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Zoonotic transmission of orthohantaviruses from rodent reservoirs to humans has been the cause of severe fatalities. Human infections are reported worldwide, but vaccines have been approved only in China and Korea. Orthohantavirus vaccine development has been pursued with no sense of urgency due to the relative paucity of cases in countries outside China and Korea. However, the orthohantaviruses continuously evolve in hosts and thus the current vaccine may not work as well against some variants. Therefore, a more effective vaccine should be prepared against the orthohantaviruses. In this review, we discuss the issues caused by the orthohantavirus vaccine. Given the pros and cons of the orthohantavirus vaccine, we suggest strategies for the development of better vaccines in terms of pandemic preparedness.

Of various rodent-borne hantaviruses, Seoul orthohantavirus (SEOV) causes haemorrhagic fever with renal syndrome (HFRS), as does Hantaan orthohantavirus (HTNV). Given global-scale of cases of human infection with SEOV, it is of great clinical importance to distinguish SEOV from other HFRS-causing hantaviruses. In May 2019, a middle-aged patient who had lived in a suburban area of Chungcheong Province, Republic of Korea and enjoyed outdoor activities was transferred to Asan Medical Center in Seoul, Republic of Korea with HFRS; his symptoms included high fever and generalized myalgia. The rapid diagnostic test performed immediately after his transfer detected HTNV-specific antibodies, and the patient was treated accordingly. However, two consecutive IFAs performed at ten-day intervals showed no HTNV-specific immunoglobulin (Ig) G. During continuous supportive care, next-generation sequencing successfully identified viral genomic sequences in the patient’s serum, which were SEOV and not HTNV. Phylogenetic analysis grouped the L, M, and S genes of this SEOV strain together with those of rat- or human-isolated Korean strains reported previously. Given global outbreaks and public health threats of zoonotic hantaviruses, a causative pathogen of hantavirus HFRS should be identified correctly at the time of diagnosis and by point-of-care testing., Author summary Rodent-borne Seoul orthohantavirus (SEOV) has provoked human cases from Asia to the Americas and Europe whereas most orthohantaviruses cause regional cases. Despite this, SEOV gets less attention than other orthohantaviruses. In Korea, 2019, a middle-aged man was initially diagnosed with Hantaan orthohantavirus (HTNV) and treated accordingly. However, next-generation sequencing identified SEOV, not HTNV, in the patient’s serum. Given its global outbreaks and public health threats, zoonotic SEOV should be diagnosed correctly on point of care to reduce unnecessary medical costs.

Squirrel adenovirus (SqAdV) was reported previously. However, only partial sequences of its hexon and polymerase genes have been revealed. For the first time, we report the full-length genome of SqAdV including the complete hexon and penton base genes. From internal body organs of 59 red squirrels archived in Korea Bank for Pathogenic Viruses, the hexon, penton base, and full-length genome of SqAdV were determined by a PCR method. Of the internal body organs examined, the spleen showed the highest detection rate (25.42%) for SqAdV whereas the kidney and lung exhibited 18.64% and 3.39% rates, respectively. Based on the phylogenetic relationships of the hexon and penton base genes, SqAdV appears to belong to the genus Mastadenovirus, and, at least in our study, the hexon of SqAdV exhibits the closest relationship to that of an alpaca AdV. Compared with the hexon, the penton base of SqAdV appears to be genetically more divergent from that of other mastadenoviruses. It was also revealed that the full-length SqAdV genome retained AT nucleotide content similar level to AT-rich atadenoviruses, which is unusual for mastadenoviruses. Our results emphasize that SqAdV is classified into the genus Mastadenovirus and demonstrate the AT-biased nucleotide constitution of SqAdV.

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness. ispartof: PLoS One vol:12 issue:3 ispartof: location:United States status: published

Human parainfluenza viruses (HPIV) are important causes of respiratory tract infections in young children. To characterize the molecular epidemiology of an HPIV outbreak occurring in Korea during 2006, genetic analysis of 269 cell culture isolates from HPIV-infected children, was conducted using nested reverse transcription-PCR (RT-PCR). HPIV-1 was detected in 70.3% of tested samples (189/269). The detection rate of HPIV-2 and HPIV-3 was 1.5% (4/269) and 9.3% (25/269), respectively. Mixed HPIV-1, -2 and -3 infections were detected in 19.0% (51/269): HPIV-1 and HPIV-2 in 15, HPIV-1 and HPIV-3 in 26, HPIV-2 and HPIV-3 in 6, and HPIV-1, -2 and -3 in 4. Of these positive samples for three different types HIPV-1, -2, and -3, two each representative strains were selected, the full length of hemagglutinin-neuraminidase (HN) gene for HPIV was amplified by RT-PCR, and sequenced. Multiple alignment analysis, based on reference sequence of HPIV-1, -2, and -3 strains available in GenBank, showed that the identity of nucleotide and deduced amino acid sequences was 92.4–97.6% and 92.7–97.9%, respectively, for HPIV-1, 88.5–99.8% and 88.6–100% for HPIV-2, and 96.3–99.5% and 95.0–99.3% for HPIV-3, respectively. Phylogenetic analysis showed that HPIV-1, -2, and -3 strains identified in this study were closely related among the strains in the same type with no significant genetic variability. These results show that HPIV of multiple imported sources was circulating in Korea. J. Med. Virol. 86:1041–1047, 2014. © 2014 Wiley Periodicals, Inc.

