Kumiko Yoshimatsu

Outbreak of Leptospirosis after Flood, the Philippines, 2009

After a typhoon in September 2009, an outbreak of leptospirosis occurred in Metro Manila, the Philippines; 471 patients were hospitalized and 51 (10.8%) died. A hospital-based investigation found risk factors associated with fatal infection to be older age, hemoptysis, anuria, jaundice, and delayed treatment with antimicrobial drugs.

Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies

SummaryTo investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2±2.1 to 21.5±2.8 days) being significantly shorter than that of the control group (25.8±1.8, p<0.01, Mann-Whitney,U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the antivirus antibody.

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

SummaryRecombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.

Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

ABSTRACT A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

Pathogenicity of Hantaan Virus in Newborn Mice: Genetic Reassortant Study Demonstrating that a Single Amino Acid Change in Glycoprotein G1 Is Related to Virulence

ABSTRACT Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.

Extensive Host Sharing of Central European Tula Virus

ABSTRACT To examine the host association of Tula virus (TULV), a hantavirus present in large parts of Europe, we investigated a total of 791 rodents representing 469 Microtus arvalis and 322 Microtus agrestis animals from northeast, northwest, and southeast Germany, including geographical regions with sympatric occurrence of both vole species, for the presence of TULV infections. Based on serological investigation, reverse transcriptase PCR, and subsequent sequence analysis of partial small (S) and medium (M) segments, we herein show that TULV is carried not only by its commonly known host M. arvalis but also frequently by M. agrestis in different regions of Germany for a prolonged time period. At one trapping site, TULV was exclusively detected in M. agrestis, suggesting an isolated transmission cycle in this rodent reservoir separate from spillover infections of TULV-carrying M. arvalis. Phylogenetic analysis of the S and M segment sequences demonstrated geographical clustering of the TULV sequences irrespective of the host, M. arvalis or M. agrestis. The novel TULV lineages from northeast, northwest, and southeast Germany described here are clearly separated from each other and from other German, European, or Asian lineages, suggesting their stable geographical localization and fast sequence evolution. In conclusion, these results demonstrate that TULV represents a promiscuous hantavirus with a large panel of susceptible hosts. In addition, this may suggest an alternative evolution mode, other than a strict coevolution, for this virus in its Microtus hosts, which should be proven in further large-scale investigations on sympatric Microtus hosts.

In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus

Summary. Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED50) of 2500 μg/ml, while pretreatment with LF was highly efficacious having an ED50 of 39 μg/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED50: 10 μg/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 105 foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED50 of 312.5 μg/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF.

Mouse Mx2 protein inhibits hantavirus but not influenza virus replication.

Summary. The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.

