Hiroshi Ushirogawa

Novel antiviral activity of neuraminidase inhibitors against an avian influenza a virus

BackgroundNeuraminidase (NA) inhibitors used for influenza therapy are believed to prevent the release of progeny virus from the surface of an infected cell. In this study, we found that NA inhibitors have a novel antiviral function against an avian influenza virus.ResultsMadin-Darby canine kidney cells, commonly used for the isolation and propagation of the influenza virus, were infected with an avian influenza viral strain A/chicken/German/N/49(H10N7) (H10/chicken) or a human influenza viral strain A/Osaka/981/98(H3N2) (H3/Osaka) virus. Cells were incubated in a medium without or with a NA inhibitor, oseltamivir carboxylate (GS4071), from 1 to 13 h post infection (p.i.). Infected cells were washed 12 h p.i. to remove GS4071, incubated for 1 h without GS4071, and assayed for virus production. Incubation with GS4071 decreased the production of infectious viruses. When H10/chicken virus-infected cells were incubated with GS4071 from 12 to 13 h p.i. (i.e., 1 h before the virus production assay), the inhibitory effect was clearly observed, however, the same was not evident for H3/Osaka virus-infected cells. Furthermore, viral protein synthesis in infected cells was not affected by GS4071. Using a scanning electron microscope, many single spherical buds were observed on the surface of H3/Osaka virus-infected cells incubated without GS4071, whereas many aggregated particles were observed on the surface of cells incubated with GS4071. However, many long tubular virus-like structures, with no aggregated particles, were observed on the surface of H10/chicken virus-infected cells incubated with GS4071. The same results were obtained when another NA inhibitor, zanamivir, was used.ConclusionsThese results indicate that NA inhibitors interfered with virus particle formation in the H10/chicken virus-infected cells, in which the inhibitor caused the formation of long tubular virus-like structures instead of spherical virus particles.

The pandemic (H1N1) 2009 influenza virus is resistant to mannose-binding lectin

BackgroundMannose-binding lectin (MBL) is an important component of innate immunity because it promotes bacterial clearance and neutralization of human influenza A viruses. Since a majority of humans have no neutralizing antibody against the pandemic (H1N1) 2009 influenza (pandemic 2009) virus, innate immunity may be crucial and MBL susceptibility may therefore influence viral pathogenesis.ResultsWe examined MBL susceptibility of influenza A viruses and observed that the pandemic 2009 virus was resistant to MBL, whereas all seasonal influenza A viruses tested were susceptible. The mortality of mice infected with a seasonal H1N1 influenza virus was evidently enhanced on transient blockage of MBL activity by simultaneous inoculation of mannan, whereas mannan inoculation had no effect on mice infected with a pandemic 2009 virus. This indicates that MBL protects mice against infection with the seasonal virus but not against that with the pandemic 2009 virus.ConclusionsThese results indicate that the pandemic 2009 virus is not susceptible to MBL, an important component of innate immunity.

Re-emergence of H3N2 strains carrying potential neutralizing mutations at the N-linked glycosylation site at the hemagglutinin head, post the 2009 H1N1 pandemic

BackgroundSeasonally prevalent H1N1 and H3N2 influenza A viruses have evolved by antigenic drift; this evolution has resulted in the acquisition of asparagine (N)-linked glycosylation sites (NGSs) in the globular head of hemagglutinin (HA), thereby affecting the antigenic and receptor-binding properties, as well as virulence. An epidemiological survey indicated that although the traditional seasonal H1N1 strain had disappeared, H3N2 became predominant again in the seasons (2010–11 and 2011–12) immediately following the H1N1 pandemic of 2009. Interestingly, although the 2009 pandemic H1N1 strain (H1N1pdm09) lacks additional NGSs, clinically isolated H3N2 strains obtained during these seasons gained N (Asn) residues at positions 45 and 144 of HA that forms additional NGSs.MethodsTo investigate whether these NGSs are associated with re-emergence of H3N2 within the subtype, we tested the effect of amino acid substitutions on neutralizing activity by using the antisera raised against H3N2 strains with or without additional NGSs. Furthermore, because the N residue at position 144 of HA was identified as the site of mismatch between the vaccine and epidemic strains of 2011–2012, we generated mutant viruses by reverse genetics and tested the functional importance of this particular NGS for antibody-mediated neutralization by intranasal inoculation of mice.ResultsThe results indicated that amino acid substitution at residue 144 significantly affected neutralization activity, acting as an escape mutation.ConclusionsOur data suggest that the newly acquired NGSs in the HA globular head may play an important role in the re-emergence of endemic seasonal H3N2 strain by aiding the escape from humoral immunity.

