Research Articles Abstracts - January 10, 2009 Issue

[In this post: (1) Research Articles Abstracts.

Contents:

(1.1) Pathological lesions and viral localization of Influenza A (H5N1) virus in experimentally infected Chinese rhesus macaques: implications for pathogenesis and viral transmission.
(1.2) Intracellular localization of influenza C virus NS2 protein (NEP) in infected cells and its incorporation into virions.
(1.3) A Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for the Confirmation of Respiratory Syncytial Virus and Influenza Viruses Detected by Antigen Immunoassay Testing. (1.4) Induction of proinflammatory cytokines in primary human macrophages by influenza A virus (H5N1) is selectively regulated by IFN regulatory factor 3 and p38 MAPK.
(1.5) Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.
(1.6) Plasmacytoid dendritic cells are dispensable during primary influenza virus infection.
(1.7) Respiratory syncytial virus persistence in the lungs correlates with airway hyperreactivity in the mouse model.
(1.8) The MAPK-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.
(1.9) Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections.
(1.10) HLA class I molecules consistently present internal influenza epitopes.
(1.11) Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3.
(1.12) Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using pyrosequencing method.

To see the original abstract please follow this LINK. EDITED.]
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(1.1): Arch Virol. 2009 Jan 8. [Epub ahead of print]

Pathological lesions and viral localization of Influenza A (H5N1) virus in experimentally infected Chinese rhesus macaques: implications for pathogenesis and viral transmission.

Chen Y, Deng W, Jia C, Dai X, Zhu H, Kong Q, Huang L, Liu Y, Ma C, Li J, Xiao C, Liu Y, Wei Q, Qin C. - Department of Pathology, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 5, Panjiayuan, Nanli, Chaoyang District, 100021, Beijing, People's Republic of China.

Chinese rhesus macaques infected with influenza virus A/Tiger/Harbin/01/2002 (H5N1) developed acute interstitial pneumonia with diffuse alveolar damage. The results of virus isolation, reverse transcriptase polymerase chain reaction, immunohistochemistry, and in situ hybridization showed that the lung was the major target organ of the H5N1 virus infection.
No virus was detected in the extrapulmonary organs.
The results of immunohistochemistry and in situ hybridization also showed that pneumocytes and macrophages of the lower airway, not the ciliary epithelium of the trachea and bronchi, were the chief target cells in the lung tissue of the infected Chinese rhesus macaque.
Our data indicate that the Chinese rhesus macaque is suitable as a new primate model for H5N1 virus research, especially for the study of H5N1 virus transmission.
The predilection of the H5N1 virus to infect the lower airway suggests that the failure of the virus to attach to the ciliary epithelium of the trachea and bronchi may be a limiting factor in human-to-human transmissibility of the H5N1 virus.

PMID: 19130169 [PubMed - as supplied by publisher]
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(1.2): Arch Virol. 2009 Jan 8. [Epub ahead of print]

Intracellular localization of influenza C virus NS2 protein (NEP) in infected cells and its incorporation into virions.

Kohno Y, Muraki Y, Matsuzaki Y, Takashita E, Sugawara K, Hongo S. - Department of Bacteriology, Faculty of Medicine, Yamagata University, Iida-Nishi, Yamagata, 990-9585, Japan.

RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2.
The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP).
In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions.
Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection.
Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography.
Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope.
Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.

PMID: 19130168 [PubMed - as supplied by publisher]
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(1.3): J Clin Microbiol. 2009 Jan 7. [Epub ahead of print]

A Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for the Confirmation of Respiratory Syncytial Virus and Influenza Viruses Detected by Antigen Immunoassay Testing.
Liao RS, Tomalty LL, Majury A, Zoutman DE. - Division of Medical Microbiology and Infection Control, Department of Pathology and Molecular Medicine, and Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.

