Päivi Klemola

Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland

BACKGROUND A nationwide outbreak of hand, foot and mouth disease (HFMD) occurred in Finland in autumn 2008. The outbreak was untypical since a considerable number of clinically diagnosed patients were adults. Furthermore, many of the patients suffered from onychomadesis several weeks after the acute phase of HFMD. OBJECTIVES Detection, identification and phylogenetic analysis of human enteroviruses (HEV) that caused the outbreak. STUDY DESIGN A total of 420 clinical specimens were obtained from 317 HFMD cases all over the country. The presence of HEV in the specimens was analysed by virus isolation and/or direct real-time RT-PCR; selected HEV strains were further typed by molecular methods. The genetic similarities of HEV strains were assessed by phylogenetic analyses on partial VP1 sequences. RESULTS HEV were detected in 212 HFMD cases, including both children and adults, throughout Finland. Two HEV types, coxsackieviruses A6 (CV-A6) and A10 (CV-A10), were identified as the causative agents of the outbreak. One genetic variant of CV-A6 predominated, but, additionally, three other genetically distinct CV-A6 strains were found. All CV-A10 strains segregated into one genetic cluster distinct from previously reported CV-A10 sequences. CONCLUSIONS The Finnish 2008 HFMD outbreak was caused by two infrequently detected, co-circulating, coxsackie A viruses. Our data suggest endemic circulation of both CV-A types in Northern Europe and that the outbreak was due to the emergence of new genetic variants of these viruses.

Cellular tropism of human enterovirus D species serotypes EV-94, EV-70, and EV-68 in vitro: implications for pathogenesis.

Enterovirus 94 (EV‐94) is an enterovirus serotype described recently which, together with EV‐68 and EV‐70, forms human enterovirus D species. This study investigates the seroprevalences of these three serotypes and their abilities to infect, replicate, and damage cell types considered to be essential for enterovirus‐induced diseases. The cell types studied included human leukocyte cell lines, primary endothelial cells, and pancreatic islets. High prevalence of neutralizing antibodies against EV‐68 and EV‐94 was found in the Finnish population. The virus strains studied had wide leukocyte tropism. EV‐94 and EV‐68 were able to produce infectious progeny in leukocyte cell lines with monocytic, granulocytic, T‐cell, or B‐cell characteristics. EV‐94 and EV‐70 were capable of infecting primary human umbilical vein endothelial cells, whereas EV‐68 had only marginal progeny production and did not induce cytopathic effects in these cells. Intriguingly, EV‐94 was able to damage pancreatic islet β‐cells, to infect, replicate, and cause necrosis in human pancreatic islets, and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells. These results suggest that HEV‐D viruses may be more prevalent than has been thought previously, and they provide in vitro evidence that EV‐94 may be a potent pathogen and should be considered a potentially diabetogenic enterovirus type. J. Med. Virol. 82:1940–1949, 2010.

Diabetogenic Effects of the Most Prevalent Enteroviruses in Finnish Sewage

Common enterovirus infections appear to initiate or facilitate the pathogenetic processes leading to type 1 diabetes (T1D) and also sometimes precipitate the clinical disease. We have recently demonstrated that (1) enterovirus‐positive islet cells were seen on postmortem pancreatic specimens of several T1D patients but not in the corresponding samples of nondiabetic controls, and (2) several different enteroviruses can be associated with T1D. Enterovirus infections are transmitted from person to person by fecal–oral or respiratory routes, which means that infections usually start from the respiratory or gastrointestinal mucosa. Regardless of the clinical symptoms of the disease, viral replication continues in the submucosal lymphatic tissue for several weeks, up to a couple of months, and during that time the virus is excreted into the feces and translocated to the environment. Monitoring of sewage samples for enteroviruses can be used as a tool in epidemiologic studies of enterovirus. Finland has successfully used environmental control data in poliovirus surveillance for decades. About 24 samples have been collected annually from the Helsinki region, which covers about 20% of the population. In the present study, we have reanalyzed the sewage samples of the years 1993–2004 for nonpolio enteroviruses by inoculating them into five different continuous cell lines known to cover a wide range of serotypes. Isolated strains were identified by RT‐PCR and VP1 sequencing. The most commonly detected serotypes were coxsackie B viruses (CBV1–5) and echoviruses (E6, 7, 11, 25, 30). Diabetogenic effects of the most prevalent enterovirus serotypes were studied in primary human β cells.

