Coxsackievirus A6 and Hand, Foot, and Mouth Disease, Finland

Article (PDF Available)inEmerging Infectious Diseases 15(9):1485-8 · September 2009with94 Reads
DOI: 10.3201/eid1509.090438 · Source: PubMed
During fall 2008, an outbreak of hand, foot, and mouth disease (HFMD) with onychomadesis (nail shedding) as a common feature occurred in Finland. We identified an unusual enterovirus type, coxsackievirus A6 (CVA6), as the causative agent. CVA6 infections may be emerging as a new and major cause of epidemic HFMD.


Coxsackievirus A6
and Hand, Foot,
and Mouth
Disease, Finland
Riikka Österback, Tytti Vuorinen, Mervi Linna,
Petri Susi, Timo Hyypiä, and Matti Waris
usual enterovirus type, coxsackievirusA6 (CVA6), as the
causative agent. CVA6 infections may be emerging as a
and, foot, and mouth disease (HFMD) is a common
childhood illness characterized by fever and vesicular
eruptions on hands and feet and in the mouth (Figure 1). It
is caused by members of the family Picornaviridae in the
genus Enterovirus. Complications are rare, but pneumonia,
meningitis, or encephalitis may occur. Outbreaks of HFMD
have been mainly caused by 2 types of enterovirus A spe-
cies, coxsackievirus (CV) A16 (CVA16) or enterovirus 71
(1). Some outbreaks have been associated with CVA10, but
only sporadic cases involving other members of the entero-
virus A species have been reported (2,3).
During fall 2008, a nationwide outbreak of HFMD oc-
curred in daycare centers and schools in Finland, starting in
August and continuing at least until the end of the year and
possibly into the following year. From vesicle uid speci-
mens of hospitalized children, we identied the etiologic
agent as coxsackievirus A6.
The Study
In August 2008, vesicle uid specimens were collected
from 2 children and 1 parent with HFMD at the Central
Hospital of Seinäjoki, Southern Ostrobothnia. Specimens
were sent to the Department of Virology, University of
Turku, for identication of the causative agent. After detec-
tion of CVA6 in these index cases, the virus was also found
in specimens obtained from the Pirkanmaa Hospital Dis-
trict (Tampere), Turku University Hospital (Turku), Pori
Central Hospital (Pori), and Central-Ostrobothnia Central
Hospital (Kokkola) (Table).
Nucleic acids were extracted from specimens by using
the NucliSens EasyMag automated extractor (bioMèrieux,
Boxtel, the Netherlands). When the extracts were analyzed
for enteroviruses by using real-time reverse transcriptase–
PCR (RT-PCR) specic for the 5′ noncoding region (NCR)
of picornaviruses (4), amplicons with melting points indis-
tinguishable from each other and typical to enteroviruses
were obtained.
To identify the enterovirus type in the specimens, RT-
PCR, specic for a partial sequence of the viral protein
1 (VP1) region, was performed by using the COnsensus-
DEgenerate Hybrid Oligonucleotide (CODEHOP) Prim-
ers (
(5). The amplicons were separated by agarose gel electro-
phoresis, puried with the QIAquick PCR Purication Kit
(QIAGEN, Hilden, Germany), and sequenced in the DNA
Sequencing Service Laboratory of the Turku Centre for
Biotechnology. The virus type in the 3 index specimens, 3
samples of vesicular uids, and 1 throat swab was success-
fully identied with sequencing and BLAST (www.ncbi. analysis as CVA6. Phylogenetic rela-
tionships of the sequences were examined by using CVA6
(Gdula strain), CVA16 (G10), and enterovirus 71 (BrCr)
prototype strains as well as selected clinical CVA6 isolates
obtained from GenBank. Sequence alignments were gen-
erated with the ClustalW program (
alw), and the phylogenetic tree was computed by using the
Jukes-Cantor algorithm and the neighbor-joining method.
Phylogenetic analyses were conducted by using MEGA4
software ( and the bootstrap con-
sensus tree inferred from 1,000 replicates (6) (Figure 2).
