Takaaki Koma

H5N1 Avian Influenza Virus Induces Apoptotic Cell Death in Mammalian Airway Epithelial Cells

ABSTRACT In recent years, the highly pathogenic avian influenza virus H5N1 has raised serious worldwide concern about an influenza pandemic; however, the biology of H5N1 pathogenesis is largely unknown. To elucidate the mechanism of H5N1 pathogenesis, we prepared primary airway epithelial cells from alveolar tissues from 1-year-old pigs and measured the growth kinetics of three avian H5 influenza viruses (A/Crow/Kyoto/53/2004 [H5N1], A/Duck/Hong Kong/342/78 [H5N2], and A/Duck/Hong Kong/820/80 [H5N3]), the resultant cytopathicity, and possible associated mechanisms. H5N1, but not the other H5 viruses, strongly induced cell death in porcine alveolar epithelial cells (pAEpC), although all three viruses induced similar degrees of cytopathicity in chicken embryonic fibroblasts. Intracellular viral growth and the production of progeny viruses were comparable in pAEpC infected with each H5 virus. In contrast, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were detected only in H5N1-infected pAEpC, and the activities of caspases 3, 8, and 9 were significantly elevated in pAEpC infected with H5N1, but not with H5N2 and H5N3. These results suggest that only H5N1 induces apoptosis in pAEpC. H5N1 cytopathicity was inhibited by adding the caspase inhibitor z-VAD-FMK; however, there were no significant differences in viral growth or release of progeny viruses. Further investigations using reverse genetics demonstrated that H5N1 hemagglutinin protein plays a critical role in inducing caspase-dependent apoptosis in infected pAEpC. H5N1-specific cytopathicity was also observed in human primary airway epithelial cells. Taken together, these data suggest that avian H5N1 influenza virus leads to substantial cell death in mammalian airway epithelial cells due to the induction of apoptosis.

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.

Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

Truncated Hantavirus Nucleocapsid Proteins for Serotyping Sin Nombre, Andes, and Laguna Negra Hantavirus Infections in Humans and Rodents

ABSTRACT Sin Nombre virus (SNV), Andes virus (ANDV), and Laguna Negra virus (LANV) have been known as the dominant causative agents of hantavirus pulmonary syndrome (HPS). ANDV and LANV, with different patterns of pathogenicity, exist in a sympatric relationship. Moreover, there is documented evidence of person-to-person transmission of ANDV. Therefore, it is important in clinical medicine and epidemiology to know the serotype of a hantavirus causing infection. Truncated SNV, ANDV, and LANV recombinant nucleocapsid proteins (trNs) missing 99 N-terminal amino acids (trN100) were expressed using a baculovirus system, and their applicability for serotyping SNV, ANDV, and LANV infection by the use of enzyme-linked immunosorbent assays (ELISA) was examined. HPS patient sera and natural-reservoir rodent sera infected with SNV, ANDV, and LANV showed the highest optical density (OD) values for homologous trN100 antigens. Since even patient sera with lower IgM and IgG antibody titers were serotyped, the trN100s are therefore considered useful for serotyping with early-acute-phase sera. In contrast, assays testing whole recombinant nucleocapsid protein antigens of SNV, ANDV, and LANV expressed in Escherichia coli detected homologous and heterologous antibodies equally. These results indicated that a screening ELISA using an E. coli-expressed antigen followed by a serotyping ELISA using trN100s is useful for epidemiological surveillance in regions where two or more hantavirus species cocirculate.

Neutrophil Depletion Suppresses Pulmonary Vascular Hyperpermeability and Occurrence of Pulmonary Edema Caused by Hantavirus Infection in C.B-17 SCID Mice

ABSTRACT Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice. IMPORTANCE Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

Innate Immune Response to Arenaviral Infection: A Focus on the Highly Pathogenic New World Hemorrhagic Arenaviruses

Abstract Arenaviruses are enveloped, negative-stranded RNA viruses that belong to the family Arenaviridae. This diverse family can be further classified into OW (Old World) and NW (New World) arenaviruses based on their antigenicity, phylogeny, and geographical distribution. Many of the NW arenaviruses are highly pathogenic viruses that cause systemic human infections characterized by hemorrhagic fever and/or neurological manifestations, constituting public health problems in their endemic regions. NW arenavirus infection induces a variety of host innate immune responses, which could contribute to the viral pathogenesis and/or influence the final outcome of virus infection in vitro and in vivo. On the other hand, NW arenaviruses have also developed several strategies to counteract the host innate immune response. We will review current knowledge regarding the interplay between the host innate immune response and NW arenavirus infection in vitro and in vivo, with emphasis on viral-encoded proteins and their effect on the type I interferon response.

Highly pathogenic New World and Old World human arenaviruses induce distinct interferon responses in human cells

ABSTRACT The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, a proinflammatory response, or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Junin virus (JUNV), induced an IFN response in human A549 cells. Here, we report that Machupo virus (MACV), another highly pathogenic NW arenavirus, also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited an IFN response in human primary dendritic cells and A549 cells. Coinfection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 h after MACV infection, while the replication levels of MACV and LASV were not affected by virus coinfection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with the NW arenaviruses JUNV and MACV are quite different from responses to infections with the OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis. IMPORTANCE Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or proinflammatory cytokine production. In contrast, infections with the highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We here demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses, in spite of higher replication levels, and blocked the MACV-triggered IFN response in a coinfection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with the NW arenaviruses JUNV and MACV and to infection with the OW arenavirus LASV and provides important insights into the virus-host interaction and pathogenesis.

Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.

Serological evidence of infection with rodent-borne hepatitis E virus HEV-C1 or antigenically related virus in humans

Zoonotic potential of a rat-derived hepatitis E virus (HEV), designated as HEV-C1, remains unknown. To evaluate the risk for HEV-C1 infection in humans, paired sera of 208 hospitalized febrile patients collected from 2001 to 2003 in Hanoi, Vietnam, were examined for IgG antibodies to HEV-C1 and genotype 1 HEV (HEV-1), which is common in humans. IgG antibodies to virus-like particles (VLPs) of HEV-C1 and/or HEV-1 were detected from 99 of the 208 convalescent sera in enzyme-linked immunosorbent assay (ELISA). IgG antibody titers to HEV-C1 antigen in 3 of the 99 sera were more than 8-fold higher than those to HEV-1 antigen. IgM antibodies to HEV-C1 antigen were detected in acute sera from 2 of the 3 patients in ELISA and Western blotting. However, no HEV genome was detected. Clinical information was available for 1 of the 2 patients. Hepatic enzymes, aspartate aminotransferase and alanine aminotransferase, were mildly elevated (156 IU/l and 68 IU/l, respectively), and hepatomegaly was detected by ultrasonography. The patient recovered from the illness after 17 days. These results indicated that HEV-C1 or its variants infect humans in Vietnam and may cause acute febrile illness with mild liver dysfunction.

Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats.

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.