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain

ABSTRACT Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production. IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Massaliatrema misgurni n. sp.(Trematoda: Heterophyidae) whose metacercariae encyst in loaches (Misgurnus anguillicaudatus)

Hitherto unknown metacercariae were found encysted in loaches (Misgurnus anguillicaudatus) from China. They were experimentally fed to golden hamsters, and gravid adults were recovered 1 week post-infection from their small intestines. A new species, Massaliatrema misgurni n. sp. (Heterophyidae), is described from the adults. This new species is different from M. gyrinicola Dollfus and Timon-David, 1960, in having a smaller acetabulum/oral sucker ratio, less branched vitellaria widely entering the intercecal anteroacetabular area and an almost median seminal receptacle; and from M. yamashitai Kamiya and Ohbayashi, 1975, in having a larger acetabulum/oral sucker ratio, with the seminal vesicle situated in the uterine loop and the vitellaria entering the intercecal anteroacetabular area. This report is the first record of M. anguillicaudatus as a second intermediate host of the genus Massaliatrema Dollfus and Timon-David, 1960.

The CC chemokine ligand (CCL) 1, upregulated by the viral transactivator Tax, can be downregulated by minocycline: possible implications for long-term treatment of HTLV-1-associated myelopathy/tropical spastic paraparesis

BackgroundChemokine (C-C motif) ligand 1 (CCL1) is produced by activated monocytes/ macrophages and T-lymphocytes, and acts as a potent attractant for Th2 cells and a subset of T-regulatory (Treg) cells. Previous reports have indicated that CCL1 is overexpressed in adult T-cell leukemia cells, mediating an autocrine anti-apoptotic loop. Because CCL1 is also known as a potent chemoattractant that plays a major role in inflammatory processes, we investigated the role of CCL1 in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP).ResultsThe results showed that: (1) CCL1 was preferentially expressed in HAM/TSP-derived HTLV-1-infected T-cell lines, (2) CCL1 expression was induced along with Tax expression in the Tax-inducible T-cell line JPX9, (3) transient Tax expression in an HTLV-1-negative T-cell line activated the CCL1 gene promoter, (4) plasma levels of CCL1 were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with multiple sclerosis and HTLV-1-infected asymptomatic healthy carriers, and (5) minocycline inhibited the production of CCL1 in HTLV-1-infected T-cell lines.ConclusionsThe present results suggest that elevated CCL1 levels may be associated with the pathogenesis of HAM/TSP. Although further studies are required to determine the in vivo significance, minocycline may be considered as a potential candidate for the long-term treatment of HAM/TSP via its anti-inflammatory effects, which includes the inhibition of CCL1 expression.

Establishment of the complete life cycle of Spirometra (Cestoda: Diphyllobothriidae) in the laboratory using a newly isolated triploid clone

Methods to maintain the life cycle of pathogenic organisms become powerful tools for studying molecular and cellular bases of infectious diseases. Spirometra erinaceieuropaei is a parasitic tapeworm that causes sparganosis in humans. Because S. erinaceieuropaei has a complex life cycle with different stages and host species requirements, there have been no reports to establish the complete life cycle in the laboratory. In this study, using Cyclops as the first intermediate host, mouse as the experimental second intermediate host, and dog as the final host, we succeeded in maintaining S. erinaceieuropaei in the laboratory. By repeating the established life cycle five times, we obtained a clonal population of S. erinaceieuropaei from a single adult worm. A karyotype study showed that the chromosome of this clone is triploid (3n=27), indicating that a genetically uniform strain is established by apomictic reproduction. The strain was named Kawasaki triploid (Kt). A partial sequence of mitochondrial cytochrome c oxidase subunit 1 gene of the strain Kt showed more than 98% similarity with those of S. erinaceieuropaei isolates from Australia, China, and South Korea, and the resultant phylogeny indicated that the strain Kt is a member of a distinctive clade from East Asia and Oceania. Our system will be particularly useful for studies of S. erinaceieuropaei infection and human sparganosis.