Expression and antigenicity of recombinant human respiratory syncytial virus glycoproteins having different affinity tags.

Human respiratory syncytial virus (HRSV) is a main cause of lower respiratory tract infections in infants and the elderly. Glycoprotein (G) is major antigen on the viral surface, and plays a key role for virus entry. Therefore, purification of the glycoprotein of HRSV is critical for the development of HRSV vaccine and serological diagnosis. In this study, we report the design and characterization of glycoprotein engineered rationally to enhance the protein solubility and to facilitate efficient purification. We permuted HRSV glycoproteins with two tags: (i) an immunoglobulin (Ig) M signal peptide and a protein A B domain tag to render HRSV glycoprotein secret into the culture media and (ii) a foldon and 6 × histidine tag with or without transmembrane domain. Three recombinant baculoviruses were constructed: (i) transmembrane-truncated HRSV glycoprotein (amino acid positions 66-298) inserted with the N-terminal IgM signal peptide and protein A B domain (MG-GΔTM), (ii) truncated HRSV glycoprotein (amino acid positions 66-298) fused with a C-terminal foldon and 6 × histidine tag (GΔTM-FH), and (iii) full-length HRSV glycoprotein (amino acid positions 1-298) fused with a C-terminal foldon and 6 × histidine tag (G-FH). Highly soluble recombinant MG-GΔTM protein was clearly purified using one-step affinity chromatography with IgG-sepharose resin, whereas the recombinant G-FH protein and truncated GΔTM-FH were purified partially using nickel-resin. Although, the antigenicity of GΔTM-FH was stronger than highly mannose-rich MG-GΔTM protein, MG-GΔTM induced neutralizing antibodies efficiently in the mice to protect from infectious HRSV.

Molecular epidemiology of a post-influenza pandemic outbreak of acute respiratory infections in Korea caused by human adenovirus type 3.

An outbreak of upper respiratory tract infections associated with human adenovirus (HAdV) occurred on a national scale in Korea from September to December 2010, following a major H1N1 influenza pandemic. Data from the Korea Influenza and Respiratory Surveillance System (KINRESS) showed an unusually high positive rate accounting for up to 20% of all diagnosed cases. To determine the principal cause of the outbreak, direct polymerase chain reaction (PCR) amplification followed by sequence analysis targeting parts of the hexon gene of HAdV was performed. Serotypes of 1,007 PCR-diagnosed HAdV-positive samples from patients with an acute upper respiratory tract illness were determined and epidemiological characteristics including major aged group and clinical symptoms were analyzed. The principal symptom of HAdV infections was fever and the vulnerable aged group was 1-5 years old. Based on sequence analysis, HAdV-3 was the predominant serotype in the outbreak, with an incidence of 74.3%. From the beginning of 2010 until May, the major serotypes were HAdV-1, 2, and 5 (70-100%) in any given period. However, an outbreak dominated by HAdV-3 started between July and August and peaked in September. Phylogenetic analysis revealed that there was no genetic variation in HAdV-3. The results demonstrated that an outbreak of upper respiratory illness followed by H1N1 influenza pandemic in Korea was caused mainly by emerged HAdV-3. J.

Complete genome sequence of human respiratory syncytial virus genotype A with a 72-nucleotide duplication in the attachment protein G gene.

The complete genome sequence of human respiratory syncytial virus genotype A (HRSV-A) with a 72-nucleotide duplication in the C-terminal part of the attachment protein G gene was determined and analyzed. The genome was 15,277 bp in length, and 0.46 to 6.03% variations were identified at the nucleotide level compared with the previously reported complete genome of HRSV-A. Characterization of the genome will improve understanding of the diversity of the HRSV-A major antigens and enable an in-depth analysis of its genetics.

Prevalence and molecular epidemiology of human coronavirus HKU1 in patients with acute respiratory illness.

