Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity.

The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is involved in the pathological reaction to SARS and is a key antigen for the development of a sensitive diagnostic assay. However, the antigenic properties of this N protein are largely unknown. To facilitate the studies on the function and antigenicity of the SARS-CoV N protein, 6x histidine-tagged recombinant SARS-CoV N (rSARS-N) with a molecular mass of 46 and 48kDa was successfully produced using the recombinant baculovirus system in insect cells. The rSARS-N expressed in insect cells (BrSARS-N) showed remarkably higher specificity and immunoreactivity than rSARS-N expressed in E. coli (ErSARS-N). Most of all, BrSARS-N proteins were expressed as a highly phosphorylated form with a molecular mass of 48kDa, but ErSARS-N was a nonphosphorylated protein. In further analysis to determine the correlation between the phosphorylation and the antigenicity of SARS-N protein, dephosphorylated SARS-N protein treated with protein phosphatase 1 (PP1) remarkably enhanced the cross-reactivity against SARS negative serum and considerably reduced immunoreactivity with SARS-N mAb. These results suggest that the phosphorylation plays an important role in the immunoreactivity and specificity of SARS-N protein. Therefore, the BrSARS-N protein may be useful for the development of highly sensitive and specific assays to determine SARS infection and for further research of SARS-N pathology.

Preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein.

Severe acute respiratory syndrome-coronavirus nucleocapsid (SARS-CoV N) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. We have used recombinant N protein expressed in insect cells to generate 17 mAbs directed against this protein. We selected five mAbs that could be used in various diagnostic assays, and all of these mAbs recognized linear epitopes. Three IgG(2b) mAbs were recognized within the N-terminus of N protein, whereas the epitope of two IgG(1) mAbs localized within the C-terminus. These mAbs were found to have significant reactivity with both non-phosphorylated and phosphorylated N proteins, which resulted in high reactivity with native N protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. Therefore, these results suggested that these mAbs would be useful in the development of various diagnostic kits and in future studies of SARS-CoV pathology.

Molecular characterization of the full-length genome of the Japanese encephalitis viral strain K87P39.

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain K87P39, isolated from a pool of circulating Culex tritaeniorhynchus mosquitoes in Korea. In comparison with 27 fully sequenced JEV genomes currently available, we found that the 10968-nucleotide RNA genome of K87P39 has a nine-nucleotide deletion in the 3' nontranslated variable region and that its single open reading frame has a total of eight amino acid substitutions. The K87P39 isolate is highly similar to other JEV isolates, and homology ranges from 97.9 to 89.0% at the nucleotide level, and 99.1 to 96.7% at the deduced amino acid level. Phylogenetic analyses using the full-length sequence of the 27 available JEV genomes showed that the K87P39 strain is most closely related to six Chinese SA14 derivatives and that it is distantly related to the Australian FU, Korean K94P05 and Japanese Ishikawa strains. In addition, we also found that phylogenetic relationships based on the full-length genome are highly similar to those based on the E gene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. We therefore performed a more extensive E gene-based phylogenetic analysis on a selection of 70 JEV isolates available from GenBank, which represent a temporally and geographically wide variety of JEV strains.

Molecular characterization of two genotypes of mumps virus circulated in Korea during 1998-2001.

Sequence analyses of the entire small hydrophobic (SH) and hemagglutinin-neuraminidase (HN) genes of mumps viruses circulated in Korea from 1998 to 2001 showed that these isolates were grouped into two genotypes, H and I. While genotype I was predominant throughout the country during this period, genotype H was found in the restricted region, 1999. The nucleotide and deduced amino acid sequences of Korean isolates showed the type-specific changes including the signature motif at positions 28-30 in the SH gene and the neutralizing epitopes in the HN gene. Particularly, Asian strains including Korean isolates and European strains differed from 2.3 to 3.8% at the nucleotide sequence level in the SH gene although they belonged to the same genotype H. Furthermore, none of Korean isolates were genetically related to the vaccine strains used in Korea. The results provide important information to understand the epidemiology of mumps infection and to facilitate the development of more efficient vaccine program in Korea.

An HIV type 1 subtype B founder effect in Korea: gp160 signature patterns infer circulation of CTL-escape strains at the population level.

HIV-1 subtype B predominates in the Republic of Korea. Phylogenetic analyses of sequences for complete nef genes and env gene fragments encoding the V3 loop have identified a major monophyletic Korean subclade that is distinct from Western subtype B sequences in the Los Alamos HIV Sequence Database. This was investigated further by sequence analysis of complete env genes recovered from the DNA of peripheral blood mononuclear cells for matched groups of Koreans, four patients per group, previously assigned as being infected with either Korean or Western strains. The phylogenetic classifications were confirmed and analysis of the translation products identified 32 amino acid signature pattern differences, dispersed throughout gp160, which differentiate the two subclades. Twenty-three of these positions map to epitopes recognized by HLA-I-restricted cytotoxic T-lymphocytes (CTL) as catalogued in the Los Alamos HIV Immunology Database. The remaining nine map at or close to sites predicted to be targets for immunoproteasomes that are involved in producing peptides that bind to MHC Class I. These results suggest that a founder effect in the Korean population is based on the spread of CTL-escape/host-adapted HIV-1 strains.

Isolation of Japanese encephalitis and Getah viruses from mosquitoes (Diptera: Culicidae) collected near Camp Greaves, Gyonggi Province, Republic of Korea, 2000.

As part of an evaluation of the ecology of arthropod-borne diseases in the Republic of Korea (ROK), we examined 8,765 mosquitoes captured in Paju County, Gyonggi Province, ROK, for the presence of viruses. Mosquitoes were captured in propane lantern/human-baited Shannon traps, Mosquito Magnet traps, or American Biophysics Corporation (East Greenwich, RI) miniature light traps with or without supplemental octenol bait and/or dry ice. Mosquitoes were identified to species, placed in pools of up to 40 mosquitoes each, and tested on Vero cells for the presence of virus. A total of 15 virus isolations were made from 293 pools of mosquitoes. Viruses were identified by reverse transcriptase-polymerase chain reaction and sequencing and consisted of 14 isolations of Japanese encephalitis (JE) virus and one isolation of Getah (GET) virus. All JE isolates were from Culex tritaeniorhynchus Giles, and the isolate of GET was from Aedes vexans (Meigen). The minimum field infection rate for JE in Cx. tritaeniorhynchus was 3.3 per 1,000, whereas the GET virus infection rate for Ae. vexans was 0.2 per 1,000. Isolation of JE and GET indicated that both viruses were actively circulating in northern Gyonggi Province, ROK. The lack of human cases of JE among the Korean population probably is because of an effective government-mandated vaccination program. The reason for no cases among >10,000 United States military and others that reside or train nearby is unknown, but may be related to personnel protection measures (permethrin-impregnated uniforms and use of deet repellent), adult mosquito control, mosquito selection of nonhuman hosts (unpublished data), and the low symptomatic to asymptomatic ratio of disease in adults.