EmergingInfectiousDiseases••Vol.15,No.9,September2009 1485
Author afliations: University ofTurku,Turku, Finland (R. Öster-
a6.5-year-old boy from Turku,Finland, withcoxsackievirus (CV)
A6 infection. Several of his ngernails shed 2 months after the
Seinäjoki,Finland,2 months after hand,foot and mouthdisease
Phylogenetic analysis placed all CVA6 strains from
the HFMD outbreak in 1 cluster (97%–100% identity),
whereas the nucleotide identities between those isolates
and CVA6 prototype strains Gdula, CAV16, G-10, and en-
terovirus 71 BrCr were 82.5%–83.2%, 55.6%–56.6%, and
55.6%–57.3%, respectively. The closest preceding CVA6
strain was isolated from cerebrospinal uid in the United
Kingdom in 2007 and had 92%–94% nucleotide identity
with the strains described here (7).
To improve the detection of the novel CVA6 strains
in clinical specimens, we designed specic VP1 primers
from the aligned sequences. CVA6vp1 reverse primer (5′-
ward primer (5′-CGTCAAAGCGCATGTATGTT-3′) gen-
erated a 199-bp amplicon. First, cDNA was synthesized in
a 20-μL reaction mixture containing 1 μmol/L CVA6vp1
reverse primer, 2.5 mmol/L of each dNTP, 20 U Rever-
tAid H Minus M-MuLV reverse transcriptase (Fermentas,
St. Leon-Rot, Germany), reaction buffer (Fermentas), 4 U
RiboLock RNase inhibitor (Fermentas), and 5 μL RNA in-
cubated at 42ºC for 1 h. Then, 5 μL of cDNA was added
to 20 μL of master mixture containing 0.4 μmol/L each of
the CVA6vp1 primers and Maxima SYBR Green qPCR
Master Mix (Fermentas). PCR with melting curve analy-
sis was performed in a Rotor-Gene 6000 real-time instru-
ment (Corbett Research, Mortlake, Victoria, Australia) by
1486 EmergingInfectiousDiseases••Vol.15,No.9,September2009
Table. Laboratory findings in clinical specimens and epidemiologic data for patients with CVA6 infections, Finland, 2008*
City or place,
date Sex/age, y Specimen type Disease or signs
RT-PCR VP1 sequence
5ƍ NCR
Fin/Se8717 2008 Aug M/1.3 Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Se8781 2008 Aug F/34 Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Se8841 2008 Aug M/0.9 Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Se8865 2008 Aug Feces HFMD Pos NR CVA6
Fin/Se8913 2008 Sep M/10 Throat swab HFMD Pos NR CVA6
Fin/Se8925 2008 Sep M/0.9 Throat swab HFMD Pos NR CVA6
Fin/Se8926 2008 Sep Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Se8927 2008 Sep F/1.3 Throat swab HFMD Pos NR CVA6
Fin/Se8928 2008 Sep Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Se8931 2008 Sep M/0.8 Throat swab NA Pos NR CVA6
Fin/Tu8859 2008 Sep M/5.8 Throat swab Fever, tonsillitis Pos CVA6 CVA6
Fin/Tu8866 2008 Sep M/3.1 Throat swab HFMD Pos NR CVA6
Fin/Tu81027 2008 Oct M/1.8 Throat swab HFMD Pos NR CVA6
Fin/Tu81042 2008 Oct M/1.8 Throat swab Fever, eczema Pos NR CVA6
Fin/Tu81038 2008 Oct F/2.2 Throat swab Fever, seizure Pos NR CVA6
Fin/Tu81274 2008 Nov M/6.5 Vesicle fluid HFMD Pos ND CVA6
Fin/Tu81309 2008 Dec F/3.7 Vesicle fluid HFMD Pos ND CVA6
Fin/Tu81321 2008 Dec F/1.2 Throat swab HFMD Pos ND NR
Fin/Tu963 2009 Jan M/1.2 Throat swab HFMD Pos ND CVA6
