The complete nucleotide sequence of a variant of coxsackievirus A24, an agent causing acute hemorrhagic conjunctivitis

The complete nucleotide sequence was determined for the cDNAs that represent the RNA genome of the standard strain of a variant of coxsackievirus A24, the EH24/70, one of the agents causing acute hemorrhagic conjunctivitis. The genome is 7461 nucleotide long and is polyadenylated at the 3-end terminus. Following a 750-nucleotide 5-noncoding region, there was a long open reading frame of 6642 nucleotides, which serve to encode a viral polyprotein consisting of 2214 amino acids. Comparison of the deduced amino acid sequence of the polyprotein with those of known enteroviruses allowed us to predict the possible cleavage sites. The overall structure and the organization of the RNA genome is typical for an enterovirus. Based on the similarity of the nucleotide sequence of the 5 and 3 noncoding regions, together with the amino-acid sequence of the encoded proteins, EH24/70 appeared to be closely related to polioviruses and coxsackievirus A21.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession number D90457.     

Two outbreaks of acute hemorrhagic conjunctivitis in Africa due to genotype III coxsackievirus A24 variant

Reported here are two outbreaks of acute hemorrhagic conjunctivitis that occurred in the Democratic Republic of the Congo and in Morocco in the summers of 2003 and 2004, respectively, with a large impact on public health. Virus was isolated from the conjunctival swabs of 30 Congolese and 20 Moroccan patients. Enterovirus-specific cytopathic effect was observed in all samples. None of the strains could be typed using a conventional neutralization assay with the Melnick intersecting pools; however, by sequencing the VP1 region, the viruses could be identified as coxsackie A24 variants. Phylogenetic analysis of the 3C protease region revealed that these strains were closely related to each other as well as to genotype III isolates detected in Korea in 2002, thus proving their worldwide spread. This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Africa since 1987 and the first ever from Morocco. An erratum to this article can be found at     

Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR

Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.     

An outbreak of acute hemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant in Pakistan

Acute hemorrhagic conjunctivitis (AHC) is a self-limiting viral infection of the eyes but having epidemic potential. In winter 2004-2005, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Islamabad, Pakistan. The etiological agent was confirmed as coxsackievirus A24 variant (CA24v) by virus isolation and sequencing of a part of the VP1/VP3 gene. Phylogenetic analysis in VP1 region showed that Pakistan isolates has closest matches both in Asia and Europe while in VP1/VP3 region they were more closely related to Chinese strains, suggesting their common source in Asia which is constantly evolving to cause AHC outbreaks in susceptible hosts in different parts of the world.     

The nucleotide sequence of 3C proteinase region of the coxsackievirus A24 variant: Comparison of the isolates in Taiwan in 1985–1988

Acute hemorrhagic conjunctivitis caused by coxsackievirus A24 variant (CA24v) first appeared in Taiwan in October 1985, followed by two other sequential epidemics in 1986 and 1988. In order to know the evolutionary relationship of the CA24v strains isolated in Taiwan, we first determined the nucleotide sequence of the 3C proteinase (3C^pro) region of the prototype strain (EH24/70), isolated in Singapore in 1970, by molecular cloning. The nucleotide sequence of the 3C^pro region thus sequenced showed striking homology with polioviruses and coxsackievirus A21.Viral RNA of eight isolates obtained from the three epidemics was reverse transcribed, amplified by the polymerase chain reaction, and cloned into M13 phage for the production of ssDNA for nucleotide sequencing by the dideoxy chain termination method. When the number of nucleotide difference was taken as a genetic distance between isolates, all isolates showed a very similar distance from the EH24/70, the earliest isolate of CA24v, indicating that they evolved at a constant evolutionary rate. Phylogenetic analysis by the unweighted pairwise grouping method of arithmetic average (UPGMA) indicated that the six isolates collected in 1985 and 1986 were closely related, while two 1988 isolates were more distant from them. The branching time between these two groups was estimated to be May 1984, 18 months before the first recognition of the CA24v epidemic in Taiwan.This is the first report of the nucleotide sequence of CA24v genome RNA and of an evolutionary analysis of the virus using the nucleotide sequence.     

北京市2010-2013年急性出血性结膜炎的病原学分析

目的 确定北京市急性出血性结膜炎的病原学及流行病学特征.方法 2010-2013年7月至10月在北京地区所有区县监测点收集急性结膜炎患者眼拭子标本,通过RT-PCR分别鉴定柯萨奇病毒A24变种(CVA24v)和肠道病毒70(EV70).分析急性出血性结膜炎的时间分布、患者性别、年龄及临床症状特征,利用系统发育树分析CA24v的VP1区、3Cpro区基因特征.结果 随机抽取的1144件急性结膜炎样本中,4.5％(52/1144)为CVA24v阳性,未检测到EV70.CVA24v相关急性出血性结膜炎在9月达到高峰.各年龄组发病差异有统计学意义,10-19岁年龄组发病率最高.CA24v和非CA24v急性结膜炎的症状构成比无统计学差异.北京市2007-2012年分离的CA24v株VP1、3Cpro的核苷酸序列同源性分别为95.2％-100％和92.8％-100％.结论 2010-2013年CVA24v是引起北京市急性出血性结膜炎的最主要病原体.     