In addition to influenza A subtypes, two distinct lineages of influenza B virus also cause seasonal epidemics to humans. Recently, Dudas et al. have done evolutionary analyses of reassortment patterns of the virus and suggested genetic lineage relationship between PB1, PB2, and HA genes. Using genetic plasmids and reassortant viruses, we here demonstrate that a homologous lineage PB1-PB2 pair exhibits better compatibility than a heterologous one and that the lineage relationship between PB1 and HA is more important for viral replication than that between PB2 and HA. However, co-adaptation of PB1-PB2-HA genes appears to be affected by complete gene constellation. ispartof: Scientific Reports vol:6 issue:1 pages:27480- ispartof: location:England status: published

Human metapneumovirus (HMPV) has been described as an important etiologic agent of upper and lower respiratory tract infections, especially in young children and the elderly. Most of school-aged children might be introduced to HMPVs, and exacerbation with other viral or bacterial super-infection is common. However, our understanding of the molecular evolution of HMPVs remains limited. To address the comprehensive evolutionary dynamics of HMPVs, we report a genome-wide analysis of the eight genes (N, P, M, F, M2, SH, G, and L) using 103 complete genome sequences. Phylogenetic reconstruction revealed that the eight genes from one HMPV strain grouped into the same genetic group among the five distinct lineages (A1, A2a, A2b, B1, and B2). A few exceptions of phylogenetic incongruence might suggest past recombination events, and we detected possible recombination breakpoints in the F, SH, and G coding regions. The five genetic lineages of HMPVs shared quite remote common ancestors ranging more than 220 to 470 years of age with the most recent origins for the A2b sublineage. Purifying selection was common, but most protein genes except the F and M2-2 coding regions also appeared to experience episodic diversifying selection. Taken together, these suggest that the five lineages of HMPVs maintain their individual evolutionary dynamics and that recombination and selection forces might work on shaping the genetic diversity of HMPVs.

Background:Borna disease virus (BDV) predominantly infects horses and sheep, causing a broad range of behavioural disorders. It is controversial whether BDV infects humans and causes psychiatric disorders.Objectives:We searched for BDV-derived nucleic acids in blood of race horses and jockeys riding the horses.Methods:We assayed for the BDV genome in RNA extracted from peripheral blood mononuclear cells (PBMC) of 39 race horses and 48 jockeys. Two polymerase chain reaction protocols [one-tube reverse transcription–polymerase chain reaction (RT-PCR) and two-step RT-PCR] were used to assay BDV p24 and p40 transcripts.Results:The p24 and p40 viral nucleic acid sequences were not detected in the PBMC RNAs from any of the race horses or jockeys.Conclusions:These data do not support an epidemiological association between BDV infection, race horses and humans.

Background:It is controversial whether Borna disease virus (BDV) infects humans and causes psychiatric disorders.Objectives:The relationship between BDV infection and schizophrenia with deficit syndrome was investigated.Study design:Using the Schedule for the Deficit Syndrome, 62 schizophrenic in-patients were selected from three psychiatric hospitals. RNA was extracted from peripheral blood mononuclear cells and analyzed using nested reverse transcriptase-polymerase chain reaction with primers to detect BDV p24 and p40.Results and conclusions:BDV transcripts were not detected in samples from any of the 62 schizophrenic patients. These data do not support an etiologic association between BDV infection and the deficit form of schizophrenia.

Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.

We have previously identified a defensin-like peptide (BmDp, Genbank accession number DQ118523), in the larvae of a silkworm, Bombyx mori (B. mori) using wdS20658 cDNA in a Bombyx EST database (Silk Base: http://www.ab.a.u-tokyo.ac.jp/silkbase ). A full-length of BmDp cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli cells in order to obtain a functional peptide. Amino acid sequence comparison of cysteine-rich defensin-like peptides with those of the BmDp showed to be identical protein as BmDefensinA designated as BmdefA gene deposited in the DDBJ database under the accession number AB367525. Sequence alignments revealed that the BmDp belongs to the insect defensin family and is close to galiomicin and spodoptericin, a defensin and a defensin-like protein having antimicrobial activities in phylogenetic analysis. Thus, we evaluated the effect of immune challenge and antimicrobial activity to understand the nature of this anionic BmDp. Semiquantitative RT-PCR analysis showed that BmDp gene expression was inducible by bacterial injection and peaked at a highest level at 8h after bacterial injection. This result suggests that BmDp is related with immune response against bacteria. A purified recombinant GSTBmDp fusion protein did not show direct antimicrobial activity against bacteria and fungi, which is differed from cationic antibacterial insect defensins and neutral antifungal defensin-like peptides.

