and Hand, Foot,
Riikka Österback, Tytti Vuorinen, Mervi Linna,
Petri Susi, Timo Hyypiä, and Matti Waris
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 fluid speci-
mens of hospitalized children, we identified the etiologic
agent as coxsackievirus A6.
and, foot, and mouth disease (HFMD) is a common
childhood illness characterized by fever and vesicular
In August 2008, vesicle fluid 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 identification 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) specific for the 5′ noncoding region (NCR)
of picornaviruses (4), amplicons with melting points indis-
tinguishable from each other and typical to enteroviruses
To identify the enterovirus type in the specimens, RT-
PCR, specific for a partial sequence of the viral protein
1 (VP1) region, was performed by using the COnsensus-
DEgenerate Hybrid Oligonucleotide (CODEHOP) Prim-
(5). The amplicons were separated by agarose gel electro-
phoresis, purified with the QIAquick PCR Purification 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 fluids, and 1 throat swab was success-
fully identified with sequencing and BLAST (www.ncbi.
nlm.gov/BLAST) 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 (www.ebi.ac.uk/clust-
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 (www.megasoftware.net) and the bootstrap con-
sensus tree inferred from 1,000 replicates (6) (Figure 2).
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 fluid 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 specific VP1 primers
from the aligned sequences. CVA6vp1 reverse primer (5′-
ACTCGCTGTGTGATGAATCG-3′) and CVA6vp1 for-
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
Table. Laboratory findings in clinical specimens and epidemiologic data for patients with CVA6 infections, Finland, 2008*
City or place,
identification date Sex/age, y Specimen type
Fin/Se8717 2008 Aug M/1.3 Vesicle fluid
Fin/Se8781 2008 Aug F/34 Vesicle fluid
Fin/Se8841 2008 Aug M/0.9 Vesicle fluid
Fin/Se8865 2008 Aug † Feces
Fin/Se8913 2008 Sep M/10 Throat swab
Fin/Se8925 2008 Sep M/0.9 Throat swab
Fin/Se8926 2008 Sep † Vesicle fluid
Fin/Se8927 2008 Sep F/1.3 Throat swab
Fin/Se8928 2008 Sep † Vesicle fluid
Fin/Se8931 2008 Sep M/0.8 Throat swab
Fin/Tu8859 2008 Sep M/5.8 Throat swab
Fin/Tu8866 2008 Sep M/3.1 Throat swab
Fin/Tu81027 2008 Oct M/1.8 Throat swab
Fin/Tu81042 2008 Oct M/1.8 Throat swab
Fin/Tu81038 2008 Oct F/2.2 Throat swab
Fin/Tu81274 2008 Nov M/6.5 Vesicle fluid
Fin/Tu81309 2008 Dec F/3.7 Vesicle fluid
Fin/Tu81321 2008 Dec F/1.2 Throat swab
Fin/Tu963 2009 Jan M/1.2 Throat swab
Fin/Tu/IB 2009 Feb F/5.7 Nail
Fin/Po8959 2008 Oct M/4.8 Throat
Fin/Po81375 2008 Dec M/0.5 Vesicle fluid
Fin/Po81376 2008 Dec † Throat
Fin/Po81324 2008 Dec M/10 Feces
Fin/Po81325 2008 Dec M/10 Feces
Disease or signs
*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 fluid samples, 23 throat swabs,
2 tracheal aspirates, 5 fecal samples, and 5 cerebrospinal
fluid specimens from 43 patients yielded amplicons with
similar melting points as the originally identified CVA6
strains in 5′ NCR RT-PCR. All specimens were subjected to
the specific CVA6-VP1 real-time RT-PCR, and a positive
result was obtained for 11 vesicle fluid samples, 14 throat
swabs, 2 tracheal aspirates, and 4 fecal samples (Table).
The virus in 1 throat swab was identified 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-specific 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 fingernails 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
identified as CVA6 by the specific 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 identified 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-
fied more viruses with the designated CVA6-VP1 specific
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 specific 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.
