Masahiro Kajihara

Characterization of H5N1 highly pathogenic avian influenza virus strains isolated from migratory waterfowl in Mongolia on the way back from the southern Asia to their northern territory

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.

Characterization of the envelope glycoprotein of a novel filovirus, lloviu virus.

ABSTRACT Lloviu virus (LLOV), a novel filovirus detected in bats, is phylogenetically distinct from viruses in the genera Ebolavirus and Marburgvirus in the family Filoviridae. While filoviruses are known to cause severe hemorrhagic fever in humans and/or nonhuman primates, LLOV is biologically uncharacterized, since infectious LLOV has never been isolated. To examine the properties of LLOV, we characterized its envelope glycoprotein (GP), which likely plays a key role in viral tropism and pathogenicity. We first found that LLOV GP principally has the same primary structure as the other filovirus GPs. Similar to the other filoviruses, virus-like particles (VLPs) produced by transient expression of LLOV GP, matrix protein, and nucleoprotein in 293T cells had densely arrayed GP spikes on a filamentous particle. Mouse antiserum to LLOV VLP was barely cross-reactive to viruses of the other genera, indicating that LLOV is serologically distinct from the other known filoviruses. For functional study of LLOV GP, we utilized a vesicular stomatitis virus (VSV) pseudotype system and found that LLOV GP requires low endosomal pH and cathepsin L, and that human C-type lectins act as attachment factors for LLOV entry into cells. Interestingly, LLOV GP-pseudotyped VSV infected particular bat cell lines more efficiently than viruses bearing other filovirus GPs. These results suggest that LLOV GP mediates cellular entry in a manner similar to that of the other filoviruses while showing preferential tropism for some bat cells.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus

ABSTRACT Until the recent emergence of two human-pathogenic tick-borne phleboviruses (TBPVs) (severe fever with thrombocytopenia syndrome virus [SFTSV] and Heartland virus), TBPVs have been neglected as causative agents of human disease. In particular, no studies have addressed the global distribution of TBPVs, and consequently, our understanding of the mechanism(s) underlying their evolution and emergence remains poor. In order to provide a useful tool for the ecological and epidemiological study of TBPVs, we have established a simple system that can detect all known TBPVs, based on conventional reverse transcription-PCR (RT-PCR) with degenerate primer sets targeting conserved regions of the viral L genome segment. Using this system, we have determined that several viruses that had been isolated from ticks decades ago but had not been taxonomically identified are novel TBPVs. Full-genome sequencing of these viruses revealed a novel fourth TBPV cluster distinct from the three known TBPV clusters (i.e., the SFTS, Bhanja, and Uukuniemi groups) and from the mosquito/sandfly-borne phleboviruses. Furthermore, by using tick samples collected in Zambia, we confirmed that our system had enough sensitivity to detect a new TBPV in a single tick homogenate. This virus, tentatively designated Shibuyunji virus after the region of tick collection, grouped into a novel fourth TBPV cluster. These results indicate that our system can be used as a first-line screening approach for TBPVs and that this kind of work will undoubtedly lead to the discovery of additional novel tick viruses and will expand our knowledge of the evolution and epidemiology of TBPVs. IMPORTANCE Tick-borne phleboviruses (TBPVs) have been largely neglected until the recent emergence of two virulent viruses, severe fever with thrombocytopenia syndrome virus and Heartland virus. Little is known about the global distribution of TBPVs or how these viruses evolved and emerged. A major hurdle to study the distribution of TBPVs is the lack of tools to detect these genetically divergent phleboviruses. In order to address this issue, we have developed a simple, rapid, and cheap RT-PCR system that can detect all known TBPVs and which led to the identification of several novel phleboviruses from previously uncharacterized tick-associated virus isolates. Our system can detect virus in a single tick sample and novel TBPVs that are genetically distinct from any of the known TBPVs. These results indicate that our system will be a useful tool for the surveillance of TBPVs and will facilitate understanding of the ecology of TBPVs.

Inhibition of Marburg Virus Budding by Nonneutralizing Antibodies to the Envelope Glycoprotein

ABSTRACT The envelope glycoprotein (GP) of Marburg virus (MARV) and Ebola virus (EBOV) is responsible for virus entry into host cells and is known as the only target of neutralizing antibodies. While knowledge about EBOV-neutralizing antibodies and the mechanism for the neutralization of infectivity is being accumulated gradually, little is known about antibodies that can efficiently regulate MARV infectivity. Here we show that MARV GP-specific monoclonal antibodies AGP127-8 (IgG1) and MGP72-17 (IgM), which do not inhibit the GP-mediated entry of MARV into host cells, drastically reduced the budding and release of progeny viruses from infected cells. These antibodies similarly inhibited the formation of virus-like particles (VLPs) consisting of GP, the viral matrix protein, and nucleoprotein, whereas the Fab fragment of AGP127-8 showed no inhibitory effect. Morphological analyses revealed that filamentous VLPs were bunched on the surface of VLP-producing cells cultured in the presence of the antibodies. These results demonstrate a novel mechanism of the antibody-mediated inhibition of MARV budding, in which antibodies arrest unformed virus particles on the cell surface. Our data lead to the idea that such antibodies, like classical neutralizing antibodies, contribute to protective immunity against MARV and that the “classical” neutralizing activity is not the only indicator of a protective antibody that may be available for prophylactic and therapeutic use.