Soldiers from the Republic of Korea and the United States conducting peacetime military operations at various training sites and multiple range complexes located near the demilitarized zone separating North and South Korea are exposed to rodents and their potentially disease-carrying ectoparasites. These diseases include scrub typhus, murine typhus, and leptospirosis. Many of the training sites are rural or semi-rural, surrounded or co-located with various forms of agriculture, and are infested with rodents and insectivores (as well as their ectoparasites), which are commonly found in association with unmanaged tall grasses, scrub, and crawling vegetation habitats. For 5 years, rodents and insectivores were collected seasonally (spring, summer, fall, and winter) at firing points 10 and 60 near the demilitarized zone and serologically tested for the presence of scrub typhus, murine typhus, and leptospirosis antibodies. Of the nine species of small mammals collected, Apodemus agrarius, the common striped field mouse and known reservoir of scrub typhus, was the most frequently collected (90.6%). Only four of the nine species captured, A. agrarius (60.9%), Micromys minutus (100%), Mus musculus (55.6%), and Rattus norvegicus (46.7%), were positive for scrub typhus. Of all the small mammals captured, only A. agrarius was positive for murine typhus (0.3%) and leptospirosis (1.3%). Seasonal and annual prevalence rates based on weight and sex are presented.

Four US soldiers admitted to the Brian Allgood Army Community Hospital, Seoul, Korea, were diagnosed with hemorrhagic fever with renal syndrome, an acute febrile disease, from 27 October to 13 November 2005. Epidemiological investigations were conducted and rodents live trapped at military training sites where the soldiers had recently trained. Total RNA was extracted from patient sera and lung tissues of Apodemus agrarius, the primary reservoir of Hantaan virus (HTNV), amplified by RT-PCR, sequenced, and identified as HTNV. Phylogenetic relationships based on comparison of the 281-nt region of the G2 glycoprotein-encoding M segment of the patients and Murinae rodent-borne hantaviruses were conducted. HTNV amplified from A. agrarius captured at Firing Point 60 corresponded to patient 1, while HTNV amplified from A. agrarius captured at Twin Bridges Training Area corresponded to patients 2-4. These results strongly support that transmission sites were linked with patient and reservoir host HTNV strains.

Throughout Korea, small mammals are hosts to a number of disease-causing agents that pose a health threat to U.S. and Korean military forces while they conduct field-training exercises. A seasonal rodent-borne disease surveillance program was established at two firing points (FP), FP-10, and FP-60, and conducted over five years from 2001 through 2005 in response to hantavirus cases among U.S. soldiers. The ecology of these sites consisted primarily of tall grasses associated with semi-permanent and temporary water sources (drainage ditches and a small stream) and dry-land agriculture farming. Eight species of rodents and one species of insectivore were collected, including Apodemus agrarius, Micromys minutus, Mus musculus, Rattus norvegicus, Tscherskia triton, Microtus fortis, Myodes regulus, and Crocidura lasiura. The striped field mouse, A. agrarius, (primary reservoir for Hantaan virus, the causative agent of Korean hemorrhagic fever), was the most frequently collected, representing 90.6% of the 1,288 small mammals captured at both sites. Reported herein are the ecological parameters, seasonal population densities, and seasonal population characteristics associated with small mammals collected at two military training sites in the Republic of Korea.

Acute-phase sera from >5 % of cases of haemorrhagic fever with renal syndrome occurring annually in Korea have been found to exhibit a fourfold or higher antibody titre to Puumala virus (PUUV) than to Hantaan virus (HTNV) by double-sandwich IgM ELISA, suggesting the existence of a PUUV-related hantavirus. Based on the phylogenetic relationships among arvicolid rodents, the royal vole (Myodes regulus) was targeted as a likely reservoir host of hantavirus. Using RT-PCR, a genetically distinct hantavirus, designated Muju virus (MUJV), was detected in lung tissue of royal voles, captured in widely separated geographical regions in Korea during 1996–2007. Pairwise analysis of the full-length S (1857 nt) and M (3634 nt) segments of MUJV indicated approximately 77 % sequence similarity with PUUV. At the amino acid level, MUJV differed from PUUV by 5.5–6.9 % (nucleocapsid) and 10.0–11.6 % (Gn and Gc envelope glycoproteins). Interstrain variation of MUJV sequences from royal voles captured in different regions suggested geographic-specific clustering. Neutralizing antibody titres against PUUV were two- to sixfold higher than to HTNV in sera of MUJV-infectedMyodes regulus. Although virus isolation attempts were unsuccessful, the collective data indicate that MUJV is a distinct hantavirus species.

