Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (Hantavax™) in healthy adults.

BACKGROUND: Hemorrhagic fever with renal syndrome is a serious health problem in Eurasian countries, including Korea and China. This study evaluated the long-term immunogenicity and safety of formalin-inactivated Hantaan virus vaccine (Hantavax™). METHODS: A phase III, multi-center clinical trial was undertaken to evaluate the immunogenicity and safety of Hantavax™ (three-dose schedule at 0, 1, and 13 months) among healthy adults. Immune response was assessed using the plaque reduction neutralizing antibody test (PRNT) and immunofluorescent antibody assay (IFA). Antibody levels were measured pre-vaccination and at 2, 13, 14, 25, 37, and 49 months after the initial vaccination. Systemic and local adverse events were assessed. RESULTS: A total of 226 healthy subjects aged 19-75 years were enrolled. Following two primary doses of Hantavax™, the seroconversion rate was 90.14% by IFA, but it was only 23.24% by PRNT50. With booster administration, seropositive rates were 87.32% and 45.07% at one month post-vaccination according to IFA and PRNT50, respectively. In young adults (19-39 years), the seropositive rate according to PRNT50 reached about 60% after booster vaccination. The mean duration of seropositive response was 735 days for PRNT50 and 845 days for IFA. Solicited local and systemic adverse events occurred in 47.79% and 25.22% of study subjects, respectively, and most were grade 1. CONCLUSION: Hantavax™ showed a booster effect and immunogenicity lasting two years with a three-dose schedule. The neutralizing antibody response was quite poor with two primary doses, so an early booster vaccination at 2-6 months might be warranted to provide timely protection to high-risk subjects.

Lethal disease in infant and juvenile Syrian hamsters experimentally infected with Imjin virus, a newfound crocidurine shrew-borne hantavirus.

To gain insights into the pathogenicity of Imjin virus (MJNV), a newfound hantavirus isolated from the Ussuri white-toothed shrew (Crocidura lasiura), groups of Syrian hamsters (Mesocricetus auratus) of varying ages (<1, 5, 10, 14, 21, 35 and 56 days) were inoculated by the intraperitoneal route with 1000 pfu of MJNV strains 04-55 and 05-11. MJNV-infected Syrian hamsters, aged 21 days or less, exhibited reduced activity, weight loss, respiratory distress, hind-limb paralysis and seizures. Death ensued 1 to 6 days after onset of clinical disease. MJNV RNA was detected in brain and other major organs by RT-PCR and real time-PCR. Histopathological examination showed alveolar hemorrhage, interstitial pneumonia and severe pulmonary congestion; focal hepatic necrosis and portal inflammation; and acute meningoencephalitis. By immunohistochemistry, MJNV antigen was detected in pulmonary microvascular endothelial cells and glial cells. Older hamsters (35 and 56 days of age) developed subclinical infection without histopathological changes. Future studies are warranted to determine the pathophysiologic bases for the differential age susceptibility of Syrian hamsters to lethal MJNV disease.

Intestinal nematodes from small mammals captured near the demilitarized zone, Gyeonggi province, Republic of Korea.

A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.

Combination effects of peramivir and favipiravir against oseltamivir-resistant 2009 pandemic influenza A(H1N1) infection in mice.

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.

Muju virus, harbored by Myodes regulus in Korea, might represent a genetic variant of Puumala virus, the prototype arvicolid rodent-borne hantavirus.

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.

Whole-genome sequence of muju virus, an arvicolid rodent-borne hantavirus.

The complete genome sequence of Muju virus was determined from lung tissue samples of three royal voles (Myodes regulus) captured in Gangwon province in the Republic of Korea. Since few whole genome sequences of hantaviruses are available, this sequence may help to clarify the molecular phylogeny of arvicolid rodent-borne hantaviruses.

Divergent ancestral lineages of newfound hantaviruses harbored by phylogenetically related crocidurine shrew species in Korea.