In 2005, human coronavirus HKU1 (HCoV-HKU1) was isolated and identified from a 71-year-old man with pneumonia in Hong Kong. To identify and classify genotypes of HCoV-HKU1 in Korea, a sensitive, specific, and quantitative real-time polymerase chain reaction (PCR) assay was developed and analyzed the sequences of HCoV-HKU1 isolated in Korea. A total of 1,985 respiratory specimens taken from patients with acute respiratory illness were tested for HCoV-HKU1 from January 2007 to May 2008. The major clinical symptoms associated with HCoV-HKU1 infection were examined statistically and sequence variations of the RNA-dependent RNA polymerase (RdRp), spike, and nucleocapsid genes were also analyzed. Fifty cases (2.5%) HCoV-HKU1 were identified by real-time PCR and viral loads ranged from 6.7 × 10(4) to 1.6 × 10(9) copies/ml. The clinical symptoms of HCoV-HKU1 infection included rhinorrhea (72%), cough (64%), nasal congestion (56%), fever (32%), sputum (30%), sore throat (18%), chills (16%), postnasal discharge (14%), and tonsillar hypertrophy (10%). There was a seasonal distribution of HCoV-HKU1 infection, peaking in winter and spring. Both genotypes A and B were detected but no recombination between them was found. This is the first report on the identification and genotyping of HCoV-HKU1 as a causative agent of acute respiratory illness in Korea. The data suggest that at least two genotypes, A and B, of HCoV-HKU1 with scattered silent mutations were circulating in Korea from 2007 to 2008.

Development of real-time PCR assays for detection and quantification of human bocavirus.

BACKGROUND: Human bocavirus (HBoV) is a parvovirus that has been recently detected in patients with respiratory illness. OBJECTIVES: We developed a sensitive, specific, and quantitative real-time PCR assay based on the TaqMan method for HBoV detection and quantification in respiratory specimens. STUDY DESIGN: Three individual real-time PCR assays were designed to amplify HBoV NS1, NP-1, and VP1 genes. For clinical evaluation, 506 nasal aspirates obtained from patients with acute respiratory tract infections during December 2006 to May 2007 were tested. RESULTS: Each assay had a broad dynamic range (50 x 10(7) to 5 x 10(7)copies of plasmid DNA) and high inter- and intra-assay reproducibility. The detection limit of each assay was 10 genome copies per reaction, and no crossreactivity with other major respiratory viruses or bacteria was detected. Clinical evaluation revealed that 11 (2.1%) of 506 patients diagnosed with upper respiratory tract infections, pneumonia, bronchitis, pharyngitis, or sinusitis had HBoV detected by all three assays, with viral loads ranging from 8.2 x 10(4) to 8.1 x 10(9)copies/ml of specimen. CONCLUSIONS: The three assays for HBoV diagnosis and quantification are highly sensitive, specific real-time tools for the reliable epidemiological and pathogenetic study of HBoV infection.

Complete genome sequences of human respiratory syncytial virus genotype a and B isolates from South Korea.

There is a paucity of complete genome sequence information for human respiratory syncytial virus (HRSV). To this end, we sequenced the complete genome sequences of HRSV genotype A (HRSV-A/IC688/12) and genotype B (HRSV-B/GW0047/14 and HRSV-B/IC0027/14). This information will increase the understanding of HRSV genetic diversity, evolution, pathogenicity, antigenicity, and transmissibility.

Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene.

Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011-2012 to 94.6% in 2012-2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10(-3) nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.

Molecular Classification of Human Adenovirus Type 7 Isolated From Acute Respiratory Disease Outbreak (ARD) in Korea, 2005-2006.

OBJECTIVES: To assess the genomic characteristics of human adenoviruses (HAdVs) that caused small-scale epidemics in Korea and compare sequence analysis and restriction fragment length polymorphism (RFLP). METHODS: Two hundred sixty-two throat swabs were collected from geographically distinct two cohabitation facilities during outbreaks in August 2005 and February-May 2006. 148 isolates were obtained using the adenocarcinomic human alveolar basal epithelial cells (A549 cells) from 262 specimens. The sequences of 448 bp partial hexon gene of isolates were analized and compared with serotype results using neutralizing test. The hexon (1.2 kb), fiber, and E4 ORF 6/7 34.7 kDa protein (2.1 kb) genes were further analysed in 10 randomly selected specimens. RFLP of the genomic DNA for genotyping was also performed and compared with sequence information. RESULTS: All the isolates were localized into the same cluster when phylogenetic tree was generated based on hexon gene using Clustal W. While fiber and E4 ORF 6/7 34.7 kDa protein genes were analysed, the tree was divided into two clusters. Interestingly, isolates with same genetic characteristics of hexon gene did not show identical RFLP patterns in accordance with their origin of episode, rather phylogenetic analysis of fiber and E4 ORF 6/7 34.7 kDa protein genes were correlated with RFLP patterns. CONCLUSION: These results indicate that serotype classification based on hexon gene might not be enough to discriminate HAdV serotype, and additional genetic characteristics including fiber and/or E4 ORF 6/7 should be recruited to dispose subgroup of HAdV serotype.