Fin/Tu/IB 2009 Feb F/5.7 Nail Recent HFMD Pos ND CVA6
Fin/Po8959 2008 Oct M/4.8 Throat Fever, vomiting Neg ND CVA6
Fin/Po81375 2008 Dec M/0.5 Vesicle fluid HFMD Pos ND CVA6
Fin/Po81376 2008 Dec Throat HFMD Pos ND CVA6
Fin/Po81324 2008 Dec M/10 Feces Fever, eczema Pos ND NR
Fin/Po81325 2008 Dec M/10 Feces Fever, HFMD
Fin/Ta8966 2008 Sep M/3.1 Tracheal aspirate NA Pos NR CVA6
Fin/Ta81145 2008 Sep M/2.1 Feces NA Pos NR NR
Fin/Ta81074 2008 Oct M/0.3 Tracheal aspirate NA Pos ND CVA6
Fin/Ta81125 2008 Oct F/0.7 Vesicle fluid HFMD Pos CVA6 CVA6
Fin/Ta81126 2008 Oct M/0.3 Vesicle fluid HFMD Pos NR CVA6
Fin/Ta81216 2008 Nov F/2.0 Throat swab NA Pos ND CVA6
Fin/Ta81252 2008 Nov M/6.1 Throat swab NA Pos ND CVA6
Fin/Ko937 2009 Jan M/10 Vesicle fluid HFMD Pos ND CVA6
*CVA6, coxsackievirus A6; VP1, virus protein 1; RT-PCR, reverse transcriptase–PCR; NCR, noncoding region; HFMD, hand, foot, and mouth disease;
pos, positive; neg, negative; NA, not available; ND, not done; NR, no result (VP1 sequencing was attempted without result).
†Same patient as on the line above.
using the following cycling conditions: initial denaturation
at 95ºC for 10 min, 45 cycles at 95ºC for 15 s, 60ºC for 30
s, and at 72ºC for 45 s, followed by generation of melting
curve from 72°C to 95°C with temperature increments of
0.5°C/s. Partial 5′ NCR sequence of the strains in clinical
specimens was determined as described (4) and compared
with the known sequences by using BLAST (http://blast.
During autumn 2008, a total of 47 acute-phase speci-
mens, including 12 vesicle uid samples, 23 throat swabs,
2 tracheal aspirates, 5 fecal samples, and 5 cerebrospinal
uid specimens from 43 patients yielded amplicons with
similar melting points as the originally identied CVA6
strains in 5′ NCR RT-PCR. All specimens were subjected to
the specic CVA6-VP1 real-time RT-PCR, and a positive
result was obtained for 11 vesicle uid samples, 14 throat
swabs, 2 tracheal aspirates, and 4 fecal samples (Table).
The virus in 1 throat swab was identied as CVA6 from
the result of 5′ NCR sequencing alone. None of the CVA6-
positive specimens were positive by an RT-PCR assay with
CVA16- and EV71-specic primers (8). Attempts to culti-
vate the virus from 8 CVA6 RT-PCR-positive specimens
were unsuccessful, whereas the prototype strain could be
propagated in rhabdomyosarcoma cells.
Onychomadesis was 1 characteristic feature in patients
during this HFMD outbreak; parents and clinicians reported
that their children shed ngernails and/or toenails within
1–2 months after HFMD (Figure 1). Only a few published
reports of nail matrix arrest in children with a clinical his-
tory of HFMD exist in the medical literature (9–11). We
obtained shed nails from 2 siblings who had HFMD 8 weeks
before the nail shedding. The nail fragments were stored at
–70°C for a few weeks and treated with proteinase K be-
fore nucleic acid extraction. The extracts were enterovirus
positive in 5′ NCR RT-PCR. The virus in one of them was
identied as CVA6 by the specic RT-PCR and yielded a 5′
NCR sequence that was similar to the novel CVA6 strains.