Spurred by the recent isolation of a novel hantavirus, named Imjin virus (MJNV), from the Ussuri white-toothed shrew (Crocidura lasiura), targeted trapping was conducted for the phylogenetically related Asian lesser white-toothed shrew (Crocidura shantungensis). Pair-wise alignment and comparison of the S, M and L segments of a newfound hantavirus, designated Jeju virus (JJUV), indicated remarkably low nucleotide and amino acid sequence similarity with MJNV. Phylogenetic analyses, using maximum likelihood and Bayesian methods, showed divergent ancestral lineages for JJUV and MJNV, despite the close phylogenetic relationship of their reservoir soricid hosts. Also, no evidence of host switching was apparent in tanglegrams, generated by TreeMap 2.0ß.

Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro.

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-ß 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.

Ecological surveillance of small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005.

A seasonal rodent-borne disease surveillance program was established at Dagmar North Training Area located near the demilitarized zone, Republic of Korea, from 2001 through 2005. Selected habitats surveyed included earthen banks separating rice paddies, fighting positions along a 5 m rock-faced earthen berm, and extensive tall grasses with various degrees of herbaceous and scrub vegetation associated with dirt roads, rice paddies, ditches, ponds, or the Imjin River. Of the nine species of small mammals captured, the striped field mouse (Apodemus agrarius), the primary reservoir for Hantaan virus, was the most frequently collected, representing 92.5% of the 1,848 small mammals captured. Males were captured similarly to females during the spring and summer seasons but were captured less frequently during the fall and winter seasons. Gravid rates were highest in the fall (25.5-57.3%) with the lowest rates during the summer (0.0-2.2%). Capture rates were the lowest along earthen banks separating rice paddies (5.5%) and highest in unmanaged tall grasses and crawling vegetation (15.3-43.5%). An increased knowledge of ecological factors that impact the abundance and distribution of small mammals and the associated ectoparasites and pathogens they harbor is critical for developing accurate disease risk assessments and mitigation strategies for preventing vector- and rodent-borne diseases among soldiers training in field environments.

Small-molecule probe using dual signals to monitor leucine aminopeptidase activity.

Leucine aminopeptidases (LAPs) are widely distributed in organisms from bacteria to humans, and play crucial roles in cell maintenance and cell growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this Letter, we report a small-molecule probe which exhibits colorimetric and fluorogenic changes according to LAP activity.

Genetic diversity of Imjin virus in the Ussuri white-toothed shrew (Crocidura lasiura) in the Republic of Korea, 2004-2010.

Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues of the Ussuri white-toothed shrew (Crocidura lasiura) captured near the demilitarized zone in the Republic of Korea. To clarify the genetic diversity of MJNV, partial M- and L-segment sequences were amplified from lung tissues of 12 of 37 (32.4%) anti-MJNV IgG antibody-positive Ussuri white-toothed shrews captured between 2004 and 2010. A 531-nucleotide region of the M segment (coordinates 2,255 to 2,785) revealed that the 12 MJNV strains differed by 0-12.2% and 0-2.3% at the nucleotide and amino acid levels, respectively. A similar degree of nucleotide (0.2-11.9%) and amino acid (0-3.8%) difference was found in a 632-nucleotide length of the L segment (coordinates 962 to 1,593) of nine MJNV strains. Phylogenetic analyses, based on the partial M and L segments of MJNV strains generated by the neighbor-joining and maximum likelihood methods, showed geographic-specific clustering, akin to the phylogeography of rodent-borne hantaviruses.

Inhibitory effect of breast milk on infectivity of live oral rotavirus vaccines.

