Human Enterovirus Strains Research Articles

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

Human enteroviruses (HEVs) include many different types that cause a wide range of diseases, and an effective method of genus-level identification has therefore significant clinical implications. However, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), the gold-standard method, still has shortfalls in diagnostic sensitivity and timeliness. Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay (RT-RAP) to detect HEV fragment within an hour. The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains. Among 15 types of HEV (species A-C), the sensitivity of RT-RAP was approximately 2–8 folds lower than that of the qRT-PCR in 9 types, and no-cross reaction with other viruses was observed. RT-RAP was further applied to analyze CSF and fecal specimens; the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results. These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.

High rate of human enteroviruses among clinically suspected meningitis cases at selected Hospitals in Addis Ababa, Ethiopia.

BackgroundBecause of limited infrastructure and skilled human capital, the etiology of meningitis is rarely identified in developing countries like Ethiopia. This results in unnecessary antibiotics use, economic crisis, hospitalization, and related nosocomial infections. Thus, we aimed to assess the epidemiology of human enteroviruses (HEVs) among clinically suspected meningitis cases in Addis Ababa, Ethiopia.MethodA cross-sectional study was conducted from January to August 2020 at selected Hospitals in Addis Ababa, Ethiopia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted on cerebrospinal fluid (CSF) collected from 146 clinically suspected meningitis and bacterial culture-negative patients. SPSS v 21.0 was used for data analysis and bivariate correlation was done for the association between variables of interest.ResultsHEVs were detected in 39 (26.7%) of the 146 clinically suspected meningitis cases. Most of the HEVs cases 28 (71.9%) were detected in younger-aged infants less than 1 year. The most commonly observed clinical manifestations were vomiting (75.5%) followed by fever (56.8%) and impaired consciousness or irritability (50.7%). The mean length of hospital stay for patients with enteroviral meningitis was 9 days. Many patients with HEVs were recovered with sequelae (46.2%), and HEVs has contributed for one out of the nine meningeal deaths (11.1%).ConclusionsHEVs were found to be the commonest cause of morbidity and mortality in all age groups. Many of the patients were mistreated with antibiotics and hospitalized. The detection of HEVs in 26.7% of clinically suspected meningitis cases indicated the need for molecular tests in investigating the etiology of meningitis. Therefore, we suggest the introduction of molecular tests as a routine practice in referral hospitals and the need to further characterize circulating HEVs strains.

Next generation sequencing of human enterovirus strains from an outbreak of enterovirus A71 shows applicability to outbreak investigations

BackgroundThe most recent documented Australian outbreak of enterovirus A71 (EV-A71) occurred in Sydney from 2012 to 2013. Over a four-month period more than 100 children presented to four paediatric hospitals with encephalitic presentations including fever and myoclonic jerks. The heterogeneous presentations included typical encephalomyelitis, and cardiopulmonary complications. ObjectivesTo characterise the genomes of enterovirus strains circulating during the 2013 Sydney EV-A71 outbreak and determine their phylogeny, phylogeography and association between genome and clinical phenotype. Study designWe performed an analysis of enterovirus (EV) positive specimens from children presenting to hospitals in the greater Sydney region of Australia during the 2013 outbreak. We amplified near full-length genomes of EV, and used next generation sequencing technology to sequence the virus. We used phylogenetic/phylogeographic analysis to characterize the outbreak viruses. ResultsWe amplified and sequenced 23/63 (37 %) genomes, and identified the majority (61 %) as EV-A71. The EV-A71 sequences showed high level sequence homology to C4a genogroups of EV-A71 circulating in China and Vietnam during 2012-13. Phylogenetic analysis showed EV-A71 strains associated with more severe symptoms, including encephalitis or cardiopulmonary failure, grouped together more closely than those from patients with hand, foot and mouth disease. Amongst the non-EV-A71 sequences were five other EV subtypes (representing enterovirus subtypes A and B), reflecting the diversity of EV co-circulation within the community. ConclusionsThis is the first Australian study investigating the near full-length genome of EV strains identified during a known outbreak of EV-A71. EV-A71 sequences were very similar to strains circulating in Asia during the same time period. Whole genome sequencing offers additional information over routine diagnostic testing such as characterisation of emerging recombinant strains and inform vaccine design.