Enterovirus CVA6 was a primary pathogen associ-
ated with HFMD during a nationwide outbreak in Finland
in autumn of 2008. HFMD epidemics have primarily been
associated with CVA16 or enterovirus 71 infections; those
caused by enterovirus 71 have occurred more frequently
in Southeast Asia and Australia in recent years (12). Re-
portedly, CVA10 has been found in minor outbreaks; other
coxsackievirus A types have been found in only sporadic
cases of HFMD (2,3). In general, CVA6 infections have
been seldom detected and mostly in association with her-
pangina (13,14). In Finland, CVA6 has been identied only
on 4 occasions over 8 years during enterovirus surveillance
from 2000 to 2007 (15).
Although the CODEHOP primers were elementary
for rapid genotyping of the novel CVA6 strains, we identi-
ed more viruses with the designated CVA6-VP1 specic
primers. Onychomadesis was a hallmark of this HFMD
outbreak. To our surprise, we detected CVA6 also in a frag-
ment of shed nail. The same virus could have given rise to
the outbreak in Spain in 2008 (10). Supposedly, virus rep-
lication damages nail matrix and results in temporary nail
dystrophy. Whether nail matrix arrest is specic to CVA6
infections remains to be shown. This study demonstrates
that CVA6, in addition to CVA16 and enterovirus 71, may
be emerging as a primary cause of HFMD.
We thank Heidi Berghäll for nail samples, Harry Kujari for
photographs, and Tiina Ylinen for assistance in the laboratory.
Ms Österback is a doctoral candidate at the University of
Turku in Finland. Her research interests are laboratory diagnostics
and molecular epidemiology of viruses.
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Page 1 of 1
CVA6 AB114097
CVA6 AB114095
CVA6 AB114096
CVA6 AB114101
CVA6 AB114099
CVA6 AB140094
CVA6 AB114100
CVA6 AB114116
CVA6 AB114111
CVA6 AB114113
CVA6 AB114115
CVA6 AB114112
CVA6 AB162726
CVA6 AB114114
CVA6 FJ525951
Fin/Ta81125 FJ870502
Fin/Se8928 FJ870503
Fin/Se8781 FJ870504
Fin/Se8717 FJ870505
Fin/Se8841 FJ870506
Fin/Se8926 FJ870507
Fin/Tu8859 FJ870508
CVA6 Gdula AY421764
CVA6 AY919579
CVA6 AY919527
CAV16 G-10 U05876
EV71 BrCr U22521
Finland 2008
UK 2007
Japan 1999–2003
Bangladesh 1999–2002
Figure2. Phylogenetic analysisofcoxsackievirus(CV)A6partial
(289 bp) viral protein 1 sequences showing the relationships
between the recent clinical CVA6 samples isolated in Finland
of CVA6, CVA16, and enterovirus (EV) 71. GenBank accession
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Address for correspondence: Matti Waris, Department of Virology,
University of Turku, Kiinamyllynkatu 13 20520 Turku, Finland; email:
1488 EmergingInfectiousDiseases••Vol.15,No.9,September2009
    • "Except in a few countries, CA6 has been infrequently detected until recent years. However, since 2008, this virus has caused major outbreaks of HFMD in some countries of eastern Asia and Europe and, more recently, in the United States [29][30][31][32][33][34][35]. Sporadic cases of CA6 infection had been reported in areas such as Guangdong, Shandong in China. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by enteroviruses. A passive surveillance system has been implemented in Shanghai Pudong since 2008 and etiology surveillance since 2009.We characterized the epidemiology and the etiology of HFMD in Pudong from 2008-2013. Methodology: Health care providers were required to report any clinically diagnosed HFMD to Pudong District Center for Disease Control and Prevention. For all severe cases and randomly selected mild HFMD cases, throat or rectal swabs or feces were collected for enterovirus detection by real time reverse transcription polymerase chain reaction. Results: A total of 50,149 cases were reported, with average 8,508 per year (range: 3,577-13,202) and average incidence of 167.5/100,000 persons (range: 81.4-254.1/100,000 persons). HFMD was more likely to occur in children under five years of age (85.6%), while severe cases were more likely to happen in children under three years of age (63.9%). Every year in May or June, HFMD peaked in the region; two peaks were observed from 2011 to 2013.The most common etiologic agents are EV71 and CA16.Different types of enterovirus circulate in different years. EV71 was the predominant pathogen in severe cases. The proportions of EV71 in severe cases was higher than in mild cases at the children's medical center (p<0.001). Conclusions: HFMD remains an important public health issue in Shanghai. HFMD pathogen surveillance is required for more types of enteroviruses besides EV71 and CA16, which would give a better picture of the etiology of HFMD.