BACKGROUND: Live oral rotavirus vaccines have been less immunogenic and efficacious among children in poor developing countries compared with middle income and industrialized countries for reasons that are not yet completely understood. We assessed whether the neutralizing activity of breast milk could lower the titer of vaccine virus and explain this difference in vitro. METHODS: Breast milk samples were collected from mothers who were breast-feeding infants 4 to 29 weeks of age (ie, vaccine eligible age) in India (N = 40), Vietnam (N = 77), South Korea (N = 34), and the United States (N = 51). We examined breast milk for rotavirus-specific IgA and neutralizing activity against 3 rotavirus vaccine strains-RV1, RV5 G1, and 116E using enzyme immunoassays. The inhibitory effect of breast milk on RV1 was further examined by a plaque reduction assay. FINDINGS: Breast milk from Indian women had the highest IgA and neutralizing titers against all 3 vaccine strains, while lower but comparable median IgA and neutralizing titers were detected in breast milk from Korean and Vietnamese women, and the lowest titers were seen in American women. Neutralizing activity was greatest against the 2 vaccine strains of human origin, RV1 and 116E. This neutralizing activity in one half of the breast milk specimens from Indian women could reduce the effective titer of RV1 by ∼2 logs, of 116E by 1.5 logs, and RV5 G1 strain by ∼1 log more than that of breast milk from American women. INTERPRETATION: The lower immunogenicity and efficacy of rotavirus vaccines in poor developing countries could be explained, in part, by higher titers of IgA and neutralizing activity in breast milk consumed by their infants at the time of immunization that could effectively reduce the potency of the vaccine. Strategies to overcome this negative effect, such as delaying breast-feeding at the time of immunization, should be evaluated.

Serological surveillance of scrub typhus, murine typhus, and leptospirosis in small mammals captured at firing points 10 and 60, Gyeonggi province, Republic of Korea, 2001-2005.

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.

Seasonal and environmental determinants of leptospirosis and scrub typhus in small mammals captured at a U.S. military training site (Dagmar North), Republic of Korea, 2001-2004.

OBJECTIVE: This study sought to identify seasonal and environmental determinants of scrub typhus, murine typhus, and leptospirosis in small mammals trapped at Dagmar North training area, Gyeonggi Province, South Korea. METHODS: Small mammals received titer assays to the aforementioned diseases. Logistic regression analyses were conducted to determine whether associations existed between risk of small-mammal infection and independent variables such as season of capture, habitat, small-mammal species, and sex. RESULTS: Murine typhus was not detected among the animals assayed. Risk of scrub typhus infection was associated with season, habitat, and small-mammal species. Risk of leptospirosis infection was associated with season and habitat. CONCLUSIONS: These findings indicate determinants of infection exist for scrub typhus and leptospirosis at this training site. This information can be used for developing appropriate preventive medicine plans and coordinating troop activity during periods of reduced exposure decreasing the likelihood of disease transmission to humans.

Hemorrhagic fever with renal syndrome in 4 US soldiers, South Korea, 2005.

Four US soldiers acquired hemorrhagic fever with renal syndrome while training near the Demilitarized Zone, South Korea, in 2005. Hantaan virus sequences were amplified by reverse transcription-PCR from patient serum samples and from lung tissues of striped field mice (Apodemus agrarius) captured at training sites. Epidemiologic investigations specified the ecology of possible sites of patient infection.

Echinostome infections in the striped-field mouse, Apodemus agrarius, and the Ussuri white-toothed shrew, Crocidura lasiura, caught near the demilitarized zone, Gyeonggi-do (Province), Republic of Korea.

A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.

Characterization of Imjin virus, a newly isolated hantavirus from the Ussuri white-toothed shrew (Crocidura lasiura).

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.

Ecological surveillance of small mammals at Firing Points 10 and 60, Gyeonggi Province, Republic of Korea, 2001-2005.

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.

Muju virus, a novel hantavirus harboured by the arvicolid rodent Myodes regulus in 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-infected Myodes regulus. Although virus isolation attempts were unsuccessful, the collective data indicate that MUJV is a distinct hantavirus species.

Seroepidemiological survey of rodents collected at a U.S. military installation, Yongsan Garrison, Seoul, Republic of Korea.

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.