RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Molecular Identification and Phylogenetic Analyses of Coxsackievirus A24v Causing an Outbreak of Acute Hemorrhagic Conjunctivitis in Jiangxi, China, in 2010 / 病毒学报

To identify the cause of an outbreak of acute hemorrhagic conjunctivitis (AHC) in Jiangxi (China) in 2010, 20 eye conjunctival swabs were first collected from AHC patients. Then, viruses were isola- ted and tested for human enterovirus 70, coxsackievirus A24 variant (CV-A24v) and adenovirus using the polymerase chain reaction. All CV-A24v isolates underwent sequencing of 3C and VP1 coding regions. Then, a phylogenetic tree was constructed for Jiangxi CV-A24v and worldwide CV-A24v based on,3C and VP1 regions, respectively. Ten out of 20 specimens were positive for CV-A24v, implying that the outbreak was caused by CV-A24v. The phylogenetic tree based on the 3C region showed that Jiangxi CV- A24v belonged to cluster 5 in genotype IV (GIV-C5) with strains isolated throughout the world after 2010, and were divided further into A and B lineages. Phylogenetic analyses of the VP1 region showed that all of the worldwide CV-A24v strains isolated after 2000 could be divided into five groups (1-5). Jiangxi CV-A24v was classified into group 5 and also divided further into A and B lineages upon analyses of the 3C region. These data suggested that CV-A24v causing AHC outbreaks in China in 2010 belonged to GIV-C3 and GIV-C5. At least two transmission lineages were circulated in Jiangxi in 2010. The classification of CV-A24v isolated after 2010 worldwide using the phylogenetic tree based on the VP1 region was almost consistent with that based on the 3C region and also had significant chronological clustering.

Identification and genetic characterization of coxsackievirus A24 isolated from patients with acute hemorrhagic conjunctivitis in Shandong Province / 病毒学报

To identify the pathogen of acute hemorrhagic conjunctivitis (AHC) in Shandong Province in 2010, eye mucous swab samples were collected from 26 patients in Qingdao and Linyi City. Real time-PCR assays for EV70, CVA24 and Adenovirus were performed on these samples. The result showed 17 samples (65.39%) were CVA24 positive while all the samples for HEV70 and Adenovirus detection were negative, which implied that CVA24 was the causative pathogen of this outbreak. A total of 10 virus strains isolated on Hep-2 cells were identified as CVA24 through VP1 amplification and nucleotide sequence analysis. The nucleotide and amino acid homologies on VP1 region among these isolates were 99.3%-100.0% and 99.5%-100.0%, respectively, and the strains aggregated together to one clade in phylogenetic tree. These results showed that the CVA24 circulating in Qingdao and Linyi City belonged to one transmission chain. Shandong CVA24s segregated into 5 different clades, and great nucleotide divergence was observed be tween AHC isolates and others.

Prevalence of prolonged and chronic poliovirus excretion among persons with primary immune deficiency disorders in the Philippines

OBJECTIVES: As part of the global initiative to eradicate poliovirus infections this study aims to: (1) estimate the prevalence of vaccine-derived poliovirus excretion among persons diagnosed with primary immune (B-cell or combined B/T-cell) deficiency disorders (PIDD) in the Philippines; (2) describe clinical features of these PIDD patients excreting poliovirus; (3) genetically characterize vaccine-derived polioviruses isolated from persons with PIDDs; and (4) determine the duration of poliovirus excretion among subjects who tested positive for vaccine-derived poliovirus excretion. METHODS: Seventy-one (71) Filipino patients (ages 0-35 years of age) with PIDD were recruited retrospectively and prospectively over a period of 16 months. The study participants, after informed consent and administration of a questionnaire for baseline data, underwent further testing of quantitative immunoglobulin levels (IgG, IgA, and IgM) and stool poliovirus isolation using two stool samples. Stool specimens which tested positive for the poliovirus were sent to the Regional Reference Laboratory in Australia for further characterization by Intratypic Differentiation (ITD) and Vaccine-derived polioviruses (VDPV) real-time PCR. These participants were then monitored on a monthly basis until laboratory tests identified two sequential months of negative poliovirus stool specimens. RESULTS: Seventy-one (71) patients underwent interview and quantitative serum immunoglobulin testing. However, one patient expired prior to stool isolate collection. This study, then, documented that none of the remaining 70 Filipino individuals (0-35 years old) with confirmed or suspected PIDDs chronically excreted immunodeficiency-associated vaccine-derived poliovirus (IVDPV). One patient who was a recent OPV-recipient excreted poliovirus Sabin-like 1 transiently (less than 1 month) and two patients excreted non polio-enteroviruses. CONCLUSIONS: Chronic and prolonged poliovirus excretion appears to be uncommon among Filipino patients with diagnosed Primary Immunodeficiency Disease Disorders. However, as part of the continuing global initiative for poliovirus eradication, vigilance is still necessary in patients with primary immunodeficiency diseases. Adequate identification of these patients followed by monitoring their capacity for viral excretion and environmental contamination may be necessary to achieve this goal.