Bla NDM-1-positive Klebsiella pneumoniae from environment, Vietnam

To the Editor: The blaNDM-1 gene, which produces the New Delhi metallo-β-lactamase (NDM-1) enzyme, confers resistance to the carbapenem class of antimicrobial drugs and can be transferred among different types of bacteria. NDM-1 was identified in 2008 in Sweden from a patient from India who had been hospitalized in New Delhi (1). Since that report, blaNDM-1–positive bacteria have been identified from patients in several countries; most of these patients had a direct link with the Indian subcontinent (2). The spread of blaNDM-1 among bacterial pathogens is of concern not only because of resistance to carbapenems but also because such pathogens typically are resistant to multiple antimicrobial drug classes, which leaves few treatment choices available (3–5). In 2011, spread of blaNDM-1–positive bacteria in an environmental setting in New Delhi was reported (6). The possible appearance of bacteria harboring blaNDM-1 in Vietnam is of concern because cultural and economic links between Vietnam and India are strongly established, including extensive person-to-person exchanges that could enable easy exchange of pathogens. In addition, Vietnam faces a serious problem of antimicrobial drug resistance because drugs are freely available and used in an indiscriminate fashion. Thus, once blaNDM-1–positive bacteria colonize persons in Vietnam, they would be able to spread easily and pose a serious public health threat. During September 2011, we collected paired swab samples (1 for PCR, 1 for culture) of seepage water from 20 sites (rivers, lakes, and water pools in streets) within a 10-km radius of central Hanoi, Vietnam. Samples were transported in Transystem (COPAN Italia S.p.A, Brescia, Italy) to preserve bacteria and DNA. The 20 PCR swab specimens were squeezed out into 0.5-mL volumes of sterile water and centrifuged at 3,000 × g for 30 seconds; 1 μL of the resulting suspension was then used as PCR template to detect blaNDM-1 as described (7). Two samples were positive for blaNDM-1; these 2 samples were collected from the same river (Kim Nguu River) but at sites 3 km apart. To isolate and identify the phenotype and genotype of blaNDM-1–positive bacteria, we repeatedly spread the 20 culture swab specimens onto Muller-Hinton agar (Nissui, Tokyo, Japan) containing 100 mg/L vancomycin (Nakalai, Kyoto, Japan) plus 0.5 mg/L meropenem (LKT Laboratories, St. Paul, MN, USA) until single colonies were obtained. Each colony was then subcultured by plating onto MacConkey agar (Nihon Seiyaku, Tokyo, Japan) containing 0.5 mg/L meropenem to ensure culture purity; colonies were identified by using API 20E strips (bioMerieux, Basingstoke, UK). MICs of these isolates for 13 antimicrobial drugs were calculated by using Etest (bioMerieux), and susceptibility data were interpreted by using Clinical and Laboratory Standards Institute guidelines (www.clsi.org). We harvested several species of bacteria from the 2 seepage samples positive for blaNDM-1: Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. fluorescens/putida, and P. luteola. These isolates were placed onto media containing 0.5 mg/L meropenem, and bacterial DNA was extracted and used for the template for PCR analysis to detect blaNDM-1 as described (7). blaNDM-1 was detected in 3 K. pneumoniae isolates from each of the 2 positive samples (6 isolates total); this result was confirmed by sequencing. All 6 isolates were highly resistant to all β-lactam antimicrobial drugs, including carbapenems (Table). To detect another β-lactamase, multiplex PCRs were carried out as described (8); genetic variants blaTEM, blaSHV, blaOXA, blaCTX-M, blaIMP, blaVIM, and blaKPC were not detected in any of the isolates other than K. pneumoniae. All 6 K. pneumoniae isolates were positive for blaTEM and blaCTX-M variants by PCR; these variants were confirmed as blaTEM-1 and blaCTX-M-3 by sequencing. Table Resistance to 13 antimicrobial drugs of blaNDM-1–positive Klebsiella pneumoniae isolates from the Kim Nguu River, Hanoi, Vietnam* Aminoglycosides are often used in the management of severe infectious diseases caused by gram-negative pathogens. 16S rRNA methylases were found to confer high levels of resistance to aminoglycosides such as amikacin, tobramycin, and gentamicin. The 6 K. pneumoniae isolates we found were highly resistant to gentamicin (MIC >1,024 mg/L) and tobramycin (MIC 256–>1,024 mg/L) (Table). Therefore, we screened genetic elements of 16S rRNA methylases (rmtB, rmtC, and armA) by PCR and detected rmtB in all 6 isolates (9). Multilocus sequence typing was applied for these 6 isolates; all were identified as K. pneumoniae sequence type 283 (10), which had not been reported as harboring blaNDM-1. The azide-resistant Escherichia coli strain J53 has been used as recipient for conjugation assay, which had been reported previously (6), but we found no transconjugant strain with blaNDM-1 on MacConkey agar containing 100 mg/L sodium azide and 0.5 mg/L meropenem. Our results show that blaNDM-1–positive K. pneumoniae sequence type 283 is present in the Kim Nguu River, which flows through the central part of Hanoi at 2 sites. The isolates we obtained were also positive for 2 other β-lactamases, blaTEM-1 and blaCTX-M-3, were highly resistant to aminoglycosides related to rmtB, and showed mild elevation of MIC against ciprofloxacin up to 1.5 mg/L. Wide-scale surveillance of environmental and clinical samples in Vietnam and establishment of a strategy to prevent further spread of blaNDM-1 are urgently needed.

Development of Serological Assays for Thottapalayam Virus, an Insectivore-Borne Hantavirus

ABSTRACT Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a “spillover” from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and polyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.