Isolation of Coxsackievirus A24 variant from patients with hemorrhagic conjunctivitis in Cuba, 2008-2009

Background

An outbreak of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009.

Objective

To determinate the etiological agent associated with the Cuban outbreaks of acute hemorrhagic conjunctivitis during 2008 and 2009.

Study design

Conjunctival swabs and/or faecal samples from 382 patients with clinical diagnosis suggestive of acute hemorrhagic conjunctivitis were subject to viral culture in HEp-2 human laryngeal epidermoid carcinoma cells. Positive samples were identified by a specific Coxsackievirus A24 variant PCR and the 3C protease region of 16 isolates was sequenced for phylogenetic analysis.

Results

Enterovirus cytopathic effect was observed in 138 cases (36%). A higher percent of CA24v was recovered from faecal samples, 19 out of 45 cases (42.2%), than from conjunctival swabs, 127 out of 355 samples (35.8%). All isolates were identified as Coxsackievirus A24 variant. Phylogenetic analysis revealed that 2008 and 2009 Cuban outbreaks were caused by the same virus strains and that isolates were closely related to those from Taiwan (2006-2007), China (2007-2008) and Singapore (2005) with a bootstrap value of 71%.

Conclusions

Outbreaks of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009 were caused by Coxsackievirus A24 variant. The faecal-oral route is another mode of transmission of CA24v in the acute hemorrhagic conjunctivitis outbreaks. Phylogenetic analysis of Cuban CA24v strains involved in an acute hemorrhagic conjunctivitis outbreak in 2008 and 2009 confirms a new introduction of the CA24 variant into the Americas from South-east Asia.     

Outbreak of acute hemorrhagic conjunctivitis in Maharashtra and Gujarat states of India, caused by Coxsackie virus A-24 variant

Acute hemorrhagic conjunctivitis is associated with enteroviruses. Among these, Coxsackie A-24 variant (CA-24) and Enterovirus-70 (EV-70) are known to cause epidemics and pandemics. An outbreak of acute hemorrhagic conjunctivitis occurred in August-September 2003 in Maharashtra and Gujarat states of India. The present investigation was carried out to determine the viral etiological agent associated with the epidemic. Virus isolates were obtained from 11 eye swabs of conjunctivitis patients using HeLa/ Hep-2 cell lines. The isolates were characterized by serological and mouse pathogenecity tests, RT-PCR using enterovirus common primers (VP4-VP2), CA-24 specific primers (3C-proteinase region), EV-70 primers (VP-3) followed by sequencing, and phylogenetic analysis. The virus was characterized as a Coxsackie A-24 variant (CA-24v) and none of the isolates were found to be positive for EV-70. Sequencing of the PCR products derived from all the 11 isolates revealed 98.4% (SE 0.20) nucleotide identity within the Indian strains and 98.6% (0.50) and 94.4% (0.30) nucleotide identity respectively with the West Indies and Asian strains reported worldwide. The findings suggest that the outbreak of acute hemorrhagic conjunctivitis that occurred in Maharashtra and Gujarat states of India during August-September 2003 was caused by the Coxsackie A-24 variant (CA-24v).     

1997年青岛市急性出血性结膜炎病原学研究

目的了解１９９７年青岛市流行的急性出血性结膜炎（简称ＡＨＣ）的病毒病原。方法在ＡＨＣ流行时,有选择地采集部分ＡＨＣ病人眼拭子标本,通过多种细胞进行病毒分离,并对分离出的病毒进行中和试验鉴定。结果经用ＣＡ２４ｖ、ＥＶ７０、ＣＡ２４和ＣＡ２４ｖ北京地方株病毒抗血清和Ａｄ３、Ａｄ７、Ａｄ１１病毒抗血清中和试验鉴定,分离的毒株为ＣＡ２４ｖ和腺病毒。结论１９９７年青岛市发生的ＡＨＣ多数是由腺病毒引起的,少数为柯萨奇病毒Ａ２４ｖ引起。     

Molecular identification of coxsackievirus A24 variant,isolated from an outbreak of acute hemorrhagic conjunctivitis in Singapore in 2005

Summary An outbreak of acute hemorrhagic conjunctivitis (AHC) was reported in Singapore military camps in the year 2005. A total of 103 conjunctival swab specimens were collected from military personnel diagnosed clinically with AHC. PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by virus isolation. Molecular typing using a partial VP1 gene confirmed a variant of coxsackievirus A24 (CA24v) as the most likely etiological agent for the outbreak. Full-length genome sequencing was carried out on 2 selected virus strains, DSO-26SIN05 and DSO-52SIN05. Sequence comparison and phylogenetic analyses of the VP4, VP1 and 3Cpro gene regions were performed, clustering the Singapore CA24v strains with viruses originating from Asia in the post-2000 era. In addition, we report evolution rates of 4.2 × 10⁻³ and 1.0 × 10⁻³ nucleotide/year, respectively, for the VP4 capsid and 3Cpro gene regions. Our result shows a focal evolutionary point around 1965–1966, suggesting that the CA24v virus has been evolving constantly since its emergence in Singapore, nearly 40 years ago.     

