Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004

doi:10.1016/j.jcv.2008.07.017

Journal of Clinical Virology

Volume 43, Issue 3, November 2008, Pages 323-327

https://doi.org/10.1016/j.jcv.2008.07.017 Get rights and content

Abstract

Background

Coxsackievirus A24 variant is one of the major etiological agents involved in acute haemorrhagic conjunctivitis.

Study design

An outbreak of acute hemorrhagic conjunctivitis occurred in the Southeast of Spain between September and November 2004. Cellular and molecular methods were used to identify and characterize the viral agent associated with the epidemic.

Results

Enterovirus was detected in the conjunctival swabs of 35 patients. None of the viruses isolated could be typed by conventional neutralization assays; however, amplification and sequencing of the 3′-end VP1 region of 19 of the samples identified coxsackievirus A24 variant as the serotype causing the outbreak. Phylogenetic analysis of the 5′-half VP1 region of the genome revealed that Spanish sequences, like other strains isolated during outbreaks of hemorrhagic conjunctivitis in American and African countries in 2003 and 2004, were closely related to the isolates detected in Korea (2002), thus proving their worldwide spread.

Conclusions

This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Spain. Molecular typing in combination with phylogenetic analysis is useful to study the enterovirus epidemiology associated with epidemics.

Introduction

Acute haemorrhagic conjunctivitis (AHC) is an epidemic form of highly contagious eye disease, caused mostly by two human enterovirus (HEV) serotypes, enterovirus 70 and coxsackievirus A24 variant (CV-A24v), and less frequently by adenoviruses.¹ CV-A24v is an antigenic variant of the CV-A24 strain, both classified as members of the HEV-C group,² and was first isolated from an AHC outbreak in Singapore in 1970.³ CV-A24v caused large outbreaks in India and Southeast Asia until the mid-1980s, and then spread rapidly to Japan, Taiwan and other regions, including the Americas.4, 5, 6, 7, 8 During the same period there were also outbreaks in African countries.⁹

Three genotypes, designated I–III, of the CV-A24v were described, and identified by phylogenetic analysis of the 3C protease region of the genome.⁸ In recent years, several published reports with molecular studies of outbreaks caused by CV-A24v occurred in Korea (2002), India (2003), French Guiana and West Indies (2003), and in Africa (2003 and 2004).10, 11, 12, 13, 14, 15 The phylogenetic analyses published revealed that all these strains were closely related and formed a separate cluster within genotype III.

During the autumn of 2004, an outbreak of AHC occurred in Spain, with several cases declared in five Southeastern cities. This report identifies CV-A24v as the causative agent of the epidemic and is the first in Spain to describe the virological and phylogenetic analysis of an AHC outbreak caused by this HEV serotype.

Section snippets

Clinical samples from AHC cases

Between September and November 2004, 79 conjunctival swabs from patients with clinical diagnosis of AHC were received for analysis in the National Centre for Microbiology. The samples were collected in the Hospital from five cities in the Southeast of Spain: Ceuta, Melilla, El Ejido, Cartagena and Valencia.

Virus detection and isolation

The samples were directly screened by molecular methods for enterovirus, herpesvirus and adenovirus. First, virus RNA was extracted from 100 μl clinical samples using an automated method

Virus detection in clinical samples

Of 79 conjunctival swabs received, 35 (44%) were positive for HEV by RT-PCR in the 5′-non-coding region (NCR). In one sample, adenovirus was detected, while in the rest, the causative agent of the AHC was unidentifiable. The rate between the number of HEV detected and the clinical samples analysed from each city is shown in Table 1.

Serotype identification

After inoculation into three cell lines, CPE was observed only in 11 of the 23 (48%) cell cultures inoculated (nine in HEF and two in Hep-2). The mean virus

Discussion

In this report, a CV-A24 variant could be identified as the causative agent of an outbreak of AHC in Spain during the autumn of 2004 by sequence analysis of the VP1 region of the genome. Although other studies of recent outbreaks10, 11, 13, 14, 15 have used the 3C genomic region for molecular analysis, the VP1 region is demonstrably more informative for molecular epidemiological studies than the 3C region for two reasons: due to the high recombination rates among enterovirus, which could

Acknowledgements

The work was supported by the DGVI 1429/05-2 grant from the Instituto de Salud Carlos III (Spanish Ministry of Health).

We thank Teresa de Miguel, Hortensia Pozo, and Isidoro Bustillo for their technical help and Ingrid M. Outschoorn with the English. Also we thank Dr. López-Barba from Hospital Ingesa (Ceuta), Dr. Galán from Hospital Comarcal de Melilla, Dr. Cabezas from Hospital de El Ejido (Almería), Dr. Ginés from Dirección de Salud de Cartagena (Murcia) and Dr. Gimeno from Hospital Clínico

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Cumulative data indicate that HeLa and Hep 2 have been the cells lines most widely used for the CA24-isolation.5,15,18–20 Previous studies conducted by, Lin et al.,15 Oh et al.,18 Tavares et al.,19 Cabrerizo et al.,6 Gopalkrishna et al.,2,18 showed CA24 isolation both in HeLa and Hep2 cell line, ranging from 53 to 1.1% which is in complete agreement with our results (36%). These data suggest that the frequency of CA24 isolation positivity is not entirely defined by the used cell line.
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La conjonctivite aiguë hémorragique (CAH) est une forme épidémique extrêmement contagieuse de conjonctivite caractérisée cliniquement par l’apparition brutale d’un épanchement sanguin au niveau des conjonctives. La première épidémie de CAH fut décrite en 1969 au Ghana et surnommée Apollo disease, car la rumeur populaire voulait que les atteintes oculaires soient dues au fait que les Ghanéens cherchaient à découvrir dans le ciel la capsule Apollo 11. Cette épidémie était due à l’Enterovirus 70 (EV70) qui constitue avec un variant antigénique du Coxsackievirus A24 (CVA24 v), la cause majeure des cas de CAH à travers le monde. Le mode de transmission de cette maladie fait appel aux sécrétions lacrymales des patients infectés par le biais d’une transmission interhumaine directe ou indirecte via du matériel ophtalmologique mal décontaminé ou de l’eau de baignade insuffisamment chlorée. L’hypothèse récente d’une propagation du virus par voie aérienne serait plus à même d’expliquer l’ampleur des épidémies décrites. En l’absence de traitement antiviral spécifique, l’apport d’un diagnostic étiologique rapide est essentiel pour distinguer la CAH d’autres infections oculaires curables ou afin de mettre immédiatement en place les mesures d’hygiène, seule arme susceptible de limiter la propagation de l’épidémie tant au niveau national que nosocomial. Pourtant, le diagnostic virologique reste actuellement long et laborieux, et souvent peu accessible aux pays en voie de développement, pour qui les épidémies de CAH dues aux Enterovirus (EV), sont devenues un problème de santé publique majeur et préoccupant.
Acute hemorrhagic conjunctivitis (AHC) is an epidemic form of highly contagious conjunctivitis, characterized by conjunctival hemorrhages. The first AHC outbreak was described in 1969 in Ghana, West Africa, and was called Apollo disease, from the Apollo landing on the moon. This outbreak was caused by Enterovirus 70 (EV70) together with a Coxsackievirus A24 (CVA24 v) variant, which are the major etiological agents involved in AHC outbreaks worldwide. AHC is known to be directly transmitted by close person-to-person contact or indirectly through soiled ophthalmological materials or unsafe recreational water. Recently, a possible airborne virus spread was suggested which could explain the high transmission rate of the disease. In the absence of a specific antiviral therapy, a rapid diagnosis of the causative agent is required to distinguish AHC due to enteroviruses from other ocular infectious diseases, for there are active drugs, or to quickly implement proper public health measures to limit the extension of the outbreak. However, virus identification remains difficult and time-consuming. Moreover, virological diagnosis is difficult to implement in developing countries where AHC has recently become a major problem for public health.
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Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004

Abstract

Background

Study design

Results

Conclusions

Introduction

Section snippets

Clinical samples from AHC cases

Virus detection and isolation

Virus detection in clinical samples

Serotype identification

Discussion

Acknowledgements

Clin Microbiol Infect

J Virol Methods

J Virol Methods

Eye infections

Picornaviridae

Enterovirus etiology of the 1970 Singapore epidemic of acute conjunctivitis

Intervirology

The first epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Okinawa, Japan, in 1985–1986

Jpn J Med Sci Biol

Acute non-haemorrhagic conjunctivitis due to coxsackievirus A24

Aust N Z J Ophthalmol

An epidemic of acute hemorrhagic conjunctivitis in American Samoa caused by coxsackievirus A24 variant

Am J Epidemiol