Genotype distribution of enterovirus group C in Shandong Province, China / 病毒学报

Human Enterovirus C group (HEV-C) includes 17 serotypes, which can not be serotype-identified by neutralization test using antiserum pool for NPEV. In order to elucidate the genotypes and molecular evolution of HEV-C in Shandong Province, We selected the strains isolated from AFP cases between 1994-2009 to perform reverse transcriptase-polymerase chain reactions (RT-PCR) by the primers specific for entire VP1 coding gene of HEV-C and sequencing. The phylogenetic tree was then constructed among these VP1 nucleotide sequences and other prototype strains. Totally 12 Shandong local strains were obtained and separated into 4 genotypes, CVA20, CVA21,CVA24 and EV 96. The homologous comparison and phylogenetic analysis showed Shandong strains were distinct from prototype strains in each genotype. This report showed that different genotype HEV-C strains spread widely in Shandong Province.

Outbreak of acute hemorrhagic conjunctivitis in Beijing City in 2007 caused by coxsackievirus A24 variant: molecular identification and phylogenetic analysis / 病毒学报

In 2007, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Beijing. In order to identify the etiology of this outbreak, 57 eye conjunctival swabs were collected from 57 outpatient patients, and detected for adenovirus, human enterovirus 70 (HEV70) and Coxsackievirus A24 variant (CVA24v) genes by using RT-PCR or PCR methods. The results showed that 38 were positive for CVA24v, the positive rate was 66.7%, but none was positive for HEV70 and adenovirus, showing that this outbreak was caused by CVA24v. 9 viral isolates were obtained from 57 clinical specimens by using viral isolation method, and all were identified as CVA24v by molecular typing method. All 9 CVA24v isolates were performed by VP1 sequencing, the results showed that except for strain 0744/BJ/CHN/2007, the variability at nucleotide acid level and amino acid level among other 8 CVA24v were relatively low, and the homologies were more than 99.6% and 100.0%, respectively; the homologies of nucleotide acid and amino acid between strain 0744/BJ/CHN/2007 and other 8 CVA24v were 96.8%-97.2% and 99.7%, respectively. Phylogenetic analysis of 9 CVA24v revealed that they represented the Clade 4 and Clade 5 in Group I, showed that this outbreak was caused by at least 2 viral transmission chains. Comparing to 3C region of CVA24v frequently used before, VP1 region was considered as the most rigorous target for molecular epidemiology study of CVA24v. To enhance the research of sero-epidemiology and molecular epidemiology of CVA24v and to know the genetic characterizations and molecular evolution of CVA24v are most important to prevent and control the outbreaks of AHC in China.

Retraction: Detection and Phylogenetic Analysis of Coxsackievirus A24 Variant Causing Nation-wide Epidemic of Acute Hemorrhagic Conjunctivitis in Korea, 2002 / 감염과화학요법

We have submitted and published the above paper in Infection and Chemotherapy in 2003. Another paper with a condensed but almost same content as the above paper was submitted to and published in an English journal (Acute Hemorrhagic Conjunctivitis Caused by Coxsackievirus A24 Variant, South Korea, 2002. Emerg Infect Dis 2003). Our original intent was to introduce the study to all readers because the two journals seemed to cover different spectrum of readers. However, we did not follow the necessary steps for secondary publication. So we are asking the permission of the Editor to retract the above paper. We hereby regret to have to retract the paper.

Retraction: Detection and Phylogenetic Analysis of Coxsackievirus A24 Variant Causing Nation-wide Epidemic of Acute Hemorrhagic Conjunctivitis in Korea, 2002 / 감염과화학요법

We have submitted and published the above paper in Infection and Chemotherapy in 2003. Another paper with a condensed but almost same content as the above paper was submitted to and published in an English journal (Acute Hemorrhagic Conjunctivitis Caused by Coxsackievirus A24 Variant, South Korea, 2002. Emerg Infect Dis 2003). Our original intent was to introduce the study to all readers because the two journals seemed to cover different spectrum of readers. However, we did not follow the necessary steps for secondary publication. So we are asking the permission of the Editor to retract the above paper. We hereby regret to have to retract the paper.

Viral investigation of acute hemorrhagic conjunctivitis outbreak (2003) in Nepal using molecular methods.

A nationwide outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Nepal during August to September 2003, which affected nearly half of the population. Sixty conjunctival swabs from AHC patients were collected at Tilganga Eye Center in Kathmandu. For the first time in Nepal, we demonstrated the etiologic viral agents of AHC, namely, Coxsackievirus A24 variant (CA24v) by reverse transcription real time polymerase chain reaction (PCR). Of the 60 samples, 19 were positive for CA24v. No difference in the two genders was observed. Conversely, Adenovirus (AdV) was detected in 32 samples, which suggested that the epidemic was caused by mixed infection. AdV was detected also on 10 rupee notes. Findings indicated that inadequate personal hygiene was the main cause of the spread of these highly contagious viruses in the community environment in Nepal during the summer of 2003.

Detection and Phylogenetic Analysis of Coxsackievirus A24 Variant Causing Nation-wide Epidemic of Acute Hemorrhagic Conjunctivitis in Korea, 2002 / 감염과화학요법

BACKGROUND: Nation-wide outbreak of acute hemorrhagic conjunctivitis occurred in the summer, 2002 in South Korea. We identified the causative agent of this outbreak through virus culture and molecular biological techniques. METHODS: Polymerase chain reaction (PCR) was carried out with direct conjunctival swab samples and cell culture supernatants. Conjunctival swab was done at a community based-eye clinic in Seoul, September 2002. Initial screening for adenovirus and enterovirus was performed. Nested PCR for adenovirus was done with adenovirus common primers using direct swab sample, and reverse transcription PCR (RT-PCR) for enterovirus was done with enterovirus common primers. RT-PCR with primer 188/222 for VP1 region of enterovirus was done, if initial screening test was positive. PCR product was sequenced, and homology searching, compared to prototype strains, was done for serotyping. Protease 3C region of coxsackievirus A24v was amplified and sequenced with primer D1/U2. The sequence of this region was compared to those of viral isolates, which had been obtained from several Asian outbreaks since 1970. RESULTS: Conjunctival swabs were performed in 88 patients. Thirty nine (44%) samples out of the 88 were culture positive on HeLa or MRC-5 cells. Nine (100%) out of 9 culture supernatants, randomly selected from 39 culture positve samples, were positive for coxsackievirus A24v-specific RT-PCR. Phylogenetic analysis showed that sequences from 14 culture positive supernatants, randomly selected from 39 culture positive samples, clustered into a time-related, but distinct lineage, with Asian strains. CONCLUSIONS: We identified the causative agent of the epidemic hemorrhagic conjunctivits in year 2002 as coxsackievirus A24v.

Detection and Phylogenetic Analysis of Coxsackievirus A24 Variant Causing Nation-wide Epidemic of Acute Hemorrhagic Conjunctivitis in Korea, 2002 / 감염과화학요법

BACKGROUND: Nation-wide outbreak of acute hemorrhagic conjunctivitis occurred in the summer, 2002 in South Korea. We identified the causative agent of this outbreak through virus culture and molecular biological techniques. METHODS: Polymerase chain reaction (PCR) was carried out with direct conjunctival swab samples and cell culture supernatants. Conjunctival swab was done at a community based-eye clinic in Seoul, September 2002. Initial screening for adenovirus and enterovirus was performed. Nested PCR for adenovirus was done with adenovirus common primers using direct swab sample, and reverse transcription PCR (RT-PCR) for enterovirus was done with enterovirus common primers. RT-PCR with primer 188/222 for VP1 region of enterovirus was done, if initial screening test was positive. PCR product was sequenced, and homology searching, compared to prototype strains, was done for serotyping. Protease 3C region of coxsackievirus A24v was amplified and sequenced with primer D1/U2. The sequence of this region was compared to those of viral isolates, which had been obtained from several Asian outbreaks since 1970. RESULTS: Conjunctival swabs were performed in 88 patients. Thirty nine (44%) samples out of the 88 were culture positive on HeLa or MRC-5 cells. Nine (100%) out of 9 culture supernatants, randomly selected from 39 culture positve samples, were positive for coxsackievirus A24v-specific RT-PCR. Phylogenetic analysis showed that sequences from 14 culture positive supernatants, randomly selected from 39 culture positive samples, clustered into a time-related, but distinct lineage, with Asian strains. CONCLUSIONS: We identified the causative agent of the epidemic hemorrhagic conjunctivits in year 2002 as coxsackievirus A24v.

Detection & differentiation of Coxsackie A 24 variant isolated from an epidemic of acute haemorrhagic conjunctivitis in north India by RT-PCR using a novel primer pair.

BACKGROUND & OBJECTIVES: An epidemic of acute haemorrhagic conjunctivitis (AHC) occurred in north India during July to September 1994. We report a reverse transcription-polymerase chain reaction (RT-PCR) using known and novel primers to differentiate and identify the CA 24 virus isolated from the epidemic of AHC. METHODS: Conjunctival swabs were collected from 46 patients (in 12 patients from both the eyes) yielding 58 swabs. The swabs were inoculated in RD 19S and HeLa-199 cell monolayers and observed for cytopathic effect. Serum neutralizing antibodies were tested in 17 acute and 10 convalescent phase serum samples. RT-PCR was done on 9 isolates (7 Coxsackie A 24 and 2 ECHO-1 as identified by neutralization test) using known and a novel primer. Fourteen virus isolates (9 CA 24, 3 ECHO-1 and 2 untyped) were inoculated in suckling mice and these mice were observed daily for 10 days for flaccid paralysis of hind limb or death. RESULTS: Cytopathic virus was isolated from conjunctival swabs in 21 of 46 (45.6%) patients subjected to virus isolation. Sixteen of 21 (76.2%) isolates were neutralized by CA 24 specific antisera, 3 isolates were identified as ECHO-1 with Schmidt enteroviruses antiserum pools while 2 remained untypable. Of these 21 isolates, 9 representative isolates (7 CA 24 and 2 ECHO-1) tested by RT-PCR had enterovirus common region DNA but did not show any amplification in RT-PCR with EV-70 specific primers (VP-1 and VP-3). Using CA 24 specific novel (VP 3-1) primers amplification was seen in 6 of 7 CA 24 isolates while 2 ECHO-1 remained unamplified. In contrast with 3C-proteinase region primers, only 2 of 7 CA 24 were amplified along with false amplification of both ECHO-1. Serum neutralizing antibodies were seen in 2 of 17 (11.7%) acute phase sera and 6 of the 10 (60%) convalescent phase sera while in paired sera (available in two patients) a four-fold rise in titres were observed. Hind-limb paralysis and/or death occurred in all suckling mice inoculated with CA 24 isolates while mice remained healthy after inoculation with 3 isolates of ECHO-1 and 2 untypable isolates. INTERPRETATION & CONCLUSION: The epidemic of AHC was caused by a variant of CA 24. Molecular typing can detect and differentiate between CA 24 and EV-70 viruses. Novel primer pair was found useful in the identification and confirmation of CA 24 isolates.