Hyypiä T

Linna M

Susi P

Vuorinen T

Waris M

Österback R

English

UTUPub

Coxsackievirus A6 and Hand, Foot, and Mouth Disease, FinlandRiikka Österback, Tytti Vuorinen, Mervi Linna, Petri Susi, Timo Hyypiä, and Matti WarisDuring	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	un-usual	 enterovirus	 type,	 coxsackievirus	A6	 (CVA6),	 as	 the	causative	 agent.	 CVA6	 infections	may	 be	 emerging	 as	 a	new	and	major	cause	of	epidemic	HFMD.	Hand, 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 fluid speci-mens of hospitalized children, we identified the etiologic agent as coxsackievirus A6.The StudyIn 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 were obtained.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-ers (bioinformatics.weizmann.ac.il/blocks/codehop.html) (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).	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	15,	No.	9,	September	2009	 1485 Author	 affiliations:	 University	 of	 Turku,	 Turku,	 Finland	 (R.	Öster-back,	T.	Vuorinen,	P.	Susi,	T.	Hyypiä,	M.	Waris);	and	Central	Hospi-tal	of	Seinäjoki,	Seinäjoki,	Finland	(M.	Linna)DOI:	10.3201/eid1509.090438Figure	1.	Vesicular	eruptions	in	A)	hand,	B)	foot,	and	C)	mouth	of	a	6.5-year-old	boy	 from	Turku,	Finland,	with	coxsackievirus	 (CV)	A6	 infection.	 Several	 of	 his	 fingernails	 shed	 2	 months	 after	 the	pictures	were	taken.	D)	Onychomadesis	in	a	10-year-old	boy	from	Seinäjoki,	Finland,	2	months	after	hand,	 foot	and	mouth	disease	with	CVA6	infection.	Photographs	courtesy	of	H.	Kujari	(A–C)	and	M.	Linna	(D).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 DISPATCHES1486	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	15,	No.	9,	September	2009Table. Laboratory findings in clinical specimens and epidemiologic data for patients with CVA6 infections, Finland, 2008*  City or place,  identification Sampling date Sex/age, y Specimen type  Disease or signs CVA6-VP1 RT-PCR VP1 sequence5   NCR sequence Seinäjoki         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 Turku         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 Pori         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 contact Pos ND NR Tampere         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 Kokkola         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. Coxsackievirus	A6,	Finlandusing 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.ncbi.nlm.nih.gov/Blast.cgi).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.ConclusionsEnterovirus 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. AcknowledgmentsWe 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.References  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	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	15,	No.	9,	September	2009	 1487 Page 1 of 1    CVA6 AB114097 CVA6 AB114095 CVA6 AB114096 CVA6 AB114101 CVA6 AB114099 CVA6 AB140094CVA6 AB114100 CVA6 AB114116CVA6 AB114111CVA6 AB114113 CVA6 AB114115 CVA6 AB114112 CVA6 AB162726 CVA6 AB114114 CVA6 FJ525951  Fin/Ta81125 FJ870502  Fin/Se8928 FJ870503 Fin/Se8781 FJ870504Fin/Se8717 FJ870505  Fin/Se8841 FJ870506 Fin/Se8926 FJ870507  Fin/Tu8859 FJ870508 CVA6 Gdula AY421764CVA6 AY919579CVA6 AY919527CAV16 G-10 U05876EV71 BrCr U225218864781006257754571584433348756967799 100 Finland 2008 UK 2007 Japan 1999–2003 Bangladesh 1999–2002 0.05 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	(triangles),	selected	CVA6	isolates	from	GenBank,	and	prototypes	of	 CVA6,	 CVA16,	 and	 enterovirus	 (EV)	 71.	 GenBank	 accession	numbers	are	included.	Scale	bar	indicates	nucleotide	substitutions	per position.  2.  Kamahora T, Itagaki A, Hattori N, Tsuchie H, Kurimura T. Oligo-nucleotide fingerprint analysis of coxsackievirus A10 isolated in Japan. J Gen Virol. 1985;66:2627–34. DOI: 10.1099/0022-1317-66-12-2627  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: 10.1086/525542  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/s00705-008-0266-8  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: 649–51.10.  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.x11.  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/ViewArticle.aspx?ArticleId=1891712.  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.75.16.7732-7738.200113.  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/JCM.00313-08Address for correspondence: Matti Waris, Department of Virology, University of Turku, Kiinamyllynkatu 13 20520 Turku, Finland; email: matti.waris@utu.fiDISPATCHES1488	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	15,	No.	9,	September	2009

Coxsackievirus A6 and hand, foot and mouth disease, Finland

https://www.utupub.fi/bitstream/handle/10024/171836/%c3%96sterback2009%20EID%2015%20-%20CAV6.pdf?sequence=1&isAllowed=y

Coxsackievirus A6 and hand, foot and mouth disease, Finland

Abstract

Similar works

Full text

Available Versions

UTUPub