Molecular evolution and epidemiology of echovirus 6 in Finland

Echovirus 6 (E-6) (family Picornaviridae, genus Enterovirus) is one of the most commonly detected enteroviruses worldwide. The aim of this study was to determine molecular evolutionary and epidemiologic patterns of E-6. A complete genome of one E-6 strain and the partial VP1 coding regions of 169 strains were sequenced and analyzed along with sequences retrieved from the GenBank. The complete genome sequence analysis suggested complex recombination history for the Finnish E-6 strain. In VP1 region, the phylogenetic analysis suggested three major clusters that were further divided to several subclusters. The evolution of VP1 coding region was dominated by negative selection suggesting that the phylogeny of E-6 VP1 gene is predominantly a result of synonymous substitutions (i.e. neutral genetic drift). The partial VP1 sequence analysis suggested wide geographical distribution for some E-6 lineages. In Finland, multiple different E-6 lineages have circulated at the same time.

Virus infections among young children—the first year of the INDIS study

The frequencies of early childhood infections were studied in healthy children with increased genetic risk for type 1 diabetes participating in the ongoing prospective high intensive infection follow‐up Study, INDIS, started in 2009 in Turku, Finland. Here the results obtained from 160 stool to 160 nasal swab specimens collected in parallel at times of infectious symptoms in 2009–2010 from 45 children at the age of 24 months or younger are reported. The specimens were analyzed for enteric (human enterovirus, norovirus, rotavirus, sapovirus, astrovirus) and respiratory RNA viruses (human enterovirus and rhinovirus) common in early childhood, respectively, using highly validated virus‐specific real‐time PCR methods. According to the results 96% of the children had at least one virus infection during the study period and one or several viral agents were detected in 76% of sample sets. The most prevalent viral agents were human rhinovirus, enterovirus, parechovirus, and norovirus (genotype GII) with positive specimens 57.5%, 28.8%, 19.4%, and 6.9%, respectively. Other intestinal viruses were found in less than 2% of stool specimens. Single infections covered 40.0% of the specimens while multiple infections with two or more infectious agents were detected in 36.3% of specimens and altogether 11 combinations of viruses were included in the mixed infections. Although human enterovirus is known to be a frequent finding in stool specimens, especially during early childhood, it was found in this study more frequently in nasal swab specimens. Whether this is true, more general, in countries with the high hygiene level remains to be shown. J. Med. Virol. 85:1678–1684, 2013.

Early human enterovirus infections in healthy Swedish children participating in the PRODIA pilot study

Human enteroviruses (HEV) are common, especially in childhood and during the enterovirus season, causing mainly asymptomatic infections but also mild and severe illnesses. Numerous studies have shown the association between HEV infections and type 1 diabetes. Here, the prevalence of HEV infections was studied in healthy Swedish children with increased HLA‐associated risk for type 1 diabetes participating in the PRODIA pilot study in which children were randomized to receive probiotics or placebo during the first 6 months of life. Stool specimens collected from 197 children in every 3 months from the age of 3 to 24 months were screened for HEV using traditional viral culturing method and identified with reverse transcriptase polymerase chain reaction (RT‐PCR) and sequencing of the partial VP1 coding part of the viral genome. Altogether 4.8% (52/1,094) of the specimens were HEV‐positive and 22.3% (44/197) of the children excreted HEV during the follow‐up. HEV‐A and HEV‐B were present in 2.1 and 2.7% of the specimens, respectively. HEV‐C and HEV‐D viruses were not detected. In total, 17 different HEV serotypes were detected and the most common findings were CV‐A9 (13.5%), CV‐A16 (11.5%), and CV‐A2 (9.6%). The majority of the infections (92.3%) were during the enterovirus season extending from July to December. Probiotic treatment did not affect significantly the risk of HEV infections during the 2‐year follow‐up although a trend for transient decrease for HEV positivity (HEV‐A and/or HEV‐B) by the age of 12 months was observed in children who received probiotics [OR 0.40; 95% confidence interval 0.15 to 1.08; P‐value 0.071, generalized estimating (GEE) analysis]. According to the results, HEV‐A findings were nearly as common as HEV‐B findings among the healthy children participating in this study. Also it was shown that serotypes belonging to HEV‐A species can be detected by means of viral culturing. J. Med. Virol. 84:923–930, 2012.