    Article · Jun 2016
    • "The main causative agents are CVA16 and EV71 [17]. Moreover, outbreaks caused by CVA4, CVA5, CVA6, CVA9, CVA10, CVA12, CVB1, CVB3 and CVB5 have been observed [3,9101131323334. Links have also been found between EV-4, HFMD and meningitis [22]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background To report an uncommon case of hand, foot and mouth disease, (HFMD) in an immunocompetent adult; a highly infectious disease, characterized by the appearance of vesicles on the mouth, hands and feet, associated with coxsackieviruses and enteroviruses; including a literature review. Case report A 23 year Caucasian male with no medical or surgical history, no allergies, was not taking any medication and smoked ten cigarettes a day, suffering from discomfort in the oral cavity; itching, burning and pain when swallowing associated with small erythematous lesions located on the hard palate, and small ulcers in tonsillar pillars and right buccal mucosa. Mild fever of 37.8 °C and general malaise. The patient reported he had had contact with a child diagnosed with HFMD. From his background and symptoms, the patient was diagnosed with HFMD. Following symptomatic treatment, the symptoms remitted in 7 days. Methods A literature review in MEDLINE (PubMed). The inclusion criteria were for studies on humans over the last 5 years, using the keywords HFMD. Results We found 925 articles, which were subsequently reduced to 52 documents after applying the inclusion criteria. Maculopapular lesions were found on hands and feet. Conclusions Dentists may have a key role diagnosing the disease. A surveillance system to predict future outbreaks, encourage early diagnosis, put appropriate public health measures in place and research vaccine development is vitally important in order to control the disease.
    Full-text · Article · Mar 2016
    • "In some instances, neurological manifestations are observed including aseptic meningitis, brainstem encephalitis, pulmonary edema, and polio-like paralysis [1]. HFMD is primarily caused by Enterovirus 71 (EV71; Picornaviridae; Enterovirus) and Coxsackievirus A16 (CVA16; Picornaviridae; Enterovirus), but occasionally other non-polio enteroviruses, including Coxsackievirus A6 (CVA6; Picornaviridae; Enterovirus), are involved [2]. The disease is now endemic in most Asian-Pacific countries, but serious outbreaks still occur. "
    [Show abstract] [Hide abstract] ABSTRACT: Hand, foot, and mouth disease (HFMD) has recently emerged as a major public health concern across the Asian-Pacific region. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the primary causative agents of HFMD, but other members of the Enterovirus A species, including Coxsackievirus A6 (CVA6), can cause disease. The lack of small animal models for these viruses have hampered the development of a licensed HFMD vaccine or antivirals. We have previously reported on the development of a mouse model for EV71 and demonstrated the protective efficacy of an inactivated EV71 vaccine candidate. Here, mouse-adapted strains of CVA16 and CVA6 were produced by sequential passage of the viruses through mice deficient in interferon (IFN) α/β (A129) and α/βand γ(AG129) receptors. Adapted viruses were capable of infecting 3 week-old A129 (CVA6) and 12 week-old AG129 (CVA16) mice. Accordingly, these models were used in active and passive immunization studies to test the efficacy of a trivalent vaccine candidate containing inactivated EV71, CVA16, and CVA6. Full protection from lethal challenge against EV71 and CVA16 was observed in trivalent vaccinated groups. In contrast, monovalent vaccinated groups with non-homologous challenges failed to cross protect. Protection from CVA6 challenge was accomplished through a passive transfer study involving serum raised against the trivalent vaccine. These animal models will be useful for future studies on HFMD related pathogenesis and the efficacy of vaccine candidates.
    Full-text · Article · Nov 2015
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