Multiple outbreaks of acute hemorrhagic conjunctivitis due to a variant of coxsackievirus A24: Guangdong, China, 2007

Acute hemorrhagic conjunctivitis (AHC) is usually caused by enterovirus 70, coxsackievirus A24(CA24v) and adenoviruses. Several outbreaks of AHC caused by a CA24v have occurred since it was imported into China in 1971. Multiple outbreaks of AHC reappeared in 10 cities of Guangdong during June to November in 2007. The epidemic began in the June, and spread extensively, with a peak in the September. A total of 31,659 cases were reported to center for disease control and prevention of Guangdong, it was estimated that the number of actual AHC was >200 thousands. Forty conjunctival swab specimens were collected from the cases diagnosed clinically with AHC. (RT)-PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by 16 isolates. We demonstrated the most likely etiological agent for the multiple outbreaks was a variant of coxsackievirus A24 by molecular typing using a partial VP1 sequence. Sequence comparison and phylogenetic analyses of the VP1 and 3Cpro gene regions were performed by Neighbor-joining method, the strains from different outbreaks and different geographical areas within Guangdong had no sequence divergence in 2007. The representative isolates from mainland of China including Hangzhou, Ningbo, Beijing, Yunnan, Liaoning, and Henan were analyzed in this study. Phylogenetic analysis revealed theses isolates were located in different clusters, a close phylogenetic and chronological relationship with Singaporean, South Korean and Thailand isolates had been observed. This confirms CA24v circulated in China's mainland has not evolved independently, but co-evolved with the isolates of Southeast Asia.     

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.     

Phylogenetic analysis of a coxsackievirus A24 variant: the most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981.

Nucleotide substitutions in the viral-encoded proteinase 3C (3Cpro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970-1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3Cpro was estimated to be 3.7 x 10(-3)/nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 +/- 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985-1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.     

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.     

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 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.     

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.     

Molecular epidemiology of enterovirus 71 in Taiwan

Summary.  Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10⁻³vs. 1.4 × 1010⁻³) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance. Received June 13, 2000 Accepted July 29, 2000     

柯萨奇病毒A24变种的血清抗体检测

目的　观察急性出血性结膜炎（ＡＨＣ）流行后人群中柯萨奇病毒Ａ２４ 变种（ＣＡ２４ ｖ） 感染情况（或状态）。方法　采用中和试验测定人群中血清抗体：血清作１∶１０ 稀释,病毒用１００ＴＣＩＤ５０ 在微量培养板上进行。ＣＡ２４ ｖ 病毒与血清等量混合,３７℃结合１ 小时,接种细胞,观察细胞病变。结果　ＡＨＣ流行后城市居民ＣＡ２４ ｖ 血清中和抗体阳性率为４９－６７％ ,其中１９～２５ 岁年龄组约占６９－４９％ ,统计学上有显著性（ Ｐ＜ ０－０１） 。结论　ＡＨＣ流行后人群的抗体水平,反映人群受ＣＡ２４ ｖ 感染状态、抗体阳性率与年龄有关,可能反映不同年龄人群参加社会活动多少所致     

Comparisons of the complete genomes of two Chinese isolates of a recent foot-and-mouth disease type Asia 1 virus

Summary China reported the first outbreak of foot-and-mouth disease (FMD) serotype Asia 1 in Chinese Hong Kong in March, 2005. Subsequently, this type of the virus was reported from mainland of China in April 2005. Up to September of 2006, it was detected in more than 15 areas of China. In this paper, the complete genomes of two Chinese isolates, Asia 1/HNK/CHA/05 and Asia 1/JS/CHA/05, of foot-and-mouth disease virus (FMDV) were sequenced and compared with some Chinese sequences and reference sequences from other countries. The identities between Asia 1/HNK/CHA/05 and Asia 1/JS/CHA/05 of 5′-UTR, L gene, P1 (VP1) gene, P2 gene, P3 gene, 3′-UTR are 84.8, 87.6, 86.4 (82.3%), 92.5, 92.8 and 95.3%, respectively. The data revealed that these two strains do not belong to the same genotype depending on the analysis of VP1 sequences, and neither of them have deleted bases in 5′UTR and 3A genes compared with the reference sequences. In addition, the secondary structures of their 5′UTR and 3′UTR are discussed.     

Phylogenetically different strains of a variant of coxsackievirus A 24 were repeatedly introduced but discontinued circulating in Japan

Summary Variations in the nucleotide sequence of 3 C proteinase of coxsackie-virus A 24 variant (CA 24 v) were analyzed to define the route of transmission and spread of the virus which was introduced to Japan on three separate occasions, 1985–86, 1988, and 1989. The nucleotide sequences of isolates from the same year's outbreak in Japan were identical or closely related, while the isolates from different outbreaks were less closely related to one another than to those from other countries in the same year. All Japanese isolates from Okinawa and other prefectures in 1985 and 1986 were closely related to the Taiwan strains in those same years, indicating common-source outbreaks. Two 1988 isolates from Chiba Prefecture, Japan, were closely related to those from Singapore in 1987, China in 1988 and Hong Kong in 1988. All seven Japanese isolates from Chiba Prefecture in 1989 comprised a group together with the Taiwan and Singapore strains in 1988. The results indicate that CA 24v was introduced into Japan on each occasion from the outside. Furthermore, in contrast to the explosive epidemics in Okinawa Prefecture in 1985 and 1986, the virus which was repeatedly introduced to other areas in Japan did not circulate endemically, and disappeared within a short time.     