1. McMinn PC. An overview of the evolution of enterovirus 71 and
its clinical and public health significance. FEMS Microbiol Rev.
2002;26:91–107. DOI: 10.1111/j.1574-6976.2002.tb00601.x
Page 1 of 1
CVA6 Gdula AY421764
CAV16 G-10 U05876
EV71 BrCr U22521
(289? bp)? viral? protein? 1? sequences? showing? the? relationships?
between? the? recent? clinical? CVA6? samples? isolated? in? Finland?
2. Kamahora T, Itagaki A, Hattori N, Tsuchie H, Kurimura T. Oligo- Download full-text
nucleotide fingerprint analysis of coxsackievirus A10 isolated in
Japan. J Gen Virol. 1985;66:2627–34. DOI: 10.1099/0022-1317-66-
3. Cabral LA, Almeida JD, de Oliveira ML, Meza AC. Hand, foot, and
mouth disease case report. Quintessence Int. 1998;29:194–6.
4. Peltola V, Waris M, Österback R, Susi P, Ruuskanen O, Hyypiä T.
Rhinovirus transmission within children: incidence of symptomatic
and asymptomatic infections. J Infect Dis. 2008;197:382–9. DOI:
5. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR
amplification of VP1 sequences for direct identification of all entero-
virus serotypes from original clinical specimens. J Clin Microbiol.
2006;44:2698–704. DOI: 10.1128/JCM.00542-06
6. Jukes TH, Cantor CR. Evolution of protein molecules. In: Munro
HN, editor. Mammalian protein metabolism. New York: Academic
Press; 1969. p. 21–132.
7. Leitch EC, Harvala H, Robertson I, Ubillos I, Templeton K, Sim-
monds P. Direct identification of human enterovirus serotypes in ce-
rebrospinal fluid by amplification and sequencing of the VP1 region.
J Clin Virol. 2009;44:119–24. DOI: 10.1016/j.jcv.2008.11.015
8. Xiao XL, He YQ, Yu YG, Yang H, Chen G, Li HF, et al. Simulta-
neous detection of human enterovirus 71 and coxsackievirus A16
in clinical specimens by multiplex real-time PCR with an internal
amplification control. Arch Virol. 2009;154:121–5. DOI: 10.1007/
9. Bernier V, Labrèze C, Bury F, Taïeb A. Nail matrix arrest in the
course of hand, foot and mouth diease. Eur J Pediatr. 2001;160:
10. Clementz GC, Mancini AJ. Nail matrix arrest following hand-
foot-mouth disease: a report of five children. Pediatr Dermatol.
2000;17:7–11. DOI: 10.1046/j.1525-1470.2000.01702.x
11. Salazar A, Febrer I, Guiral S, Gobernado M, Pujol C, Roig J. Ony-
chomadesis outbreak in Valencia, Spain, June 2008. Euro Surveill.
2008;13:pii:18917. Available from http://www.eurosurveillance.org/
12. McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa
MJ. Phylogenetic analysis of enterovirus 71 strains isolated during
linked epidemics in Malaysia, Singapore, and Western Australia. J
Virol. 2001;75:7732–8. DOI: 10.1128/JVI.75.16.7732-7738.2001
13. Grist NR, Bell EJ, Assaad F. Enteroviruses in human disease. Prog
Med Virol. 1978;24:114–57.
14. Yamashita T, Ito M, Taniguchi A, Sakae K. Prevalence of coxsacki-
evirus A5, A6, and A10 in patients with herpangina in Aichi Prefec-
ture, 2005. Jpn J Infect Dis. 2005;58:390–1.
15. Blomqvist S, Paananen A, Savolainen-Kopra C, Hovi T, Roivainen
M. Eight years of experience with molecular identification of human
enteroviruses. J Clin Microbiol. 2008;46:2410–3. DOI: 10.1128/
Address for correspondence: Matti Waris, Department of Virology,
University of Turku, Kiinamyllynkatu 13 20520 Turku, Finland; email: