Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 mRNA Production as Revealed by a Growth-Adaptive Mutation.

We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.


Humanized mice generated by intra-bone marrow injection of CD133-positive hematopoietic stem cells: application to HIV-1 research


Editorial: HIV/SIV basic research update


Major target for UV-induced complete loss of HIV-1 infectivity: A model study of single-stranded RNA enveloped viruses


A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families.

Members of the order Bunyavirales infect a wide variety of host species, including plants, animals and humans, and pose a threat to public health. Major families in this order have tri-segmented negative-sense RNA genomes, the 5' and 3' ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral polymerases recognize the promoter to initiate RNA synthesis is important for understanding viral replication and pathogenesis, and developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. By using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5' and 3' ends of the genome that form a specific complementary strand. Among tri-segmented bunyaviruses, members of the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure differing from that of other families. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses, and the large genome architecture might confer pathogenic advantages. The promoter list provided in this report is useful for predicting the virus family-specific replication mechanisms of bunyaviruses.


Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.


The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process.

HIV-1 Vif plays an essential role in viral replication by antagonizing anti-viral cellular restriction factors, a family of APOBEC3 proteins. We have previously shown that naturally-occurring single-nucleotide mutations in the SA1D2prox region, which surrounds the splicing acceptor 1 and splicing donor 2 sites of the HIV-1 genome, dramatically alter the Vif expression level, resulting in variants with low or excessive Vif expression. In this study, we investigated how these HIV-1 variants with poor replication ability adapt and evolve under the pressure of APOBEC3 proteins. Adapted clones obtained through adaptation experiments exhibited an altered replication ability and Vif expression level compared to each parental clone. While various mutations were present throughout the viral genome, all replication-competent adapted clones with altered Vif expression levels were found to bear them within SA1D2prox, without exception. Indeed, the mutations identified within SA1D2prox were responsible for changes in the Vif expression levels and altered the splicing pattern. Moreover, for samples collected from HIV-1-infected patients, we showed that the nucleotide sequences of SA1D2prox can be chronologically changed and concomitantly affect the Vif expression levels. Taken together, these results demonstrated the importance of the SA1D2prox nucleotide sequence for modulating the Vif expression level during HIV-1 replication and adaptation.


Species-specific valid ternary interactions of HIV-1 Env-gp120, CD4, and CCR5 as revealed by an adaptive single-amino acid substitution at the V3 loop tip.

Understanding molecular bases for viral entry into cells leads to the elucidation of one of major viral survival strategies, and thus to the development of new effective antiviral measures. As experimentally shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas its biological significance remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding would certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.


Immunological Responses to Seoul Orthohantavirus in Experimentally and Naturally Infected Brown Rats ( Rattus norvegicus)

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.


Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode.

for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.


Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity.

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.


The Glycoprotein of the Live-Attenuated Junin Virus Vaccine Strain Induces Endoplasmic Reticulum Stress and Forms Aggregates prior to Degradation in the Lysosome.

The development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in regions of endemicity within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.


Expression Level of HIV-1 Vif Can Be Fluctuated by Natural Nucleotide Variations in the vif-Coding and Regulatory SA1D2prox Sequences of the Proviral Genome.


Role for Gag-CA Interdomain Linker in Primate Lentiviral Replication.

-modulator to optimize the Gag-related viral replication process. We also have noted, during the course of conducting the research project, that HIV-1 and SIVmac, belonging to distinct primate lentiviral lineages, share a similarly biologically active linker region with each other. In this brief article, we summarize and report the results obtained by mutational studies that are relevant to the functional significance of the interdomain linker of HIV/SIV Gag-CA. Based on this investigation, we discuss about the future directions of the research in this line.


Allosteric regulation of HIV-1 capsid structure for Gag assembly, virion production, and viral infectivity by a disordered interdomain linker.

HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named interdomain linker. While multiple regions in the NTD and CTD domains are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this report, we show by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.


Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains.

adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.


CXCR4- and CCR5-Tropic HIV-1 Clones Are Both Tractable to Grow in Rhesus Macaques.

genetic manipulations and viral cell-adaptations, we have successfully generated a series of HIV-1 derivatives with CXCR4-tropism or CCR5-tropism that grow in macaque cells to various degrees. Of these viruses, those with best replicative potentials can grow comparably with a pathogenic SIVmac in macaque cells by counteracting major restriction factors TRIM5, APOBEC3, and tetherin proteins. In this study, rhesus macaques were challenged with CXCR4-tropic (MN4/LSDQgtu) or CCR5-tropic (gtu + A4CI1) virus. The two viruses were found to productively infect rhesus macaques, being rhesus macaque-tropic HIV-1 (HIV-1rmt). However, plasma viral RNA was reduced to be an undetectable level in infected macaques at 5-6 weeks post-infection and thereafter. While replicated similarly well in rhesus peripheral blood mononuclear cells, MN4/LSDQgtu grew much better than gtu + A4CI1 in the animals. To the best of our knowledge, this is the first report demonstrating that HIV-1 derivatives (variants) grow in rhesus macaques. These viruses certainly constitute firm bases for generating HIV-1rmt clones pathogenic for rhesus monkeys, albeit they grow more poorly than pathogenic SIVmac and SHIV clones reported to date.


Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses.

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.


Zika virus infection elicits auto-antibodies to C1q.

Zika virus (ZIKV) causes mostly asymptomatic infection or mild febrile illness. However, with an increasing number of patients, various clinical features such as microcephaly, Guillain-Barré syndrome and thrombocytopenia have also been reported. To determine which host factors are related to pathogenesis, the E protein of ZIKV was analyzed with the Informational Spectrum Method, which identifies common information encoded by primary structures of the virus and the respective host protein. The data showed that the ZIKV E protein and the complement component C1q cross-spectra are characterized by a single dominant peak at the frequency F=0.338, suggesting similar biological properties. Indeed, C1q-specific antibodies were detected in sera obtained from mice and monkeys infected with ZIKV. As C1q has been known to be involved not only in immunity, but also in synaptic organization and different autoimmune diseases, a ZIKV-induced anti-C1q antibody response may contribute to the neurological complications. These findings might also be exploited for the design of safe and efficacious vaccines in the future.


Virological characterization of HIV-1 CA-NTD mutants constructed in a virus-lineage reflected manner.

Capsid (CA) protein is a major virion-constituent of all retroviruses including human immunodeficiency virus type 1 (HIV-1), and is essential for early and late phases in viral replication cycle through interaction with numerous cellular factors. In particular, N-terminal domain (NTD) of HIV-1 CA has been frequently and well reported to bind to various host cell proteins that considerably affect viral replication potential. In this study, in order to better define biological bases of the CA-NTD for HIV-1 replication, we performed an extensive mutational analysis in an unprecedented manner. By aligning CA-NTD sequences derived from representative infectious molecular clones of HIV-1, HIV-2, and simian immunodeficiency virus isolated from the rhesus macaque (SIVmac), a number of amino acids specific to HIV-1 were selected, and were replaced with those from SIVmac at the corresponding sites. Mutant viruses thus generated were then examined for multi-cycle infectivity, single-cycle infectivity, and ability to produce progeny virions. While some CA-NTD mutations affected viral replication ability to varying degrees, those in helix 7 abolished viral growth potential without exception. These results highlight functional importance of non-conserved amino acids in helix 7, and give new insights into functionality of HIV-1 CA-NTD. J. Med. Invest. 65:110-115, February, 2018.

The Journal of Medical Investigation : JMI


mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region.


Merimepodib, an IMPDH inhibitor, suppresses replication of Zika virus and other emerging viral pathogens.

Zika virus (ZIKV), a member of the Flaviviridae family, has recently been linked to abnormal pregnancies, fetal death, microcephaly, and Guillain-Barré syndrome in humans. Merimepodib (MMPD, VX-497), a potent inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), has shown antiviral activity against HCV and a variety of DNA and RNA viruses in vitro. In this report, we expand the antiviral spectrum of MMPD, and demonstrate that MMPD inhibits ZIKV RNA replication with an EC

of 0.6 μM. Furthermore, MMPD reduces the virus production of ZIKV as well as several other important emerging viral pathogens such as Ebola, Lassa, Chikungunya, and Junin viruses. The inhibition can be reversed by addition of exogenous guanosine to culture media, consistent with the mechanism of action of MMPD as an IMPDH inhibitor. We also provide evidence that MMPD can be used in combination with other antivirals such as ribavirin and T-705 (favipiravir) to enhance suppression of virus production.


Complete Genome Sequences of Human Immunodeficiency Type 1 Viruses Genetically Engineered To Be Tropic for Rhesus Macaques.

We have constructed two human immunodeficiency type 1 (HIV-1) derivatives, CXCR4 tropic and CCR5 tropic, that replicate in rhesus macaques. They are genetically engineered to be resistant to macaque restriction factors against HIV-1, including TRIM5, APOBEC3, and tetherin proteins. The two HIV-1 variants described here are fundamental clones aiming for rhesus infection studies of HIV-1.


Highly pathogenic New World arenavirus infection activates the pattern recognition receptor PKR without attenuating virus replication in human cells.

The arenavirus family consists of several highly pathogenic viruses including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junín virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. NW JUNV and MACV infections readily induce an IFN response in human cells, while OW LASV infection usually triggers an undetectable or weak IFN response. JUNV induces IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs during infection and activates IFN response. Double-stranded RNA activated Protein Kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Herein we report that infection with NW arenavirus JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of translation initiation factor eIF2. Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells, but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired, but slightly augmented in wt cells compared to in PKR deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV or MACV-infected, PKR deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors including RIG-I and MDA5 is critical to the initiation of innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger IFN response in a RIG-I dependent manner. Herein, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor during infection. Interestingly, the replication of JUNV and MACV was not restricted, but rather slightly augmented in the presence of PKR. Our data provide new evidences for distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provide insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.


Novel In Vitro Screening System Based on Differential Scanning Fluorimetry to Search for Small Molecules against the Disassembly or Assembly of HIV-1 Capsid Protein.

Varieties of in vitro systems have been used to study biochemical properties of human immunodeficiency virus Gag-capsid protein (HIV Gag-CA). Recently, we have comparatively characterized HIV-1 and HIV-2 Gag-CA proteins using such technology, and have demonstrated that the NaCl-initiated CA-polymerization in vitro and the stability of CA N-terminal domain as judged by differential scanning fluorimetry (DSF) are inversely correlated. In this study, we found that ZnCl2 works as a competent initiator of the in vitro HIV-1 CA-polymerization at much lower concentrations than those of NaCl frequently used for the polymerization initiation. We also showed by DSF assays that ZnCl2 highly destabilize HIV-1 CA. Furthermore, PF74, a well-known inducer of premature HIV-1 uncoating in infected cells, was demonstrated to unusually promote the HIV-1 CA-disassembly in the presence of ZnCl2 as revealed by DSF assays. Taken together, we conclude that the DSF method may be useful as an efficient monitoring system to screen anti-HIV-1 CA molecules.


Comparison of Biochemical Properties of HIV-1 and HIV-2 Capsid Proteins.

Timely disassembly of viral core composed of self-assembled capsid (CA) in infected host cells is crucial for retroviral replication. Extensive in vitro studies to date on the self-assembly/disassembly mechanism of human immunodeficiency virus type 1 (HIV-1) CA have revealed its core structure and amino acid residues essential for CA-CA intermolecular interaction. However, little is known about in vitro properties of HIV-2 CA. In this study, we comparatively analyzed the polymerization properties of bacterially expressed HIV-1 and HIV-2 CA proteins. Interestingly, a much higher concentration of NaCl was required for HIV-2 CA to self-assemble than that for HIV-1 CA, but once the polymerization started, the reaction proceeded more rapidly than that observed for HIV-1 CA. Analysis of a chimeric protein revealed that N-terminal domain (NTD) is responsible for this unique property of HIV-2 CA. To further study the molecular basis for different in vitro properties of HIV-1 and HIV-2 CA proteins, we determined thermal stabilities of HIV-1 and HIV-2 CA NTD proteins at several NaCl concentrations by fluorescent-based thermal shift assays. Experimental data obtained showed that HIV-2 CA NTD was structurally more stable than HIV-1 CA NTD. Taken together, our results imply that distinct in vitro polymerization abilities of the two CA proteins are related to their structural instability/stability, which is one of the decisive factors for viral replication potential. In addition, our assay system described here may be potentially useful for searching for anti-CA antivirals against HIV-1 and HIV-2.


Mouse Model of Neurological Complications Resulting from Encephalitic Alphavirus Infection.

Long-term neurological complications, termed sequelae, can result from viral encephalitis, which are not well understood. In human survivors, alphavirus encephalitis can cause severe neurobehavioral changes, in the most extreme cases, a schizophrenic-like syndrome. In the present study, we aimed to adapt an animal model of alphavirus infection survival to study the development of these long-term neurological complications. Upon low-dose infection of wild-type C57B/6 mice, asymptomatic and symptomatic groups were established and compared to mock-infected mice to measure general health and baseline neurological function, including the acoustic startle response and prepulse inhibition paradigm. Prepulse inhibition is a robust operational measure of sensorimotor gating, a fundamental form of information processing. Deficits in prepulse inhibition manifest as the inability to filter out extraneous sensory stimuli. Sensory gating is disrupted in schizophrenia and other mental disorders, as well as neurodegenerative diseases. Symptomatic mice developed deficits in prepulse inhibition that lasted through 6 months post infection; these deficits were absent in asymptomatic or mock-infected groups. Accompanying prepulse inhibition deficits, symptomatic animals exhibited thalamus damage as visualized with H&E staining, as well as increased GFAP expression in the posterior complex of the thalamus and dentate gyrus of the hippocampus. These histological changes and increased GFAP expression were absent in the asymptomatic and mock-infected animals, indicating that glial scarring could have contributed to the prepulse inhibition phenotype observed in the symptomatic animals. This model provides a tool to test mechanisms of and treatments for the neurological sequelae of viral encephalitis and begins to delineate potential explanations for the development of such sequelae post infection.


Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.

Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.

Frontiers in Cellular and Infection Microbiology


Appearance of renal hemorrhage in adult mice after inoculation of patient-derived hantavirus.

Hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infection is characterized by fever, renal dysfunction and hemorrhage. An animal model mimicking symptoms of HFRS remains to be established. In this study, we evaluated the pathogenicity of an HFRS patient-derived Hantaan virus (HTNV) in adult mice. Five clones of HTNV strain KHF 83-61 BL (KHFV) that was derived from blood of an HFRS patient were obtained by plaque cloning. The pathogenicity of the virus clones was evaluated by using 6-week-old female BALB/c mice. Sequence analysis of the viral genome was performed by conventional methods. All of the mice intravenously inoculated with KHFV clone (cl)-1, -2, -3 and -5 showed signs of disease such as transient body weight loss, ruffled fur, reduced activity and remarkably prominent hemorrhage in the renal medulla at 6 to 9 days post-inoculation (dpi) and then recovered. In contrast, mice intravenously inoculated with KHFV cl-4 did not show any signs of disease. We selected KHFV cl-5 and cl-4 as representative of high-pathogenic and low-pathogenic clones, respectively. Quantities of viral RNA in kidneys of KHFV cl-5-infected mice were larger than those in KHFV cl-4-infected mice at any time point examined (3, 6, 9 and 12 dpi). The quantities of viral RNA of KHFV cl-5 and cl-4 peaked at 3 dpi, which was before the onset of disease. Sequence analysis revealed that the amino acid at position 417 in the glycoprotein Gn was the sole difference in viral proteins between KHFV cl-5 and cl-4. The result suggests that amino acid at position 417 in Gn is related to the difference in pathogenicity between KHFV cl-5 and cl-4. When the inoculum of KHFV cl-5 was pretreated with a neutralizing antibody against HTNV strain 76-118, which belongs to the same serotype as KHFV clones, mice did not show any signs of disease, confirming that the disease was caused by KHFV infection. We found that an HFRS patient-derived HTNV caused renal hemorrhage in adult mice. We anticipate that this infection model will be a valuable tool for understanding the pathogenesis of HFRS.


The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-/ Receptor.

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.


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.

The Journal of Veterinary Medical Science


Animal Model of Sensorineural Hearing Loss Associated with Lassa Virus Infection.

Approximately one-third of Lassa virus (LASV)-infected patients develop sensorineural hearing loss (SNHL) in the late stages of acute disease or in early convalescence. With 500,000 annual cases of Lassa fever (LF), LASV is a major cause of hearing loss in regions of West Africa where LF is endemic. To date, no animal models exist that depict the human pathology of LF with associated hearing loss. Here, we aimed to develop an animal model to study LASV-induced hearing loss using human isolates from a 2012 Sierra Leone outbreak. We have recently established a murine model for LF that closely mimics many features of human disease. In this model, LASV isolated from a lethal human case was highly virulent, while the virus isolated from a nonlethal case elicited mostly mild disease with moderate mortality. More importantly, both viruses were able to induce SNHL in surviving animals. However, utilization of the nonlethal, human LASV isolate allowed us to consistently produce large numbers of survivors with hearing loss. Surviving mice developed permanent hearing loss associated with mild damage to the cochlear hair cells and, strikingly, significant degeneration of the spiral ganglion cells of the auditory nerve. Therefore, the pathological changes in the inner ear of the mice with SNHL supported the phenotypic loss of hearing and provided further insights into the mechanistic cause of LF-associated hearing loss. Sensorineural hearing loss is a major complication for LF survivors. The development of a small-animal model of LASV infection that replicates hearing loss and the clinical and pathological features of LF will significantly increase knowledge of pathogenesis and vaccine studies. In addition, such a model will permit detailed characterization of the hearing loss mechanism and allow for the development of appropriate diagnostic approaches and medical care for LF patients with hearing impairment.


Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic.

Machupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that a MACV strain with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. Others and our group have identified the glycoprotein to be the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication and its ability to attenuate MACV in vivo. Recombinant MACV with the Candid#1 glycoprotein (rMACV/Cd#1-GPC) exhibited growth properties similar to those of Candid#1 and was genetically stable in vitro. In a mouse model of lethal infection, rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV strain expressing the glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model. Currently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrate for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.


Highly Pathogenic New World and Old World Human Arenaviruses Induce Distinct Interferon Responses in Human Cells.

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.


Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses.

These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.


Neutrophil depletion suppresses pulmonary vascular hyperpermeability and occurrence of pulmonary edema caused by hantavirus infection in C.B-17 SCID mice.

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.


Distinct genetic characteristics of Sri Lankan Rattus and Bandicota (Murinae, Rodentia) inferred from mitochondrial and nuclear markers.

We examined genetic variation in black rats (the Rattus rattus complex) from Kandy District, Sri Lanka using mitochondrial cytochrome b (cytb, 1140 bp) and nuclear melanocortin 1 receptor (Mc1r, 954 bp) gene sequences together with database sequences. We confirmed the existence of two divergent mitochondrial lineages in Sri Lankan black rats, with genetic distance of 2.2% and estimated divergence time of 0.3 million years ago. Because one lineage is unique to the island and the other is closely related to R. rattus populations on the Indian subcontinent, two migration events of R. rattus from the subcontinent are inferred, one ancient and one recent. Mc1r analyses revealed 12 haplotypes among the Sri Lankan black rats. A median-joining network together with other available sequences separated the 12 haplotypes into two groups, one unique to the island and the other related to previously reported R. rattus sequences. Notably, most individuals possessed various combinations of both haplotype groups which had no association with the cytb clades. These results imply that old and new R. rattus lineages are now intermingled as a result of hybridization in Sri Lanka. Specimens of the lesser bandicoot rat (Bandicota bengalensis) collected from Sri Lanka (n = 24) were shown to have no genetic variability in the cytb sequence. Our results indicate that the two most abundant groups of commensal rats in Sri Lanka, black rats and lesser bandicoot rats, are the product of contrasting evolutionary histories on different timescales.


Role of nucleocapsid protein of hantaviruses in intracellular traffic of viral glycoproteins.

To understand the role of nucleocapsid protein (NP) of hantaviruses in viral assembly, the effect of NP on intracellular traffic of viral glycoproteins Gn and Gc was investigated. Double staining of viral and host proteins in Hantaan virus (HTNV)-infected Vero E6 cells showed that Gn and Gc were localized to cis-Golgi, in which virus particles are thought to be formed. When HTNV Gn and Gc were expressed by a plasmid encoding glycoprotein precursor (GPC), which is posttranslationally cleaved into Gn and Gc, Gn was localized to cis-Golgi, whereas Gc showed diffuse distribution in the cytoplasm in 32.9% of Gc-positive cells. The ratio of the diffused Gc-positive cells was significantly decreased to 15.0% by co-expression of HTNV NP. Co-expression of HTNV GPC with NPs of other hantaviruses, such as Seoul virus, Puumala virus and Sin Nombre virus, also reduced the ratios of diffused Gc-positive cells to 13.5%, 25.2%, and 11.6%, respectively. Among amino- and carboxyl-terminally truncated HTNV NPs, NP75-429, NP116-429, NP1-333, NP1-233, and NP1-155 possessed activity to reduce the ratio of diffused Gc-positive cells, while NP155-429 and NP1-116 did not. NP30-429 has partial activity. These results indicate that amino acid region 116-155 of NP is important for the activity, although amino acid region 1-30 is partially related. Truncation of the HTNV Gc cytoplasmic tail caused an increase in diffused Gc-positive cells. In addition, the effect of coexpression of HTNV NP was weakened. These results suggest that HTNV NP has a role to promote Golgi localization of Gc through a mechanism possibly mediated by the Gc cytoplasmic tail.


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.


Epidemiology of hantavirus infection in Thousand Islands regency of Jakarta, Indonesia.

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.

The Journal of Veterinary Medical Science


A survey of rodent-borne pathogens carried by wild Rattus spp. in Northern Vietnam.

To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern.


bla(NDM-1)-positive Klebsiella pneumoniae from environment, Vietnam.


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.


Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection.

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.


Studies on hantavirus infection in small mammals captured in southern and central highland area of Vietnam.

To investigate the distribution of hantaviruses among animals in Southern and Central Highland area of Vietnam, a total of 1311 serum samples were obtained from rats and Asian house shrews (Suncus murinus) captured at 11 locations between 2006 and 2009. A total of 1066 serum samples from rats were examined for IgG antibodies against Hantaan virus, and there were 30 antibody-positive serum samples from rats that had been captured mainly in a port area and urban area in Ho Chi Minh City (HCMC) (2.8%). All of the antibody-positive rats were Rattus norvegicus, and they had Seoul virus (SEOV) genome in their lungs. SEOV sequences detected from rats captured in Southern Vietnam belonged to the same lineage as those from rats captured at Haiphong Port and a market area in Hanoi City. SEOV strain CSG5 was isolated from a rat captured at Saigon Harbor. Strain CSG5 showed a cross-neutralization pattern almost the same as that of a representative strain of SEOV. A total of 245 Asian house shrews were captured in the Central Highland area and near HCMC. Sera were examined for IgG antibodies against Thottapalayam virus (TPMV), and 32 (13.1%) of the antibody-positive shrews were mainly from the Central Highland area and showed a neutralizing antibody against TPMV. These results indicated that SEOV is distributed among R. norvegicus inhabiting harbor and urban areas of Southern Vietnam and that TPMV or an antigenically related virus is distributed among Asian house shrews in Central Highland area.


The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses.

The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.


Application of truncated nucleocapsid protein (N) for serotyping ELISA of murinae-associated hantavirus infection in rats.

Truncated recombinant nucleocapsid proteins (trNs) that lack N-terminally located cross-reactive epitopes of four Murinae rodent-associated hantaviruses, Seoul virus (SEOV), Thailand virus, Hantaan virus (HTNV) and Dobrava-Belgrade virus, were produced by using a baculovirus expression system. ELISA with the trNs as antigens enabled serotyping of immune sera from rats experimentally inoculated with the corresponding hantaviruses with cut-off OD values of 60% of those of whole N of HTNV. The trN-based ELISA could serotype 12 out of 13 sera obtained from wild rodents (Rattus norvegicus) naturally infected with SEOV using the 60% cut-off value. These results indicate that screening with whole N followed by serotyping with trNs using a cut-off OD value of 60% of that of whole N is a useful method for serological surveillance of Murinae-associated hantavirus infection among rodents.


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.

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.


Different cross-reactivity of human and rodent sera to Tula virus and Puumala virus.

Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans.

Comparative Immunology, Microbiology and Infectious Diseases


Molecular epidemiological and serological studies of hantavirus infection in northern Vietnam.

The distribution of anti-hantavirus antibodies in humans and rodents in northern Vietnam was examined. In total, 837 serum samples from healthy humans (617) and patients with fever (220), living in six different areas were screened for IgG antibodies against Hantaan or Seoul virus (SEOV) by ELISA, IFA, and Western blot analysis. Antibody-positive sera were identified in 7/617 (1.1%) healthy donors, 5/150 port workers in the port of Hai Phong, and 2/185 residents of Ha Nam Province. In comparison, positive sera were detected in 5/220 (2.3%) fever patients in the provinces of Ha Nam (1/58) and Thanh Hoa (4/146). Antibody-positive Rattus norvegicus were found in the provinces of Ha Nam (7/52) and Thanh Hoa (1/67), in Haibatrung District (7/43) in Hanoi, and in Hai Phong Port (21/62), while antibody-positive R. rattus (2/17) were found in Hai Phong Port. Part of the Gc region from the viral genome was amplified by RT-PCR using lung tissue samples from R. norvegicus in Haibatrung (2/7) and Hai Phong Port (7/9), but not from R. rattus (0/2). Viral sequences were located in the SEOV clade and formed a single lineage with Indonesian SEOV, suggesting that Vietnamese SEOV is part of a distinct lineage among Asian SEOVs.

The Journal of Veterinary Medical Science


Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody.

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.

Biochemical and Biophysical Research Communications


H5N1 avian influenza virus induces apoptotic cell death in mammalian airway epithelial cells.

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.


新世界ハンタウイルス感染症の血清型鑑別診断のための組換え核蛋白を用いた新規ELISA法の開発


A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus.

Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype.