Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR.

Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P < 0.0001), whereas influenza virus type A and human rhinovirus were detected more frequently in NPS specimens (P = 0.0001 and P = 0.0289, respectively). The possibility of false-positive adenovirus detection from saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies.

Pros and cons of VP1-specific maternal IgG for the protection of Enterovirus 71 infection.

Enterovirus 71 (EV71) causes hand, foot, and mouth diseases and can result in severe neurological disorders when it infects the central nervous system. Thus, there is a need for the development of effective vaccines against EV71 infection. Here we report that viral capsid protein 1 (VP1), one of the main capsid proteins of EV71, efficiently elicited VP1-specific immunoglobulin G (IgG) in the serum of mice immunized with recombinant VP1. The VP1-specific IgG produced in female mice was efficiently transferred to their offspring, conferring protection against EV71 infection immediately after birth. VP1-specific antibody can neutralize EV71 infection and protect host cells. VP1-specific maternal IgG in offspring was maintained for over 6 months. However, the pre-existence of VP1-specific maternal IgG interfered with the production of VP1-specific IgG antibody secreting cells by active immunization in offspring. Therefore, although our results showed the potential for VP1-specific maternal IgG protection against EV71 in neonatal mice, other strategies must be developed to overcome the hindrance of maternal IgG in active immunization. In this study, we developed an effective and feasible animal model to evaluate the protective efficacy of humoral immunity against EV71 infection using a maternal immunity concept.

Antiviral Activity of Chrysin Derivatives against Coxsackievirus B3 in vitro and in vivo.

Chrysin is a 5,7-dihydroxyflavone and was recently shown to potently inhibit enterovirus 71 (EV71) by suppressing viral 3C protease (3C(pro)) activity. In the current study, we investigated whether chrysin also shows antiviral activity against coxsackievirus B3 (CVB3), which belongs to the same genus (Enterovirus) as EV71, and assessed its ability to prevent the resulting acute pancreatitis and myocarditis. We found that chrysin showed antiviral activity against CVB3 at 10 µM, but exhibited mild cellular cytotoxicity at 50 µM, prompting us to synthesize derivatives of chrysin to increase the antiviral activity and reduce its cytotoxicity. Among four 4-substituted benzyl derivatives derived from C(5) benzyl-protected derivatives 7, 9-11 had significant antiviral activity and showed the most potent activity against CVB3 with low cytotoxicity in Vero cells. Intraperitoneal injection of CVB3 in BALB/c mice with 1×10(6) TCID50 (50% tissue culture infective dose) of CVB3 induced acute pancreatitis with ablation of acinar cells and increased serum CXCL1 levels, whereas the daily administration of 9 for 5 days significantly alleviated the pancreatic inflammation and reduced the elevation in serum CXCL1 levels. Collectively, we assessed the anti-CVB3 activities of chrysin and its derivatives, and found that among 4-substituted benzyl derivatives, 9 exhibited the highest activity against CVB3 in vivo, and protected mice from CVB3-induced pancreatic damage, simultaneously lowering serum CXCL1 levels.

Complete Genome Sequence of Rotavirus Group C Isolated in South Korea.

Rotavirus group C is the major etiological agent associated with acute gastroenteritis in all human age groups. Here, we report the complete genome sequence of human group C rotavirus (GpC-RV) isolated in South Korea.

Complete sequence analysis and antiviral screening of medicinal plants for human coxsackievirus a16 isolated in Korea.

OBJECTIVES: Coxsackievirus A group 16 strain (CVA16) is one of the predominant causative agents of hand, foot, and mouth disease (HFMD). METHODS: Using a specimen from a male patient with HFMD, we isolated and performed sequencing of the Korean CVA16 strain and compared it with a G10 reference strain. Also, we were investigated the effects of medicinal plant extract on the cytopathic effects (CPE) by CPE reduction assay against Korean CVA16. RESULTS: Phylogenetic analysis showed that the Korean CVA16 isolate belonged to cluster B-1 and was closely related to the strain PM-15765-00 isolated in Malaysia in 2000. The Korean CVA16 isolate showed 73.2% nucleotide identity to the G10 prototype strain and 98.7% nucleotide identity to PM-15765-00. Next, we assessed whether the Korean CVA16 isolate could be used for in vitro screening of antiviral agents to treat HFMD infection. Vero cells infected with the Korean CVA16 isolate showed a cytopathic effect 2 days after the infection, and the treatment of cells with Cornus officinalis, Acer triflorum, Pulsatilla koreana, and Clematis heracleifolia var. davidiana Hemsl extracts exhibited strong antiviral activity against CVA16. CONCLUSION: Collectively, our work provides potential candidates for the development of vaccine and novel drugs to treat the CVA16 strain isolated from a Korean patient.

Enteroviruses isolated from herpangina and hand-foot-and-mouth disease in Korean children.

Hand-foot-and-mouth disease (HFMD) and herpangina are commonly prevalent illness in young children. They are similarly characterized by lesions on the skin and oral mucosa. Both diseases are associated with various enterovirus serotypes. In this study, enteroviruses from patients with these diseases in Korea in 2009 were isolated and analyzed. Demographic data for patients with HFMD and herpangina were compared and all enterovirus isolates were amplified in the VP1 region by reverse transcription-polymerase chain reaction and sequenced. Among the enterovirus isolates, prevalent agents were coxsackievirus A16 in HFMD and coxsackievirus A5 in herpangina. More prevalent months for HFMD were June (69.2%) and May (11.5%), and June (40.0%) and July (24.0%) for herpangina. Age prevalence of HFMD patients with enterovirus infection was 1 year (23.1%), 4 years (19.2%), and over 5 years (19.2%). However, the dominant age group of herpangina patients with enterovirus infection was 1 year (48.0%) followed by 2 years (28.0%). Comparison of pairwise VP1 nucleotide sequence alignment of all isolates within the same serotypes revealed high intra-type variation of CVA2 isolates (84.6-99.3% nucleotide identity). HFMD and herpangina showed differences in demographic data and serotypes of isolated enteroviruses, but there was no notable difference in amino acid sequences by clinical syndromes in multiple comparison of the partial VP1 gene sequence.

Updates on the genetic variations of norovirus in sporadic gastroenteritis in Chungnam Korea, 2009-2010.

Previously, we explored the epidemic pattern and molecular characterization of noroviruses (NoVs) isolated in Chungnam, Korea in 2008, and the present study extended these observations to 2009 and 2010. In Korea, NoVs showed the seasonal prevalence from late fall to spring, and widely detected in preschool children and peoples over 60 years of age. Epidemiological pattern of NoV was similar in 2008 and in 2010, but pattern in 2009 was affected by pandemic influenza A/H1N1 2009 virus. NoV-positive samples were subjected to sequence determination of the capsid gene region, which resolved the isolated NoVs into five GI (2, 6, 7, 9 and 10) and eleven GII genotypes (1, 2, 3, 4, 6, 7, 8, 12, 13, 16 and 17). The most prevalent genotype was GII.4 and occupied 130 out of 211 NoV isolates (61.6%). Comparison of NoV GII.4 of prevalent genotype in these periods with reference strains of the same genotype was conducted to genetic analysis by a phylogenetic tree. The NoV GII.4 strains were segregated into seven distinct genetic groups, which are supported by high bootstrap values and previously reported clusters. All Korean NoV GII.4 strains belonged to either VI cluster or VII cluster. The divergence of nucleotide sequences within VI and VII intra-clusters was > 3.9% and > 3.5%, respectively. The "Chungnam(06-117)/2010" strain which was isolated in June 2010 was a variant that did not belong to cluster VI or VII and showed 5.8-8.2%, 6.2-8.1% nucleotide divergence with cluster VI and VII, respectively.

Molecular characterization and antiviral activity test of common drugs against echovirus 18 isolated in Korea.

Genetic diversity and antiviral activity for five common antiviral drugs of echovirus (ECV) 5 isolated in Korea have been described. The present study extended these tests to a Korean ECV 18 isolate. An outbreak of aseptic meningitis caused by the ECV 18 isolate was reported in Korea in 2005, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from stool specimen of a 5-year-old male patient with aseptic meningitis, the complete genome sequence was obtained and was compared it with the Metcalf prototype strain. Unlike the ECV5 isolate, the 3' untranslated region had the highest identity value (94.2%) at the nucleotide level, while, at the amino acid level, the P2 region displayed the highest identity value (96.9%). These two strains shared all cleavage sites, with the exception of the 2B/2C site, which was RQ/NN in the Metcalf strain but RQ/NS in the Korean ECV 18 isolate. In Vero cells infected with the Korean ECV 18 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC50 value >100 µg/mL. Of the five drugs, only amantadine (IC50: 4.97 ± 0.77 µg/mL, TI: 20.12) and ribavirin (IC50: 7.63 ± 0.87 µg/mL, TI: 13.11) had any antiviral activity against the Korean ECV 18 isolate in the five antiviral drugs. These antiviral activity effects were similar with results of the Korean ECV5 isolate.

Epidemics of enterovirus infection in Chungnam Korea, 2008 and 2009.

Previously, we explored the epidemic pattern and molecular characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006. The present study extended these observations to 2008 and 2009. In this study, enteroviruses showed similar seasonal prevalent pattern from summer to fall and age distribution to previous investigation. The most prevalent month was July: 42.9% in 2008 and 31.9% in 2009. The highest rate of enterovirus-positive samples occurred in children < 1-year-old-age. Enterovirus-positive samples were subjected to sequence determination of the VP1 region, which resolved the isolated enteroviruses into 10 types in 2008 (coxsackievirus A4, A16, B1, B3, echovirus 6, 7, 9, 11, 16, and 30) and 8 types in 2009 (coxsackievirus A2, A4, A5, A16, B1, B5, echovirus 11, and enterovirus 71). The most prevalent enterovirus serotype in 2008 and 2009 was echovirus 30 and coxsackievirus B1, respectively, whereas echovirus 18 and echovirus 5 were the most prevalent types in 2005 and 2006, respectively. Comparison of coxsackievirus B1 and B5 of prevalent enterovirus type in Korea in 2009 with reference strains of each same serotype were conducted to genetic analysis by a phylogenetic tree. The sequences of coxsackievirus B1 strains segregated into four distinct clusters (A, B, C, and D) with some temporal and regional sub-clustering. Most of Korean coxsackievirus B1 strains in 2008 and 2009 were in cluster D, while only "Kor08-CVB1-001CN" was cluster C. The coxsackievirus B5 strains segregated in five distinct genetic groups (clusters A-E) were supported by high bootstrap values. The Korean strains isolated in 2001 belonged to cluster D, whereas Korean strains isolated in 2005 and 2009 belonged to cluster E. Comparison of the VP1 amino acid sequences of the Korean coxsackievirus B5 isolates with reference strains revealed amino acid sequence substitutions at nine amino acid sequences (532, 562, 570, 571, 576-578, 582, 583, and 585).

Genetic diversity of a Korean echovirus 5 isolate and response of the strain to five antiviral drugs.

An outbreak of echovirus 5 (ECV 5) occurred in Korea in 2006, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from a young male patient with aseptic meningitis, we performed sequencing of the Korean ECV 5 strain and compared it with a prototype strain (Noyce). At the nucleotide level, the P1 region (85.3%) had the highest identity value; at the amino acid level, the P3 region (98.0%) had the highest identity value. The two strains shared all cleavage sites, with the exception of the VP1/2A site, which was TY/GA in the Noyce strain but TR/GA in the Korean ECV 5 isolate. In Vero cells infected with the Korean ECV 5 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC50 value >100 µg/mL. Of the five drugs, only amantadine (IC50: 1 ± 0.42 µg/mL, TI: 100) and ribavirin (IC50: 22 ± 1.36 µg/mL, TI: 4.55) had any antiviral activity against the Korean ECV 5 isolate.

Molecular and epidemiological characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006.

Enteroviruses were identified and characterized from patients with aseptic meningitis and other enterovirusrelated diseases in Chungnam, Korea from 2005 to 2006. Enteroviruses were isolated from 79 of 519 cases (15.2%) in 2005, and 37 of 386 cases (9.6%) in 2006. Based on partial VP1 sequencing, a total of 116 enterovirus isolates were resolved into 13 types. Prevalent among the Chungnam isolates were echovirus 18 and coxsackievirus B5 in 2005, and echoviruses 5 and 25 in 2006. This is the first time echoviruses 5 and 18 have been identified in Korea since enterovirus surveillance began there in 1993. The temporal distribution of enterovirus epidemics in Chungnam showed a remarkable seasonal pattern, with cases occurring during most of the three months of the summer from June to August. The highest rate of enterovirus-positive cases occurred in patients less than 1 year of age. The ratio of male to female enterovirus-positive patients was approximately 1.8:1. Comparison of the VP1 amino acid sequences of the 15 coxsackievirus B5 isolates with reference strains revealed that all Chungnam isolates are substituted at positions 23 (V23I), 19 (S19G), 75 (Y75F), and 95 (N95S). Upon comparing the nine ECV5 isolates with foreign strains, it was found that only the Chungnam isolates, with the exception of Kor06-ECV5-239cn, have P at position 153 and F at position 146. The three ECV9 isolates from 2006 show alterations at amino acids 36, 148, and 154 outside of the BC-loop and at position 84 in the BC-loop, whereas the seven isolates from 2005 and the other ECV9 strains in the database only show the alteration at position 84 (D, I, N, S). The five ECV25 isolates have an S residue at position 134, whereas most of the foreign strains have an N residue.

[Application of a diagnostic method using reverse transcription-PCR ELISA for the diagnosis of enteroviral infections].

BACKGROUND: Enteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections. METHODS: Reverse transcription (RT)-PCR ELISA kit for targeting 5' non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them. RESULTS: The detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5'NCR (P=0.016). CONCLUSIONS: Our results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.

Acute hemorrhagic conjunctivitis epidemic caused by coxsackievirus A24 variants in Korea during 2002-2003.

A variant of coxsackievirus A24 (CA24v) is one of the agents causing acute hemorrhagic conjunctivitis. There was an epidemic of acute hemorrhagic conjunctivitis caused by CA24v in Korea from 2002 to 2003. Seventy-one strains of CA24v were isolated from 159 conjunctival specimens (45%). Most of the patients were school children under the age of 20. The epidemic began in the first week of August in 2002, and spread extensively, with a peak in the third week of September. CA24v strains were also isolated from conjunctival specimens in 2003. Reverse transcription polymerase chain reaction (RT-PCR) was performed and sequencing of the 340 bp fragment of the VP1 region of the viruses. Sequencing data were multiple-aligned using CLUSTAL W (version 1.81). Phylogenetic trees were plotted using TreeView (version 1.6.6). Homologies ranged from 97.7%-100%, depending on geographical regions: from 99.4%-100% in 2002 and 98.4%-100% in 2003. A phylogenetic tree based on the nucleotide sequence homologies formed clusters depending on years rather than on geographical regions. Identities (98%-100%) were found among the Korean CA24v strains, and there was 85%-90% homology between these and the prototype strain.

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002.

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.

Identification of enteroviruses by using monoclonal antibodies against a putative common epitope.

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.