Evolutionary study on the coxsackievirus A 24 variant causing acute hemorrhagic conjunctivitis by oligonucleotide mapping analysis of RNA genome

Summary The evolution of the variant of coxsackievirus A 24 (CA 24 v) which causes acute hemorrhagic conjunctivitis was explored. Using 15 isolates obtained from Southeast Asia during the period 1970–1986, the genetic distance between isolates was estimated from pairwise comparison of nucleotide changes deduced from common spots on oligonucleotide maps of the isolates. From regression analysis of the genetic distance and the time of isolation of the isolates, the evolutionary rate of CA 24 v was estimated to be 3.44×10^–4/nucleotide/month. The phylogenetic relationship of these isolates was explored using the neighbor-joining method and the modified unweighted pair group method using arithmetic averages (UPGMA). The phylogenetic tree constructed indicates that CA 24 v appeared from one focal place in July 1968±25 months, very close to the time of the first world epidemic of, then newly recognized, acute hemorrhagic conjunctivitis.     

Molecular characterisation of a coxsackievirus A24 that caused an outbreak of acute haemorrhagic conjunctivitis, Tunisia 2003

This study reports the genetic characteristics of coxsackievirus A24 isolates from Tunisia, including a coxsackievirus A24 variant (CVA24v) that caused an outbreak of acute haemorrhagic conjunctivitis (AHC) between September and November 2003. The virus genome was detected by PCR from conjunctival swabs obtained from patients with AHC. Four virus isolates were obtained from PCR-positive samples and were serotyped by sequence analysis of the VP1 and VP4 genomic region and by seroneutralisation. Phylogenetic analysis of the VP1, VP4 and 3C genomic regions was performed. Other Tunisian CVA24 isolates from paralytic cases and healthy individuals were also amplified, sequenced and included in the phylogenetic analysis. The epidemic strain belonged to the CVA24 serotype. Phylogenetic analysis of the 3C region of the genome revealed a strong relationship between the Tunisian epidemic strain and strains that caused outbreaks in Korea (2002) and Guadeloupe and French Guiana (2003). Phylogenetic analysis of the VP1 and VP4 regions showed a clear distinction between serotype CVA24 isolates from conjunctivitis and non-conjunctivitis cases. This is the first study to report an outbreak of AHC caused by CVA24v in the North African region.     

Outbreak of acute hemorrhagic conjunctivitis in French Guiana and West Indies caused by coxsackievirus A24 variant: phylogenetic analysis reveals Asian import

An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.     

Molecular identification and phylogenetic study of coxsackievirus A24 variant isolated from an outbreak of acute hemorrhagic conjunctivitis in India in 2010

An outbreak of acute hemorrhagic conjunctivitis (AHC) occured in India between August and October 2010. Molecular typing by RT-PCR and sequencing of a partial VP1 region identified coxsackievirus A24 variant (CV A24v) as the serotype involved in this outbreak. Phylogenetic analysis based on the VP1 and 3C genes revealed that CV A24v strains associated with the 2010 AHC outbreak in India were genetically similar to strains from Central and South America that caused outbreaks of AHC in Cuba between 2008 and 2009 and Brazil in 2009. The result shows that the Indian strain of CV A24v may be responsible for the recent AHC outbreak in Marseille, France, in 2012.     

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

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.     

Acute haemorrhagic conjunctivitis: seroepidemiology of coxsackievirus A24 variant and enterovirus 70 in Singapore

Epidemics of acute haemorrhagic conjunctivitis caused by a variant of coxsackievirus A24 (CA24v) and enterovirus 70 (EV70) have occurred periodically in Singapore. A seroepidemiological survey conducted before the CA24v epidemic in 1985 and in the midst of the EV70 epidemic in 1983 showed a neutralising antibody prevalence (greater than or equal to 1:4) of 19.1% and 66.9% to CA24v and EV70, respectively. The seropositivity rate to both viruses was highest in children 10-14 years of age, but no sex or ethnic difference was noted. It appears from the data that an epidemic could be triggered when the herd immunity of the population falls below a critical level. There was a significant correlation in seroprevalence between CA24v and EV70 (P = 7.75 x 10(-3).