An Environmental Surveillance in Uruguay Reveals the Presence of Highly Divergent Types of Human Enterovirus Species C and a High Frequency of Species A and B Types.

Information about Human Enterovirus circulation in Uruguay is scarce. The aim of this study was to generate the first description about their circulation in the country through the study of sewage samples collected before and after the switch from Oral Poliovirus Vaccine to Inactivated Poliovirus Vaccine. Viruses were concentrated by an adsorption-elution to a negatively charged membrane, and real-time quantitative PCR and qualitative PCR methods were used to detect, quantify, and characterize enteroviruses. Positive samples were inoculated in RD cells and two passages were performed. Additionally, RD+ samples were subsequently passed onto L20B cells. Human Enteroviruses were detected in 67.6% of the samples, with concentrations between 4.9 and 6.6 Log10 genomic copies per liter. 10% of positive samples replicated in RD cells, of which none in L20B cells. Molecular characterization of Human Enterovirus strains directly detected from sewage sample concentrates allowed the identification of highly divergent members of species C such as Enterovirus C99 and Coxsackievirus A13, as well as the frequent detection of species A and B members (particularly Coxsackievirus A16 and Echovirus 6, respectively). Other detected types were Coxsackievirus A2, A22, B1, B5, Echovirus 5, and 9. The characterization of viruses isolated in cell culture revealed the presence of Echovirus 6 and Coxsackievirus B3. Despite the absence of poliovirus, a wide circulation of different enterovirus types was evidenced in Uruguayan sewage samples, highlighting that the local populations are exposed to different kinds of diseases originated by several human enterovirus.

An array-based melt curve analysis method for the identification and classification of closely related pathogen strains.

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5ʹ-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017. Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS. Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees. Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans.

Genotyping of enteroviruses isolated in Kenya from pediatric patients using partial VP1 region.

Enteroviruses (EV) are responsible for a wide range of clinical diseases in humans. Though studied broadly in several regions of the world, the genetic diversity of human enteroviruses (HEV) circulating in the sub-Saharan Africa remains under-documented. In the current study, we molecularly typed 61 HEV strains isolated in Kenya between 2008 and 2011 targeting the 3′-end of the VP1 gene. Viral RNA was extracted from the archived isolates and part of the VP1 gene amplified by RT-PCR, followed by sequence analysis. Twenty-two different EV types were detected. Majority (72.0 %) of these belonged to Enterovirus B species followed by Enterovirus D (21.3 %) and Enterovirus A (6.5 %). The most frequently detected types were Enterovirus-D68 (EV-D68), followed by Coxsackievirus B2 (CV-B2), CV-B1, CV-B4 and CV-B3. Phylogenetic analyses of these viruses revealed that Kenyan CV-B1 isolates were segregated among sequences of global CV-B1 strains. Conversely, the Kenyan CV-B2, CV-B3, CV-B4 and EV-D68 strains generally grouped together with those detected from other countries. Notably, the Kenyan EV-D68 strains largely clustered with sequences of global strains obtained between 2008 and 2010 than those circulating in recent years. Overall, our results indicate that HEV strains belonging to Enterovirus D and Enterovirus B species pre-dominantly circulated and played a significant role in pediatric respiratory infection in Kenya, during the study period. The Kenyan CV-B1 strains were genetically divergent from those circulating in other countries. Phylogenetic clustering of Kenyan EV-D68 strains with sequences of global strains circulating between 2008 and 2010 than those obtained in recent years suggests a high genomic variability associated with the surface protein encoding VP1 gene in these enteroviruses.

English

Human enterovirus71 (HEV71) is responsible for hand, foot and mouth diseases (HFMD). Several outbreaks of HFMD were associated with severe neurological disease and deaths. In Malaysia, outbreaks normally occur periodically every two to three years but HEV71 were isolated throughout the year from HFMD cases. From 2010 to 2012, HEV71 strains were isolated from 37 children presented with typical HFMD. All isolates were sequenced and BLAST searched. A phylogenetic tree constructed based on the complete VP1 gene showed that all isolates belonged to subgenogroup B5. This subgenogroup has been found dominant since 2003. Key words: Human enterovirus71, molecular epidemiology, gene sequences, circulating subgenogroup.

Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity

Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied.

Novel Human Enterovirus C Infection in Child with Community-Acquired Pneumonia

Novel Human Enterovirus C Infection in Child with Community-Acquired Pneumonia

Population Dynamics and Genetic Diversity of C4 Strains of Human Enterovirus 71 in Mainland China, 1998–2010

BackgroundSince 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high.Methodology/Principal FindingsTo gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010.ConclusionsEV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.

Genetic diversity and molecular characterization of enteroviruses from sewage-polluted urban and rural rivers in the Philippines

Despite the vast distribution and expansive diversity of enteroviruses reported globally, indicators defining a complete view of the epidemiology of enteroviruses in tropical countries such as the Philippines are yet to be established. Detection of enteroviruses in the environment has been one of the markers of circulating viruses in a community. This study aimed to bridge the gap in the epidemiology of enteroviruses in the Philippines by providing an overview of the occurrence of enteroviruses in both urban and rural rivers. Molecular detection directed at the VP1 region of the enterovirus genome was performed on 44 grab river water samples collected from April to December 2009. The majority of the enterovirus serotypes detected were clustered with human enterovirus C species (HEV-C; 21/42), followed by HEV-B (12/42) and HEV-A (9/42). Porcine enterovirus 9 was also found in 12 out of 44 water samples. Phylogenetic analysis indicated that the viruses detected were closely related, if not all forming a monophyletic clade, with those enteroviruses detected previously from acute flaccid paralysis cases in the country. The clustering of environmental and human enterovirus strains implies that the circulation of these strains were associated with river contamination. This study gives further evidence of the environmental persistence of enteroviruses once they are shed in feces and likewise, provides additional data which may help in understanding the epidemiology of enteroviruses in humans, highlighting the need for more studies on the potential public health risks linked with enteroviruses found in the environment and their eventual clinical consequences in the country.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-012-0776-z) contains supplementary material, which is available to authorized users.

A reverse genetic study of the adaptation of human enterovirus 71 to growth in Chinese hamster ovary cell cultures

We selected Chinese hamster ovary (CHO) cell-adapted strains of human enterovirus 71 (HEV71) belonging to sub-genogroups B5 (HEV71-B5) and C2 (HEV71-C2) by serial passage in CHO cells at a high multiplicity of infection. During the course of CHO cell passage, virus growth improved significantly, with increasing virus titres and the presence of cytopathic effect observed. A study of virus growth kinetics revealed that the CHO cell-adapted strains of HEV71-B5 (CHO-B5) and HEV71-C2 (CHO-C2) grew efficiently in CHO cells with maximum titres >100-fold higher than unadapted parental virus. Both CHO-B5 and CHO-C2 harboured single amino acid mutations within the VP2 capsid protein gene. CHO-B5 has an amino acid substitution of K149→I in VP2 and CHO-C2 has an amino acid substitution of K149→M in VP2. An isolate of sub-genogroup C4 (HEV71-C4) failed to adapt to CHO cells during serial passage. Infectious cDNA clone-derived populations of HEV71-C4 containing the mutations K149→I or K149→M in VP2 were generated by site-directed mutagenesis. Both mutations resulted in the ability of the virus to replicate efficiently in CHO cells, indicating that amino acid position 149 in VP2 is critical for the adaptation of HEV71 to growth in CHO cells.

Emergence and Transmission Pathways of Rapidly Evolving Evolutionary Branch C4a Strains of Human Enterovirus 71 in the Central Plain of China