A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], scrub typhus [Orientia tsutsugamushi], murine typhus [Rickettsia typhi], and leptospirosis [Leptospira interrogans]), as part of the U.S. military rodent surveillance and control program, was conducted from 2001 through 2005 at Yongsan Garrison, Seoul, Republic of Korea. Rodents were collected to determine the prevalence of rodent-borne diseases at a U.S. military installation in an urban environment. A total of 1,750 rodents representing three species was collected by using baited live traps (Tomahawk), glue boards, and poison baits (dead rodents observed but not assayed). The Norway rat, Rattus norvegicus (99.8%), accounted for nearly all of the rodents captured/observed. Only three roof rats, Rattus rattus (0.2%), and one house mouse, Mus musculus (0.1%), were collected. R. norvegicus rats were the only rodents that were serologically positive for SEO virus (9.6%), scrub typhus (2.8%), murine typhus (3.8%), and leptospirosis (4.6%). One of six rodents that were positive for SEO virus by immunofluorescent antibody test was positive for SEO virus antigen by reverse transcriptase-polymerase chain reaction. Infection rates for SEO virus, scrub typhus, murine typhus, and leptospirosis varied annually. Rodents were captured from 228 (20.7%) of 1,104 total buildings in Yongsan Garrison. The Yongsan commissary had the highest annual infestation rate (22 rodents per year), followed by Commisky's Club (18 rodents per year). Annual infestation rates were high for food service facilities, which often store perishable food products outdoors for short periods of time, attracting rodent populations; refuse from these facilities provides harborage and food for rodents. The effect of rodent populations outside the boundary of Yongsan Garrison was not determined.

Dopamine (DA) is an oxidant that may contribute to the degeneration of dopaminergic neurons. The present study demonstrates that DA-induced cytotoxicity in human-derived neurotypic cells, SH-SY5Y, is prevented by resveratrol, one of the major antioxidative constituents found in the skin of grapes. SH-SY5Y cells, a neuroblastoma cell line, treated with DA at 300 and 500 microM for 24 h underwent apoptotic death as determined by characteristic morphological features, including nuclear condensation, and loss of mitochondrial membrane potential (MMP). Flow cytometric analysis using Annexin V showed that DA can induce significant and severe apoptosis. Exposure to resveratrol (5 microM) for 1 h prior to the DA treatment attenuated DA-induced cytotoxicity, and rescued the loss of MMP. To investigate the apoptotic signaling pathways relevant to the restoration of DA-induced apoptosis by resveratrol, we carried out quantitative analysis of Bcl-2, caspase-3, and cleaved poly ADP-ribose polymerase (PARP) by immunoblot analysis. Resveratrol pretreatment led to a decrease in cleavage of PARP, an increase in the Bcl-2 protein, and activation of caspase-3. These results suggest that DA may be a potential oxidant of neuronal cells at biologically relevant concentrations. Resveratrol may protect SH-SY5Y cells against this cytotoxicity, reducing intracellular oxidative stress through canonical signal pathways of apoptosis and may be of biological importance in the prevention of a dopaminergic neurodegenerative disorder such as Parkinson disease.

HPV16 late gene transcription from P670 is suppressed in undifferentiated keratinocytes. To identify DNA sites involved in the negative regulation, we examined the effect of a series of substitutions in the P670 promoter region (nucleotide (nt) 106-855) on the transcription, using an expression plasmid having the promoter fragment placed to drive the firefly-luciferase gene. Twenty-base pair-long segments covering the entire promoter region were replaced with a sequence lacking any so far known factor-binding motifs to produce 38 mutants. These plasmids were introduced by transfecton into undifferentiated or partially differentiated human HaCaT and HeLa cells, and transient expression of the reporter was examined with the cell extracts. The reporter expression from the wild-type promoter region was lower, half to one-third, in the undifferentiated cells than in the partially differentiated cells, which expressed hSkn-1a, a keratinocyte specific transcription factor that activates P670, and CCAAT displacement protein (CDP), a transcriptional repressor involved in cell differentiation. Two mutants with substitutions including the putative CDP-binding sites, one from nt 562 to 567 and the other from nt 673 to 678, induced markedly enhanced reporter expression particularly in the partially differentiated cells. Electrophoretic mobility shift analysis demonstrated that bacterially produced GST-CDP bound to the two sites in a sequence-specific manner. The data strongly suggest that CDP acts as a major suppressor for P670 transcription by binding to the promoter region in the undifferentiated cells and even in the partially differentiated cells that express the activator hSkn-1a.

