is fragmental. To assess the exposure of the population to arbovirus infections in southern Iraq, we conducted a serological screening of the most common arbovirus groups using immunofluorescence, hemagglutination inhibition and neutralization tests. Serum samples of 399 adult volunteers were collected in Nasiriyah, Iraq. Antibodies were detected against West Nile virus (WNV) (11.6%), sandfly-borne Sicilian virus serocomplex (18.2%), sandfly-borne Naples virus serocomplex (7.8%), Sindbis virus (1.5%), chikungunya virus (0.5%), and Tahyna virus (2.0%). The results suggest that WNV and sandfly-borne phlebovirus infections are common in southern Iraq, and these viruses should be considered as potential causative agents in patients with febrile disease and/or neurological manifestations.

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.

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.

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.