BackgroundLarge-scale outbreaks of hand, foot, and mouth disease (HFMD) occurred repeatedly in the Central Plain of China (Shandong, Anhui, and Henan provinces) from 2007 until now. These epidemics have increased in size and severity each year and are a major public health concern in mainland China.Principal FindingsPhylogenetic analysis was performed and a Bayesian Markov chain Monte Carlo tree was constructed based on the complete VP1 sequences of HEV71 isolates. These analyses showed that the HFMD epidemic in the Central Plain of China was caused by at least 5 chains of HEV71 transmission and that the virus continued to circulate and evolve over the winter seasons between outbreaks. Between 1998 and 2010, there were 2 stages of HEV71 circulation in mainland China, with a shift from evolutionary branch C4b to C4a in 2003–2004. The evolution rate of C4a HEV71 was 4.99×10-3 substitutions per site per year, faster than the mean of all HEV71 genotypes. The most recent common ancestor estimates for the Chinese clusters dated to October 1994 and November 1993 for the C4a and C4b evolutionary branches, respectively. Compared with all C4a HEV71 strains, a nucleotide substitution in all C4b HEV71 genome (A to C reversion at nt2503 in the VP1 coding region, which caused amino acid substitution of VP1–10: Gln to His) had reverted.ConclusionsThe data suggest that C4a HEV71 strains introduced into the Central Plain of China are responsible for the recent outbreaks. The relationships among HEV71 isolates determined from the combined sequence and epidemiological data reveal the underlying seasonal dynamics of HEV71 circulation. At least 5 HEV71 lineages circulated in the Central Plain of China from 2007 to 2009, and the Shandong and Anhui lineages were found to have passed through a genetic bottleneck during the low-transmission winter season.

Human enterovirus 71 that firstly isolated in Qinghai province and their genetic features of VP1 region

To study the genetic characterizations of VP1 gene of human enterovirus 71 (HEV71) isolated from clinical specimens of Hand, Foot and Mouth Disease (HFMD) patients in Qinghai Province in 2008. 335 clinical samples including stools, throat swabs and vesicle fluids were collected from HFMD patients in Qinghai Province. Viral isolation was performed, and molecular typing was performed with the positive isolates. Then 30 identified HEV71 isolates were performed for entire VP1 coding region amplification and sequencing. Among the 355 clinical samples, 45 human enteroviruses were isolated, and among them, 30 were identified as HEV71. Then 30 HEV71 positive isolates were performed by nucleotide sequencing. It showed that there was some difference in the nucleotide and the amino acid among the 30 HEV71 strains, the homology were 95.2%-100% and 96.6%-100%, respectively. But they all closed to HEV71 strains isolated in China after 1998, and from the phylogenetic tree constructed with 30 Qinghai HEV71 strains and other 35 HEV71 strains represented all known genotype and subgenotype HEV71 strains available from GenBank, it revealed that the 30 Qinghai HEV71 strains clustered within the C4a evolution branch of C4 subgenotype. HEV71 was isolated in HFMD patients in Qinghai province, and the HEV71 strains causing HFMD outbreaks in Qinghai province in 2008 were all belong to C4a evolution branch of C4 subgenotype with several transmission chains.

RT-PCR identification and genetic characterizations of VP1 region of human enterovirus 71 isolated in Beijing in 2008.

To study the genetic characterizations of VP1 region of Human enterovirus 71 (HEV71) isolated from clinical specimens of hand, foot and mouth disease (HFMD) patients in Beijing in 2008. 285 clinical samples were collected from HFMD patients in hospitals and day-care centers in Chaoyang district. They were performed by reverse transcription-polymerase chain reaction (RT-PCR) specific for HEV71. 10 HEV71 isolates were selected for entire VP1 coding gene amplification and sequencing. 129 samples were RT-PCR positive, the positive rate is 45.26%. The homology of the nucleotide and the amino acid of the 10 strains were 94.6%-99.6% and 95.9%-100%. The phylogenetic tree revealed that 10 Beijing strains clustered within the C4a evolution branch of C4 subgenotype. RT-PCR played an important role in identifying HFMD outbreak in Beijing in 2008. The HEV71 strains were all belong to C4a evolution branch of C4 subgenotype with several transmission chains, and it showed that C4 subgenotype HEV71 spread in mainland China widely after 1998. The molecular epidemiology surveillance and the research of genetic characterizations of HEV71 should be strengthened in mainland China.

