Yoon-Seok Chung

Persistent HIV-1 replication maintains the tissue reservoir during therapy

Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

Genetic analysis of the VP1 region of Human enterovirus 71 strains isolated in Korea during 2000

Summary. We have isolated Human enterovirus 71 (EV71) from stool and CSF samples taken from patients with acute flaccid paralysis, herpangina, or hand, foot and mouth disease in 2000. Both the cell culture-neutralization test and RT-PCR were used to detect enteroviruses. Rhabdomyosarcoma (RD), HEP2c, and BGM cells were used for the isolation of viruses, and serotypes were determined by the neutralization test using EV71-specific antiserum. For genomic analysis, we amplified a 437-bp fragment of the 5′-noncoding region of the enterovirus genome and a 484-bp fragment of the VP3/VP1 region of EV71 by RT-PCR, with positive results. Products amplified using an EV71-specific primer pair were sequenced and compared with other isolates of EV71. Analysis of the nucleotide sequences of the amplified fragments showed that the EV71 isolates from patients were over 98% homologous and belonged to the genotype C.

Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

Outbreak of measles in the Republic of Korea, 2007: importance of nosocomial transmission.

BACKGROUND From 2002 through 2006, Republic of Korea conducted extensive measles elimination activities and declared elimination in 2006. An outbreak of measles involving 180 confirmed cases occurred during 2007. METHODS An outbreak investigation was performed and enhanced surveillance was implemented. Detailed case investigations and laboratory testing included serologic and molecular diagnostic methods. Cases were classified according to World Health Organization and national guidelines. RESULTS During 2007, 451 suspected cases were reported and 180 (40%) cases were confirmed as measles during epidemiologic weeks 14-42. Incidence during the outbreak was 3.7 cases per million persons, excluding imported cases. Most confirmed cases were reported from Seoul; 137 (76%) cases were among children <24 months old, 124 (69%) case patients had no history of measles vaccination, and 81 (45%) case patients resulted from nosocomial transmission in 6 hospitals. Community members, patients, and health care workers all contributed to measles virus transmission. Limited outbreak control measures were implemented; high population immunity likely accounted for the self-limited transmission during this outbreak. CONCLUSIONS Limited outbreaks of measles, in which nosocomial transmission can play an important role, may occur after countries have declared elimination. Timely and opportunistic vaccination may help prevent such outbreaks; high-quality surveillance is critical for their detection.

High isolation rate of adenovirus serotype 7 from South Korean military recruits with mild acute respiratory disease

Adenovirus is a major cause of acute respiratory disease (ARD) in military recruits. When South Korean military recruits with ARD were surveyed, adenovirus was identified in 122 (61.0%) of the 200 recruits studied. Moreover, all cases of ARD involving adenovirus were caused by serotype 7.

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

Abstract 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 (50×107 to 5×107 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 8.2×104 to 8.1×109 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.

Silencing E1A mRNA by RNA interference inhibits adenovirus replication

SummaryThe adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

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

Abstract 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, 6× 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.

Avian influenza a (H5N1) virus antibodies in poultry cullers, South Korea, 2003-2004.

Transmission of influenza (H5N1) virus from birds to humans is a serious public health threat. In South Korea, serologic investigation among 2,512 poultry workers exposed during December 2003–March 2004 to poultry with confirmed or suspected influenza (H5N1) virus infection found antibodies in 9. Frequency of bird-to-human transmission was low.

Identification of Enteroviruses by Using Monoclonal Antibodies against a Putative Common Epitope

ABSTRACT 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.