Sequence analysis of the full-length medium segment and the partial small and large segments of a hantavirus, detected by reverse transcription-PCR in lung tissues of the Chinese mole shrew (Anourosorex squamipes) captured in Cao Bang Province, Vietnam, in December 2006, indicated that it is genetically distinct from rodentborne hantaviruses.

Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HawaiiHantaan (HTN) virus, the etiologic agent ofclinically severe hemorrhagic fever with renalsyndrome (HFRS),was firstisolated in 1976 fromlung tissue of a striped-field mouse (Apodemusagrarius) captured in Songnae-ri, GyeonggiProvince, Republic of Korea. Found primarily inmountainous areas, the Korean field mouse (A.peninsulae) is the second-most dominant fieldrodent species found throughout Korea. A newhantavirus, designated Soochong (SOO), wasisolated in Vero E6 cells from four A. peninsulaecaptured in August 1997 at Mt. Gyebang inHongcheon-gun, Mt. Gachil, Inje-gun, GangwonProvince, and in September 1998 at Mt. Deogyu,Muju-gun, Jeollabuk Province. The entire S, M,andLgenomicsegmentsofSOOvirus,amplifiedby RT-PCR from lung tissues of seropositive A.peninsulaeandfromvirus-infectedVeroE6cells,diverged from HTN virus (strain 76–118) by15.6%, 22.8%, and 21.7% at the nucleotide leveland3.5%,9.5%,and4.6%attheaminoacidlevel,respectively. Phylogenetic analyses of thenucleotide and deduced amino acid sequences,using the maximum parsimony and neighbor-joining methods, indicated that SOO virus wasdistinct from A. agrarius-borne HTN virus. SOOvirussharedacommonancestrywithAmurvirusfrom Far East Russia, as well as with H5 and B78hantaviruses, previously isolated from HFRSpatients in China. Cross-focus-reduction neutra-lizating antibody tests showed that SOO virus,whichisthefirsthantavirusisolatedincellculturefrom A. peninsulae, could be classified as a newhantavirus serotype. J. Med. Virol. 78:290–297, 2006.

Phylogenetic analysis of a collection of 103 E1 gene sequences from rubella viruses isolated from 17 countries from 1961 to 2000 confirmed the existence of at least two genotypes. Rubella genotype I (RGI) isolates, predominant in Europe, Japan, and the Western Hemisphere, segregated into discrete subgenotypes; international subgenotypes present in the 1960s and 1970s were replaced by geographically restricted subgenotypes after approximately 1980. Recently, active subgenotypes include one in the United States and Latin America, one in China, and a third that apparently originated in Asia and spread to Europe and North America, starting in 1997, indicating the recent emergence of an international subgenotype. A virus that potentially arose as a recombinant between two RGI subgenotypes was discovered. Rubella genotype II (RGII) showed greater genetic diversity than did RGI and may actually consist of multiple genotypes. RGII viruses were limited to Asia and Europe; RGI viruses were also present in most of the countries where RGII viruses were isolated.

Antiviral drugs are being used for therapeutic purposes against influenza illness in humans. However, antiviral-resistant variants often nullify the effectiveness of antivirals. Combined medications, as seen in the treatment of cancers and other infectious diseases, have been suggested as an option for the control of antiviral-resistant influenza viruses. Here, we evaluated the therapeutic value of combination therapy against oseltamivir-resistant 2009 pandemic influenza H1N1 virus infection in DBA/2 mice. Mice were treated for five days with favipiravir and peramivir starting 4 hours after lethal challenge. Compared with either monotherapy, combination therapy saved more mice from viral lethality and resulted in increased antiviral efficacy in the lungs of infected mice. Furthermore, the synergism between the two antivirals, which was consistent with the survival outcomes of combination therapy, indicated that favipiravir could serve as a critical agent of combination therapy for the control of oseltamivir-resistant strains. Our results provide new insight into the feasibility of favipiravir in combination therapy against oseltamivir-resistant influenza virus infection.