Ebola and Marburg virus diseases in Africa: Increased risk of outbreaks in previously unaffected areas?

Filoviral hemorrhagic fever (FHF) is caused by ebolaviruses and marburgviruses, which both belong to the family Filoviridae. Egyptian fruit bats (Rousettus aegyptiacus) are the most likely natural reservoir for marburgviruses and entry into caves and mines that they stay in has often been associated with outbreaks of MVD. On the other hand, the natural reservoir for ebola viruses remains elusive; however, handling of wild animal carcasses has been associated with some outbreaks of EVD. In the last two decades, there has been an increase in the incidence of FHF outbreaks in Africa, some being caused by a newly found virus and some occurring in previously unaffected areas such as Guinea, Liberia and Sierra Leone, in which the most recent EVD outbreak occurred in 2014. Indeed, the predicted geographic distribution of filoviruses and their potential reservoirs in Africa includes many countries in which FHF has not been reported. To minimize the risk of virus dissemination in previously unaffected areas, there is a need for increased investment in health infrastructure in African countries, policies to facilitate collaboration between health authorities from different countries, implementation of outbreak control measures by relevant multi‐disciplinary teams and education of the populations at risk.

Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus

ABSTRACT Multiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections. IMPORTANCE Filoviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

Background: It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication. Results: A correlation between viral replication/transcription activities and nuclear/cytoplasmic shuttling of Hsp70 was observed. Conclusion: Hsp70 modulates the influenza A virus polymerase activity. Significance: This study, for the first time, suggests that Hsp70 may actually assist in influenza A virus replication. The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein

Filoviruses (viruses in the genus Ebolavirus and Marburgvirus in the family Filoviridae) cause severe haemorrhagic fever in humans and nonhuman primates. Rapid, highly sensitive, and reliable filovirus-specific assays are required for diagnostics and outbreak control. Characterisation of antigenic sites in viral proteins can aid in the development of viral antigen detection assays such immunochromatography-based rapid diagnosis. We generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of Ebola virus belonging to the species Zaire ebolavirus. The mAbs were divided into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. Using synthetic peptides corresponding to the Ebola virus NP sequence, the mAb binding sites were mapped to seven antigenic regions in the C-terminal half of the NP, including two highly conserved regions among all five Ebolavirus species currently known. Furthermore, we successfully produced species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. Our data provide useful information for the development of Ebola virus antigen detection assays.

The PB2, PA, HA, NP, and NS genes of a highly pathogenic avian influenza virus A/whooper swan/Mongolia/3/2005 (H5N1) are responsible for pathogenicity in ducks

BackgroundWild ducks are the natural hosts of influenza A viruses. Duck influenza, therefore, has been believed inapparent infection with influenza A viruses, including highly pathogenic avian influenza viruses (HPAIVs) in chickens. In fact, ducks experimentally infected with an HPAIV strain, A/Hong Kong/483/1997 (H5N1) (HK483), did not show any clinical signs. Another HPAIV strain, A/whooper swan/Mongolia/3/2005 (H5N1) (MON3) isolated from a dead swan, however, caused neurological dysfunction and death in ducks.MethodTo understand the mechanism whereby MON3 shows high pathogenicity in ducks, HK483, MON3, and twenty-four reassortants generated between these two H5N1 viruses were compared for their pathogenicity in domestic ducks.ResultsNone of the ducks infected with MON3-based single-gene reassortants bearing the PB2, NP, or NS gene segment of HK483 died, and HK483-based single-gene reassortants bearing PB2, NP, or NS genes of MON3 were not pathogenic in ducks, suggesting that multiple gene segments contribute to the pathogenicity of MON3 in ducks. All the ducks infected with the reassortant bearing PB2, PA, HA, NP, and NS gene segments of MON3 died within five days post-inoculation, as did those infected with MON3. Each of the viruses was assessed for replication in ducks three days post-inoculation. MON3 and multi-gene reassortants pathogenic in ducks were recovered from all of the tissues examined and replicated with high titers in the brains and lungs.ConclusionThe present results indicate that multigenic factors are responsible for efficient replication of MON3 in ducks. In particular, virus growth in the brain might correlate with neurological dysfunction and the disease severity.