We evaluated the Prodesse ProFlu-1 real-time reverse transcription-PCR multiplex assay with the SmartCycler instrument for the detection of human respiratory syncytial virus (RSV) and influenza A and B viruses in comparison to conventional cell culture and antigen immunoassay testing with the BD Directigen A+B and Binax NOW RSV assays over 2 successive respiratory virus seasons.
Ninety-two percent of the 361 specimens tested were nasopharyngeal aspirates obtained from individual patients, of which 119 were positive for RSV and 59 were positive for influenza virus.
The median age of the patients with specimens positive for RSV and influenza were 6.3 mo and 42.4 yr, respectively.
The specificity for all of the methods tested was >/=99% and the individual sensitivities of the NOW RSV, RSV culture, Directigen A+B, influenza culture, and the Proflu-1 PCR for influenza/RSV were: 82% (95% confidence intervals [CI]: 73-88), 57% (95% CI: 44-69), 59% (95% CI: 44-72), 54% (95% CI: 38-69), and 98% (95% CI: 93-100)/95% (95% CI: 85-99).
In a clinical setting where viral isolation is performed to confirm rapid antigen immunoassay results of these common respiratory viruses, one-step real-time reverse transcriptase PCR testing can be a more sensitive and timely confirmatory method.

PMID: 19129410 [PubMed - as supplied by publisher]
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(1.4): J Immunol. 2009 Jan 15;182(2):1088-98.

Induction of proinflammatory cytokines in primary human macrophages by influenza A virus (H5N1) is selectively regulated by IFN regulatory factor 3 and p38 MAPK.

Hui KP, Lee SM, Cheung CY, Ng IH, Poon LL, Guan Y, Ip NY, Lau AS, Peiris JS. - Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong Special Administrative Region, China.

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-alpha, IFN-beta, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease.
Here we show that TNF-alpha, IFN-beta, and IFN-lambda1 are the key mediators directly induced by the H5N1 virus in primary human macrophages.
In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3).
IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-beta, IFN-lambda1, and MCP-1 but only to a partial reduction of TNF-alpha.
IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus.
We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-beta, IFN-lambda1, and MCP-1 but only partly control TNF-alpha induction.
A more precise identification of the differences in the regulation of TNF-alpha and IFN-beta could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.

PMID: 19124752 [PubMed - in process]
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(1.5): J Immunol. 2009 Jan 15;182(2):934-47.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

Lin GH, Sedgmen BJ, Moraes TJ, Snell LM, Topham DJ, Watts TH. - Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage.
In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease.
During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival.
In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality.
Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus.
Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus.
Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose.
These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

PMID: 19124736 [PubMed - in process]
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(1.6): J Immunol. 2009 Jan 15;182(2):871-9.

Plasmacytoid dendritic cells are dispensable during primary influenza virus infection.

Wolf AI, Buehler D, Hensley SE, Cavanagh LL, Wherry EJ, Kastner P, Chan S, Weninger W. - Immunology Program, The Wistar Institute, Philadelphia, PA 19104, USA.

Plasmacytoid dendritic cells (pDC) are thought to be pivotal in the first line of defense against viral infections. Although previous studies have suggested that pDC regulate the immune response against respiratory syncytial virus, their role in pulmonary infection with influenza virus has remained unclear.
Using mice with GFP-tagged pDC, we observed a marked increase in pDC numbers in the lung airways 3 days after intranasal infection with influenza virus A/PR/8/34. To further investigate their potential involvement in the disease, we made use of pDC-deficient IkarosL/L mice. In the absence of pDC, the recruitment of T cells to the bronchoalveolar space was delayed, which could be reversed by the adoptive transfer of pDC before infection.
Surprisingly, however, when compared with wild-type animals, IkarosL/L mice revealed a similar course of disease, as determined by weight loss, viral titers, levels of neutralizing Ab, and lung pathology.
Moreover, the activation and differentiation of influenza-specific CD8+ effector T cells was unaltered in the absence of pDC, as was the generation of CD8+ memory T cells. Taken together, our study suggests that pDC regulate the accumulation of T cells in the bronchoalveolar space during early influenza virus infection, but are dispensable for the control of this disease.

PMID: 19124730 [PubMed - in process]
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(1.7): J Infect Dis. 2008 Nov 15;198(10):1435-43.