Human parainfluenza viruses (HPIV) are important causes of respiratory tract infections in young children. To characterize the molecular epidemiology of an HPIV outbreak occurring in Korea during 2006, genetic analysis of 269 cell culture isolates from HPIV-infected children, was conducted using nested reverse transcription-PCR (RT-PCR). HPIV-1 was detected in 70.3% of tested samples (189/269). The detection rate of HPIV-2 and HPIV-3 was 1.5% (4/269) and 9.3% (25/269), respectively. Mixed HPIV-1, -2 and -3 infections were detected in 19.0% (51/269): HPIV-1 and HPIV-2 in 15, HPIV-1 and HPIV-3 in 26, HPIV-2 and HPIV-3 in 6, and HPIV-1, -2 and -3 in 4. Of these positive samples for three different types HIPV-1, -2, and -3, two each representative strains were selected, the full length of hemagglutinin-neuraminidase (HN) gene for HPIV was amplified by RT-PCR, and sequenced. Multiple alignment analysis, based on reference sequence of HPIV-1, -2, and -3 strains available in GenBank, showed that the identity of nucleotide and deduced amino acid sequences was 92.4-97.6% and 92.7-97.9%, respectively, for HPIV-1, 88.5-99.8% and 88.6-100% for HPIV-2, and 96.3-99.5% and 95.0-99.3% for HPIV-3, respectively. Phylogenetic analysis showed that HPIV-1, -2, and -3 strains identified in this study were closely related among the strains in the same type with no significant genetic variability. These results show that HPIV of multiple imported sources was circulating in Korea.

Background: The prevalence of antibodies for hepatitis A virus (anti-HAV) in adolescents and young adults has decreased remarkably following the economic growth in Korea. As a result, this age group has a high risk for HAV infection paradoxically, and over 1500 cases of clinically overt hepatitis A occurred in 1998. Human isolates of hepatitis A virus (HAV) are categorized within four genotypes (I, II, III, and VII). In some geographic regions, closely related isolates cluster, suggesting endemic spread of the virus, while in other regions multiple genotypes circulate. Virtually no data are available with regard to the genetic relatedness of Korean strains of HAV. Methods and Results: A 168 base pair segment encompassing the putative VP1/2A junction of the HAV genome was amplified by RT-PCR and sequenced in sera of 18 Korean patients with a sporadic form of acute hepatitis A. Pairwise comparisons of the nucleic acid and amino acid sequences of 18 Korean isolates with one another revealed that the Korean isolates showed > 94.6% and > 96.4% identity, respectively. All of the 18 Korean isolates clustered within genotype IA, irrespective of the geographic locations and the time that hepatitis occurred. Unique amino acid sequence changes that had never been reported in genotype IA were found in nine of the 18 isolates. These changes were Gln→Ser and Lys→Arg in 2A-19 and 2A-10 amino acid positions. Conclusion: The presence of single genotype and unique mutations may be related with the circulation of endemic HAV over a long period of time in Korea.

The nucleocapsid (N) protein of the Hantaan virus (HTNV) is a major viral antigen that induces a strong antibody response during the acute phase of infection. By immunoblot analyses of the recombinant N proteins using human sera of the hemorrhagic fever with renal syndrome (HFRS), we have confirmed previous finding by other investigators of the presence of a highly antigenic region near the amino terminus of the HTNV N protein. We have further located the antigenic region within a short stretch of hydrophilic sequences between the 26 and the 46th amino acid residues. The recombinant glutathione S-transferase fusion proteins containing this region was expressed as a soluble form in a large quantity in Escherichia coli, and purified by a single-step affinity chromatography. The recombinant antigen also showed a similar, but a weaker reactivity with human antisera to Seoul virus (SEOV), the virus most closely related to HTNV.

Reovirus isolates from human, striped-field mouse (Apodemus agrarius) and Korean field mouse (A. peninsulae) in Korea showed extensive variability in the patterns of electrophoretic migration of the double-stranded RNA (dsRNA) genome segments. Hemagglutination inhibition (HI) test was performed for serotype determination of 12 reovirus isolates. To clarify genetic diversity and molecular phylogeny of Korean reoviruses, L1, S3 and S4 genomic segments of reoviruses were amplified by RT-PCR and directly sequenced. Among 12 reovirus strains, 9 strains were type 3 and 3 strains were type 2. The L1 was highly conserved showing 91.5-100%, 94.7-100% similar ities among Korean isolates, and 77.5-97.9%, 92.6-96.8% similarities compared to other reference strains of each genotypes at nucleotide and amino acid levels, respectively. In S3 and S4 segments, 84.4-99.3%, 72.3-99% nucleotide sequence similarities and 92-99.3%, 89.1-98.4% amino acid sequence similarities among Korean isolates were observed, and 70.8-93.9%, 72.3-98.7% nucleotide sequence similarities and 81.8-100%, 88.3-97.7% amino acid sequence similarities compared with other reference strains of each genotype were observed, respectively. Phylogenetic analyses based on the S3 and S4 nucleotide sequences indicate that genotypes of reovirus are more related with geographic differences rather than host species or date of isolation.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissues of striped field mice (Apodemus agrarius) captured in Songnae-ri, Kyungki-do, Korea. To clarify the genetic diversity and phylogenetic relationship among Korean strains of HTN virus, viral sequences of the partial S and M segments were amplified from lung tissues of 24 seropositive striped field mice captured between 1989 and 1998 at 11 sites in South Korea. The 771-nucleotide (nt) S segment sequences (coordinates 432 to 1202) of HTN virus strains from Yangju-kun differed by 10 to 40 nt (1.3 to 5.2%) from virus strains from Pocheon-kun, Songnae-ri and Nonsan-kun. Similar degrees of genetic variation were found in the G1 and G2 glycoprotein-encoding M segment. Phylogenetic trees, based on the partial S and M segments and generated by the maximum parsimony and neighbor-joining methods, demonstrated that virus strains from various geographic regions in South Korea showed a tendency to form two phylogenetic subgroups and were evolutionarily distinct from HTN virus strains from the People’s Republic of China.