Human rhinoviruses in INDIS-study material—evidence for recovery of viable rhinovirus from fecal specimens

The significance of human rhinoviruses (HRV) as prevailing respiratory pathogens has sharpened during the recent years followed by implementation of molecular methods in detection. Rhinoviruses are detected exceedingly in hospitalized cases of respiratory infection with varying severity, in addition to being frequent in cases of common cold. The aim of this study was to evaluate occurrence of HRV in a prospective study material. The prospective INDIS material comprises nasopharyngeal (N = 429) and fecal (N = 425) specimens from children under 11 years of age collected during any clinical infection. Validated real‐time RT‐PCR assays were applied for the detection of HRV. HRV were detected numerously not only in the nasopharyngeal specimens, but a myriad also in fecal specimens, 236 (55.0%) and 149 (35.1%), respectively, fecal findings actually beyond anticipation. A total of 13 of HRV‐positive fecal specimens were selected for genetic typing in the VP4/VP2 coding region. HRV‐A strains were detected in seven specimens: HRV‐A9, ‐A10, ‐A24, ‐A49, ‐A56 and ‐A82. HRV‐B—strains were detected three times: HRV‐B42 and ‐B79, and HRV‐C twice: HRV‐C12 and HRV‐Cpat4. HRV‐B42 also showed cytopathic effect in cell culture, confirmed by real‐time RT‐PCR and VP4/VP2 sequencing, suggesting presence of viable HRV in fecal specimens. J. Med. Virol. 85:1466–1472, 2013.

Human rhinoviruses in INDIS-study material-evidence for recovery of viable rhinovirus from fecal specimens

The significance of human rhinoviruses (HRV) as prevailing respiratory pathogens has sharpened during the recent years followed by implementation of molecular methods in detection. Rhinoviruses are detected exceedingly in hospitalized cases of respiratory infection with varying severity, in addition to being frequent in cases of common cold. The aim of this study was to evaluate occurrence of HRV in a prospective study material. The prospective INDIS material comprises nasopharyngeal (N = 429) and fecal (N = 425) specimens from children under 11 years of age collected during any clinical infection. Validated real‐time RT‐PCR assays were applied for the detection of HRV. HRV were detected numerously not only in the nasopharyngeal specimens, but a myriad also in fecal specimens, 236 (55.0%) and 149 (35.1%), respectively, fecal findings actually beyond anticipation. A total of 13 of HRV‐positive fecal specimens were selected for genetic typing in the VP4/VP2 coding region. HRV‐A strains were detected in seven specimens: HRV‐A9, ‐A10, ‐A24, ‐A49, ‐A56 and ‐A82. HRV‐B—strains were detected three times: HRV‐B42 and ‐B79, and HRV‐C twice: HRV‐C12 and HRV‐Cpat4. HRV‐B42 also showed cytopathic effect in cell culture, confirmed by real‐time RT‐PCR and VP4/VP2 sequencing, suggesting presence of viable HRV in fecal specimens. J. Med. Virol. 85:1466–1472, 2013.

Enteric viral pathogens in children with inflammatory bowel disease

The causes of exacerbations of inflammatory bowel disease (IBD) are unknown. The presence of RNA of an enterovirus, norovirus GI, norovirus GII, rotavirus, astrovirus, and sapovirus was sought in stool samples of 50 children (median age 12.9 years) undergoing gastrointestinal endoscopies for IBD or its exclusion (Crohns disease n = 18, ulcerative colitis n = 13, indeterminate colitis n = 2, non‐IBD n = 17). Viral RNA was found in three fecal samples (norovirus GII n = 2, sapovirus n = 1), all in children without IBD. Therefore, enteral viruses may play only a minor role in IBD. J. Med. Virol. 84:345–347, 2012.