Respiratory syncytial virus persistence in the lungs correlates with airway hyperreactivity in the mouse model.

Estripeaut D, Torres JP, Somers CS, Tagliabue C, Khokhar S, Bhoj VG, Grube SM, Wozniakowski A, Gomez AM, Ramilo O, Jafri HS, Mejias A. - Department of Pediatrics, University of Texas Southwestern Medical Center and Children's Medical Center Dallas, Texas 75390-9063, USA.

BACKGROUND:
Previous studies in mice showed that respiratory syncytial virus (RSV) infection was associated with RSV RNA persistence. This study was designed to characterize the significance of RSV RNA persistence and its relation to RSV-induced chronic airway disease.

METHODS:
Mice were inoculated with live RSV, UV light-treated RSV, heat-inactivated RSV, or medium. Bronchoalveolar lavage fluid samples were obtained and lung specimens were harvested on days 1, 5, and 42 after inoculation to assess lung inflammation, lung mRNA expression of interleukin (IL)-4, IL-5, IL-15, and interferon (IFN)-gamma; RSV loads were assessed by culture and real-time polymerase chain reaction (PCR) and correlated with pulmonary function.

RESULTS:
During the acute phase of infection, RSV loads as indicated by culture and PCR were significantly higher in mice inoculated with live RSV. On day 42, RSV RNA remained detectable only in mice inoculated with live or UV light-treated RSV. Lung inflammation, IFN-gamma:IL-4 mRNA expression ratios, airway obstruction (AO), and airway hyperreactivity (AHR) were significantly increased in mice inoculated with live RSV. AO on day 5 and AHR on day 42 were significantly correlated with RSV RNA copy number in lung samples.

CONCLUSIONS:
Infection with live RSV induced acute and chronic airway disease that was associated with a predominantly Th-1 immune response and RSV RNA persistence that significantly correlated with pulmonary function abnormalities.

PMID: 18828742 [PubMed - indexed for MEDLINE]
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(1.8): J Virol. 2009 Jan 7. [Epub ahead of print]

The MAPK-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.

Kakugawa S, Shimojima M, Goto H, Horimoto T, Oshimori N, Neumann G, Yamamoto T, Kawaoka Y. - Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokaneda, Minato-ku, Tokyo 108-8639, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama 332-0012, Japan; Division of Oncology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA.

Viral infections induce signaling pathways in mammalian cells that stimulate innate immune responses and affect cellular processes, such as apoptosis, mitosis, and differentiation. Here, we report that the ribosomal protein S6 kinase alpha 3 (RSK2), which is activated through the 'classical' mitogen-activated protein kinase (MAPK) pathway, plays a role in innate immune responses to influenza virus infection.
RSK2 functions in the regulation of cell growth and differentiation, but was not known to play a role in cellular antiviral response.
We have found that knockdown of RSK2 enhanced the viral polymerase activity and growth of influenza viruses.
Influenza virus infection stimulates NK-kappaB- and IFN-beta-dependent promoters. This stimulation was reduced in RSK2 knockdown cells, suggesting that RSK2 executes its effect through innate immune response pathways.
Furthermore, RSK2 knockdown suppressed influenza virus-induced phosphorylation of the double-stranded RNA-activated protein kinase, PKR, a known antiviral protein. These findings establish a role for RSK2 in the cellular antiviral response.

PMID: 19129453 [PubMed - as supplied by publisher]
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(1.9): J Virol. 2009 Jan;83(1):58-64. Epub 2008 Oct 15.

Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections.

Krenn BM, Gaudernak E, Holzer B, Lanke K, Van Kuppeveld FJ, Seipelt J. - Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.