We determined the influence of aluminum on dendritic transport, using an in vitro system of dissociated mouse hippocampal neurons. Newly synthesized RNA from dissociated mouse hippocampal neurons was more slowly transported into dendrites in the presence of aluminum chloride when compared to those without the addition of aluminum chloride to the culture medium. Suppression of dendritic transport of newly synthesized RNA may be responsible for the dendritic degeneration observed in aluminum neurotoxicity, eventually leading to neuronal degeneration.

Axin is a multifunctional protein that participates in many cellular events including Wnt signaling and cell fate determination. Aurora kinase inhibitor (AKI)-induced cell death and cell membrane rupture is facilitated in L929 cells expressing axin (L-axin cells) through the activation of poly ADP-ribose polymerase (PARP). We observed that caspase-2 activity is required for AKI-induced cell death. Inhibition of caspase-2 activity suppressed AKI-induced PARP activation and mitochondrial dysfunction, resulting in a decrease in AKI-induced cell death. When an axin mutant deleted for the glycogen synthase kinase 3β (GSK3β)-binding domain was expressed in L929 cells (L-ΔGSK cells), AKI-induced caspase-2 activation and cell death decreased. AKI treatment reduced the expression of a 32-kDa caspase-2 splicing variant (caspase-2S) in most L-axin cells, but not in L-ΔGSK cells. These results suggest that AKI-induced caspase-2 activation in L-axin cells might be due to a decrease in the expression of caspase-2S, which inhibits caspase-2 activity. In addition, AKI treatment failed to activate caspase-8 and treatment with necrostatin inhibited AKI-induced cell death in L-axin cells, suggesting that the absence of caspase-8 activation might favor necrotic cell death. Axin expression may facilitate AKI-induced caspase-2 activation followed by activation of PARP and initiation of the necrotic cell death pathway.

Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signalling and participates in various cellular events, including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6 h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of the G-protein signalling (RGS) domain were involved in the axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that viral replication enhancement might be mediated by the axin RGS domain.

Background Laboratory diagnosis of hemorrhagic fever with renal syndrome (HFRS), an infectious disease caused by rodent-borne hantaviruses in Asia and Europe, depends primarily on serological methods. Since the advent of such serodiagnostic tests, few reports are available about the clinical and molecular epidemiological features of HFRS. Objectives To investigate the epidemioclinical features of HFRS patients treated at a tertiary-care teaching hospital in Seoul over a 10-year period. Study design Medical records of HFRS patients, admitted to a tertiary-care teaching hospital during February 2002 to February 2012, were reviewed. Sera from patients were tested for Hantaan virus (HTNV) and Seoul virus (SEOV) RNA using RT-PCR. Results Among 35 HFRS patients (mean age was 44.2 ± 14.7 years), 29 were male (82.9%). Acute renal failure developed in 27 patients (77.1%), and 12 patients (34.3%) were admitted to the intensive care unit (ICU). Conjunctival injection (OR 10.32, 95% CI 1.09–97.77, P = .04) and initial serum albumin less than 3 g/dL (OR 22.83, 95% CI 1.45–359.93, P = .03) were risk factors for ICU admission. Of 35 acute-phase sera, 11 (31.4%) were positive for HTNV RNA. None were positive for SEOV RNA. Conclusions HFRS was characterized by the clinical triad of fever, renal insufficiency and gastrointestinal symptoms. Conjunctival injection and serum albumin level were related to severity. A large-scale multi-center study is needed to enhance insights into epidemioclinical characteristics of HFRS in Korea.