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1

Summary.  Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia 1 virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I–IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 × 10⁻² synonymous substitutions per nucleotide per year. Received May 7, 2001 Accepted August 1, 2001     

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.     

Genomic and antigenic characterization of viruses from the 1993 Italian foot-and-mouth disease outbreak

Summary. The origin and evolution of the type O foot-and-mouth disease viruses (FMDV) that caused the outbreak occurrence in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on the viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that these Italian viruses were grouped in the ME-SA topotype, closely related to viruses that circulated previously in the Middle East. The analysis of the nucleotide sequences in VP1, VP2 and VP3 showed a co-circulation during the epizootic of genetic variants, including viruses with amino acid replacements in VP3. For some of the isolates analyzed, values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.5 to 5.1 × 10⁻². The use of a panel of new monoclonal antibodies raised against an isolate from this outbreak, as well as monoclonal antibodies to FMDV O1-Switzerland 1965, showed differences in the reactivity pattern among some of the Italian isolates analyzed, which were consistent with the co-circulation of antigenic variants. These results support the potential for FMDV diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented.     

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector

Summary.  For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 × 10³ HA units/ml from infected Bm5 cells, 2.1× 10⁵ HA units/larvae from infected larval fat body, and 1.6× 10⁶ HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines. Received December 29, 1998/Accepted August 19, 1999     

Nucleotide sequence of the gene encoding the RNA polymerase and the 3′ non-coding region of a bovine enterovirus Japanese isolate: rapid synonymous substitutions between European and Japanese strains

Summary.  The nucleotide sequences of the genome RNA encoding the RNA polymerase and the 3′ non-coding region (NCR) of bovine enterovirus (BEV) serotype I Japanese isolate, MZ468, were determined. The genetic distance between the two BEV serotype I strains, MZ468 and VG-5-27, was calculated by pairwise comparison of nucleotide sequences. The synonymous substitution rate was high (1.40 × 10⁻²/site/year), and of the same order as those of influenza virus HA, HIV-1 gag and env, and enterovirus 70 VP1 genes. Accepted December 1, 1997 Received June 2, 1997     

Phylogenetic analyses of the matrix and non-structural genes of equine influenza viruses

Summary.  Matrix (M) and nonstructural (NS) genes of thirteen equine H3N8 and H7N7 influenza viruses were sequenced and analyzed from an evolutionary point of view. The M and NS genes of H3N8 viruses isolated between 1989 and 1993 evolved into two minor branch clusters, including isolates from Europe and the American continent, respectively. It was noteworthy to reveal that the nucleotide sequences of the M and NS genes of an earlier American strain showed highest homology to those of recent European viruses. “Frozen evolution” was observed in the M and NS genes of A/eq/LaPlata/1/88. It was also evident that the NS gene of an H7N7 virus from 1977 was very similar to that of a 1979-H3N 8 virus, while the M gene was closest phylogenetically to that of the earliest H7N7 virus isolated in 1956. Furthermore, the M2 protein of A/eq/Newmarket/1/77 virus contained a carboxyl terminal deletion of three amino acids. The evolutionary rates of the M and NS genes of H3N8 equine influenza viruses were estimated to be 5.4 × 10⁻⁴ and 5.1 × 10⁻⁴ substitutions per site per year, respectively, which were slower than those of human viruses. Received November 21, 1997 Accepted March 9, 1998     

An influenza A virus hemagglutinin (HA) epitope inserted in and expressed from several loci of the infectious bursal disease virus genome induces HA-specific antibodies

The N-terminus of infectious bursal disease virus (IBDV) VP5 has been shown to be capable of tolerating the insertion of small epitopes. The objective of the present study was to determine if IBDV genomic sites, including the 5’ end of vp5, could carry an influenza A virus hemagglutinin (HA) epitope. HA-expressing IBDVs were generated when the HA epitope was fused to the N-terminus of VP5 (HA5-IBDV) or VP4 (HA4-IBDV) or the C-terminus of VP1 (1HA-IBDV). Viral titers obtained after co-transfection with cDNA from the ha-containing segment and the complementary genomic segment were 1.3 × 10⁴, 3.7 × 10³ and 3.8 × 10⁴ pfu/ml for HA5-IBDV, HA4-IBDV and 1HA-IBDV, respectively. The HA tag expression remained stable after 10 passages when the tag gene was inserted into the vp4 and vp1 genes. HA-IBDVs did not cause pathogenicity in specific-pathogen-free (SPF) chickens. However, only HA4-IBDV and 1HA-IBDV induced HA-specific antibodies, which were measured by ELISA with a maximum optical density (OD) value of 0.701 and 0.769, respectively, at 24 days after infection. Thus, IBDV can potentially be employed as a bivalent viral vector when the epitope is fused with VP4 or VP1.