We have discovered two metal ion binding compounds, pyrithione (PT) and hinokitiol (HK), that efficiently inhibit human rhinovirus, coxsackievirus, and mengovirus multiplication. Early stages of virus infection are unaffected by these compounds.
However, the cleavage of the cellular eukaryotic translation initiation factor eIF4GI by the rhinoviral 2A protease was abolished in the presence of PT and HK. We further show that these compounds inhibit picornavirus replication by interfering with proper processing of the viral polyprotein.
In addition, we provide evidence that these structurally unrelated compounds lead to a rapid import of extracellular zinc ions into cells. Imported Zn(2+) was found to be localized in punctate structures, as well as in mitochondria.
The observed elevated level of zinc ions was reversible when the compounds were removed. As the antiviral activity of these compounds requires the continuous presence of the zinc ionophore PT, HK, or pyrrolidine-dithiocarbamate, the requirement for zinc ions for the antiviral activity is further substantiated.
Therefore, an increase in intracellular zinc levels provides the basis for a new antipicornavirus mechanism.

PMID: 18922875 [PubMed - indexed for MEDLINE]
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(1.10): Proc Natl Acad Sci U S A. 2009 Jan 2. [Epub ahead of print]

HLA class I molecules consistently present internal influenza epitopes.

Wahl A, Schafer F, Bardet W, Buchli R, Air GM, Hildebrand WH. - Department of Microbiology and Immunology.

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown.
To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*0702-presented viral peptides were directly identified following influenza infection.
After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS).
Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition.
We found that the class I molecule B*0702 presents 3-6 viral ligands following infection with different strains of influenza.
Peptide ligands derived from the internal viral nucleoprotein (NP(418-426) and NP(473-481)) and from the internal viral polymerase subunit PB1 (PB1(329-337)) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP(418-426), NP(473-481), and PB1(329-337) derived from internal viral proteins were consistently revealed by class I HLA.
In contrast, ligands derived from hemagglutinin (HA) and matrix protein (M1) were presented intermittently on a strain-by-strain basis.
When tested for immune recognition, HLA-B*0702 transgenic mice responded to NP(418-426) and PB1(329-337) consistently and NP(473-481) intermittently while ligands from HA and M1 were not recognized.
These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands.

PMID: 19122146 [PubMed - as supplied by publisher]
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(1.11): Virology. 2009 Jan 5. [Epub ahead of print]

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3.

Le Nouën C, Munir S, Losq S, Winter CC, McCarty T, Stephany DA, Holmes KL, Bukreyev A, Rabin RL, Collins PL, Buchholz UJ. - Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection.
It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability.
The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis.
The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3.
Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired.
However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.

PMID: 19128816 [PubMed - as supplied by publisher]
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(1.12): Antimicrob Agents Chemother. 2009 Jan 5. [Epub ahead of print]

Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using pyrosequencing method.

Deyde VM, Nguyen T, Bright RA, Balish A, Shu B, Lindstrom S, Klimov AI, Gubareva LV. - Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta GA, USA; National Centre for Veterinary Diagnostics, DAH11-78th lane - Giai Phong str Phuong Mai - Dong Da - Hanoi, Vietnam.

Resistance to antiviral drugs in influenza viruses can emerge following medication or may result from natural variation.
Two classes of anti-influenza drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA) (oseltamivir and zanamivir), are currently licensed.
These drugs are expected to be important in controlling early stages of a potential pandemic.
In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and to NA inhibitors in A(H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 were targeted for pyrosequencing.
The NA residues for pyrosequencing analysis included the established markers of drug-resistance, (H274 and N294) as well as residues of less certain relevance (V116, I117, Q136, K150, and I222).
A single pair of pyro RT-PCR primers was designed to allow amplification of an approximately 600nt-long amplicon of the NA gene of H5N1 viruses from various clades/subclades associated with infections in humans.
The sensitivity of the assay was demonstrated by the successful pyrosequencing of RNA extracted from samples of serially diluted (10(-5) to 10(-7)) virus stocks with initial concentrations ranging from 10(5) to 10(8) pfu/ml.
The markers of resistance were detected in samples with Ct values ranging from 32 to 37 determined by real-time RT-PCR.
The pyosequencing approach may provide a valuable tool in rapid detection of markers of drug resistance in H5N1 viruses and facilitate the elucidation of the role of such changes in natural and acquired drug resistance.

PMID: 19124660 [PubMed - as supplied by publisher
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