High prevalence of human T-cell lymphotropic virus type I (HTLV-I) infection and disease has been identified among Iranian-born Mashhadi Jews, an ethnically segregated, highly inbred population. To determine the origin and genetic diversity of HTLV-I in this group, 1,039 bp spanning selected regions of the HTLV-I gag, pol, env and pX genes were enzymatically amplified and sequenced directly from DNA of five Mashhadi Jews (three with spastic myelopathy and two asymptomatic carriers). Alignment and comparison of these sequences with cosmopolitan and Australo-Melanesian topotypes of HTLV-I indicated that the HTLV-I strains from Mashhadi Jews, which were > or = 99.9% identical among themselves, exhibited considerable sequence similarity (> or = 99%) to HTLV-I strains from southern India, suggesting a common source of infection. Phylogenetic analysis, using the maximum parsimony method, was consistent with a single-source introduction of HTLV-I into the Mashhadi Jewish community.

Sequence variants of human T cell lymphotropic virus type I (HTLV-I), genetically distinct from cosmopolitan strains of HTLV-I from Japan, the Americas, the Caribbean, and Africa, have been discovered among Melanesians in Papua New Guinea and the Solomon Islands and among Australian aboriginals. By using oligonucleotide primer pairs derived from sequences unique to the gp46- and gp21-encoding regions of the env gene of the Melanesian HTLV-I variants, HTLV-I strains from widely separated geographic regions could be grouped into either of two major geographic-specific genotypes or topotypes: Australo-Melanesian and cosmopolitan. These primers did not permit amplification of the corresponding env gene regions in strains of simian T cell lymphotropic virus type I from Asia and Africa. Phylogenetic analysis also supported two distinct lineages, consistent with evolution of HTLV-I in Australia and Melanesia independent from that in other parts of the world.

Type C retroviruses, designated simian T-cell lymphotropic virus type I (STLV-I), have been isolated from several genera of Old World monkeys and apes, but not from New World monkeys and prosimians. To determine the genomic diversity and molecular evolution of STLV-I and to clarify their genetic relationship to human T-cell lymphotropic virus type I (HTLV-I), we enzymatically amplified, then directly sequenced selected regions of the gag, pal, env, and pX genes of STLV-I strains from Asia and Africa, STLV-I strains Si-2, Matsu, and JM86 from Japanese macaques, which exhibited sequence similarities ranging from 98.5 to 99.8% among themselves, diverged by 12.9 to 13.3% from STLV-I strain MM39-83 from a naturally infected rhesus macaque, by 9.7 to 11.2% from STLV-I strains from Africa, and by 8.8 to 11.2% from HTLV-I strains originating in Japan, India, Africa, the Caribbean, the Americas, Polynesia, and Melanesia. By contrast, the interspecies nucleotide sequence similarity among African STLV-I strains from green monkey, yellow baboon, sooty mangabey, and common chimpanzee was remarkably high, ranging from 96.9 to 97.4%, and these STLV-I strains diverged by only 2.2 to 2.8% from HTLV-I strain EL from equatorial Zaire. Phylogenetic trees constructed by using the neighbor-joining and maximum parsimony methods indicated that the Asian STLV-I strains diverged from the common ancestral virus prior to African STLV-I and cosmopolitan and Melanesian HTLV-I strains. Thus, our data are consistent with an archaic presence of STLV-I in Asia, probably predating macaque speciation, with subsequent independent virus evolution in Asia and Africa.

Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-β and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.

Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1-expressing cells (L-Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2-expressing cells (L-Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub-G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L-Axin cells induced poly(ADP-ribose) polymerase (PARP) activation, which increased the poly(ADP-ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI-induced cell death. Also, AKI treatment of L-Axin cells induced mitochondrial apoptosis-inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase-3 activation. Knockdown of AIF attenuated AKI-induced cell death in L-Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition-induced cell death in a PARP- and AIF-dependent manner. J. Cell. Biochem. 112: 2392–2402, 2011. © 2011 Wiley-Liss, Inc.

Carbapenem resistance in Acinetobacter baumannii is increasing these days. We investigated the roles of outer membrane proteins and efflux pumps in carbapenem resistance of A. baumannii which showed no carbapenemase activity in modified Hodge test. Among 58 carbapenem-resistant isolates collected from the Korea University Medical Center between January 2002 and March 2006, 17 isolates showed negative results in modified Hodge test. In outer membrane protein analysis, loss of the 29-kDa protein band was related with higher imipenem minimum inhibitory concentrations especially in the presence of OXA-51–like enzymes. Efflux pump–mediated carbapenem resistance was found in one out of the 17 isolates (5.9%). All of the 58 carbapenem-resistant strains and 5 of the 10 carbapenem-susceptible strains had OXA-51–like carbapenemase genes, suggesting that OXA-51–like enzymes may be naturally existing in A. baumannii and have very weak carbapenem hydrolyzing activity.

Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew ( Suncus murinus ). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

Background Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control. Results In the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins Conclusion Our results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.