Development of a multiplex polymerase chain reaction assay for simultaneous identification of human enterovirus 71 and coxsackievirus A16

Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are two major aetiological agents of hand, foot and mouth disease (HFMD) in children. Recently there have been several large outbreaks of HFMD in Vietnam and the Asia-Pacific region. In this study, a multiplex RT-PCR assay was developed in order to detect simultaneously HEV71, CVA16 and other human enteroviruses. Enterovirus detection was performed with a mixture of three pairs of oligonucleotide primers: one pair of published primers for amplifying all known enterovirus genomes and two new primer pairs specific for detection of the VP1 genes of HEV71 and CVA16. Enterovirus isolates, CVA16 and HEV71 strains identified previously from patients with HFMD were examined to evaluate the sensitivity and specificity of the multiplex RT-PCR assay. The assay was then applied to the direct detection of these viruses in clinical specimens obtained from HFMD cases identified at Children's Hospital Number 2, Ho Chi Minh City, Vietnam. The multiplex RT-PCR assay showed 100% specificity in screening for enteroviruses and in identifying HEV71 and CVA16. Similar results were obtained when using the multiplex RT-PCR assay to screen for enteroviruses and to identify HEV71 and CVA16 in clinical specimens obtained from HFMD cases identified at the hospital. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of HEV71 or CVA16 infection in cases of HFMD and is also potentially useful for molecular epidemiological investigations.

Evolutionary trajectory of the VP1 gene of human enterovirus 71 genogroup B and C viruses

From 1963 to 1986, human enterovirus 71 (HEV71) infections in the Netherlands were successively caused by viruses of subgenogroups B0, B1 and B2. A genogroup shift occurred in 1987, after which viruses of subgenogroups C1 and C2 were detected exclusively. This is in line with HEV71 typing in Australia, Europe and the USA, but is distinct from that in the Asian Pacific region, where HEV71 subgenogroups B3-B5 and C4-C5 have caused large outbreaks since 1997. To understand these observations in HEV71 epidemiology, the VP1-encoding regions of 199 HEV71 strains isolated in the Netherlands between 1963 and 2008 were used to study the detailed evolutionary trajectory and population dynamics of HEV71. Genogroup B viruses showed an epochal evolution, whereas genogroup C viruses evolved independently, which is in line with the co-circulation of C1 and C2 viruses in the Netherlands since 1997. Considering that strains from the Netherlands are interspersed phylogenetically with GenBank reference strains, the evolution of B1-B2, C1-C2 viruses has a global nature. Phylodynamic analysis confirmed that increased reporting of HEV71 infections in 1986 and 2007 reflected true epidemics of B2 and C2 viruses, respectively. Sequence analysis of the complete capsid region of a subset of isolates revealed several (sub)genogroup-specific residues. Subgenogroup B2-specific rabbit antiserum showed cross-neutralization of B0, B1 and B2 viruses, but not of subgenogroup C1 or C2 viruses, probably explaining the global shift to genogroup C in 1987 following a B2 epidemic. Anti-C1 rabbit serum neutralized both genogroup B and C viruses. Global herd immunity against C1 and C2 viruses possibly explains why epidemics with subgenogroups B4 and C4 are restricted to the Asian Pacific region.

Respiratory echovirus 30 and coxsackievirus B5 can induce production of RANTES, MCP-1 and IL-8 by human bronchial epithelial cells

Human Enteroviruses (HEV) ( picornaviridae) are considered as one the major viral causes of childhood acute respiratory wheezing illnesses including bronchiolitis and asthma exacerbation. To identify the mechanisms that can regulate the development of airway mucosa inflammation during HEV respiratory lower tract infection, we investigated the profile and the levels of mRNA and protein secretion for CC and CXC human chemokines by HEV-infected primary human bronchial epithelial cells (SAE cells) using RT-PCR array and Bio-Plex assay. Cultures of SAE cells were infected by reference and wild-type HEV respiratory strains, demonstrating a replicative and productive viral infection. We observed that the replicative infection of the SAE cells by reference and wild-type HEV strains induced specific dose and time-dependent increases in mRNA and protein secretion only for RANTES, MCP-1 and IL-8 and not for all other CC and CXC human chemokines tested. The protein secretion of these chemokines appeared to be significantly increased at 48 or 72 h post-infection in cultures treated by low-doses of IFN-γ comparatively to mock-infected cells ( P < 0.001), and was correlated to the viral replication activity. In conclusion, our findings demonstrated a selective production of RANTES, IL-8 and MCP-1 released by HEV-infected epithelial cells of the small bronchioles along with mechanisms of amplification mediated by IFN-γ.

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.