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Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins.

Latham T, Galarza JM.

Department of Viral Vaccine Research, Wyeth-Lederle Vaccines, Pearl River, NY 10965, USA.

We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of the VLPs. Immunoprecipitation of M1 protein from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11390617&dopt=Abstract flu, influenza



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Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak.

Hinshaw VS, Nettles VF, Schorr LF, Wood JM, Webster RG.

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3015104&dopt=Abstract flu, influenza



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[Correlation coefficients of the basic indicators of cardiohemodynamics, blood gas levels and acid-base status in influenza]

[Article in Russian]

Mintser OP, Kovalenko LL.

The main parameters of the cardiohemodynamics, blood gas composition and acid-base balance were studied in 82 patients with uncomplicated influenza and in 124 patients with influenza aggravated by acute pneumonia. The disorders revealed were essentially dependent on the disease gravity and complications. To evaluate quantitatively the relationship between the respiratory and cardiovascular systems, use was made of the correlation ratios for all the pair parameters under study. The pulmonary artery pressure was highly dependent on the level of the diastolic systemic pressure in grave uncomplicated influenza (r = -0.932) and on the heart rate (r = +0.861) in influenza aggravated by pneumonia. These findings enabled the derivation of appropriate regression equations, which are simple and can thus be widely used in practice to measure the pressure in the pulmonary circulation and to correct pulmonary hypertension. The making up of the correlation schemes permitted evaluating the tendencies in the changes of the most important parameters of the cardiohemodynamics and homeostasis and forming a judgement about tension of the compensatory processes in the body during grave influenza and influenza complicated by acute pneumonia.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3099414&dopt=Abstract flu, influenza



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Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

Okuda K, Ihata A, Watabe S, Okada E, Yamakawa T, Hamajima K, Yang J, Ishii N, Nakazawa M, Okuda K, Ohnari K, Nakajima K, Xin KQ.

Department of Bacteriology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. kokuda med.yokohama-cu.ac.jp

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11395202&dopt=Abstract flu, influenza



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[Assessment of the effectiveness of inactivated influenza vaccines for adults]

[Article in Russian]

Ignat'eva GS, Shadrin AS, Maksakova VL, Efremov BV, Rudenko LG.

In 9 controlled epidemiological observations (1977-1984) the effectiveness of modern Soviet whole-virion vaccines was studied in organized groups of adults and at industrial enterprises. During the epidemic outbreaks of influenza of different etiology and intensity morbidity rate in influenza and acute respiratory diseases was shown to decrease 1.1-2.2 times among the vaccinees, depending on the correspondence of epidemic and vaccine influenza strains. The absence of influenza virus B in inactivated influenza vaccines was the reason for their low effectiveness during influenza outbreaks of mixed etiology B + A (H1N1).

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3705814&dopt=Abstract flu, influenza



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Effect of influenza vaccine in patients receiving long-term warfarin therapy.

Weibert RT, Lorentz SM, Norcross WA, Klauber MR, Jagger PI.

The effect of influenza vaccine on anticoagulant response in 12 patients receiving long-term warfarin therapy was studied. Study criteria required that all patients have stable prothrombin times and be on stable warfarin dosages before immunization. Patients were immunized with 1982-83 trivalent influenza vaccine (subvirion), types A and B 0.5 mL i.m. on day 0 of the study. Prothrombin times were determined on days--7, 0, 2, 5, 7, 14, and 21, and results were reported as ratios with control values. Influenza immunization produced a small but significant increase in the prothrombin-time ratio. Before immunization the mean ratio was 1.68, and the mean ratio increased to 1.81 after immunization. The maximal increase occurred on day 14 and represented a 7.6% increase over the baseline (day 0) value. The prothrombin-time ratio was not influenced by warfarin sodium dosage (less than or equal to 5 mg/day versus greater than 5 mg/day) or by the sex of the patient. During the 1984-85 influenza season, an additional 26 patients were immunized with the 1984-85 trivalent influenza vaccine (subvirion), types A and B. The prothrombin-time ratio on days 0 and 14 were not significantly different in these patients. Although the administration of influenza vaccine to patients receiving long-term treatment with warfarin appears to be safe, patients should be monitored after immunization for possible increases in anticoagulant response.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3720216&dopt=Abstract flu, influenza



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Early augmentation of interleukin (IL)-12 level in the airway of mice administered orally with clarithromycin or intranasally with IL-12 results in alleviation of influenza infection.

Tsurita M, Kurokawa M, Imakita M, Fukuda Y, Watanabe Y, Shiraki K.

Department of Virology, Toyama Medical and Pharmaceutical University, Toyama, Japan.

The protective role of interleukin (IL)-12 against influenza infection was assessed by analyzing the efficacies of orally administered clarithromycin (CAM) as an immunomodulator and intranasal administration of recombinant IL-12 in intranasally influenza virus-infected mice. In infected mice, CAM at 20 mg/mouse/day significantly elevated the levels of IL-12 and interferon-gamma on days 2 and 3, respectively, after infection in the bronchoalveolar lavage fluid (BALF), but the levels in the sera were not affected. The levels of IL-4, -6, and -10 were not significantly affected in the sera and BALF. Corresponding with the local elevation of IL-12 level, CAM reduced virus yield and the number of infiltrated cells in the BALF, the severity of pneumonia, and mortality of the treated mice. The potential activity of CAM as an experimental immunomodulator was verified at a dose of 20 mg/mouse/day. Intranasal administration of the optimal dose (20 ng/mouse) of IL-12 on day 2 significantly reduced virus yield in the BALF after infection. The loss of body weight was significantly suppressed by IL-12 administration. The local elevation of IL-12 level at the optimal dose and timing in influenza infection was confirmed to be effective in alleviating the influenza infection in mice treated with the two different ways. Thus, the augmentation of IL-12 production or administration of supplementary IL-12 in the respiratory tract was essential in reducing virus yield in the early phase of influenza and may be crucial for recovery from influenza infection.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11408563&dopt=Abstract flu, influenza



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Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy.

Campitelli L, Mogavero E, De Marco MA, Delogu M, Puzelli S, Frezza F, Facchini M, Chiapponi C, Foni E, Cordioli P, Webby R, Barigazzi G, Webster RG, Donatelli I.

Department of Virology, Istituto Superiore Sanita', Rome, Italy. campitel iss.it

Since the "bird flu" incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002-2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived "in toto" from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15165816&dopt=Abstract flu, influenza



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Features of patients with influenza virus infection examined in the emergency department of a university hospital in north-western Japan.

Mashiba S, Takahashi T, Hayashi N, Morita H, Gotoh T, Nakata M, Watoh Y, Kanda T, Takino M.

Department of Emergency Medicine, Kanazawa Medical University, Ishikawa, Japan. massy kanazawa-med.u.ac.jp

We aimed to determine if there were any changes in the clinical features of patients infected with influenza during an influenza epidemic in north-western Japan. We retrospectively obtained data on influenza-infected patients from an electronic database set up in the emergency department. Patients were examined in the Department of Emergency Medicine, Kanazawa Medical University in 2001, 2002 or 2003. The information collected included age, sex, time of visiting the emergency department, visiting method, grade of emergency, where they were when their symptoms started to develop, influenza-related complication(s) and outcome. The retrospective data collected for the 3 years analysed (2001-2003) were similar despite the influenza epidemic in the winter of 2002/2003. These results allow us to estimate the influenza-related total medical costs and total work burden for staff in the emergency department of a university hospital during any future influenza epidemics.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15174228&dopt=Abstract flu, influenza



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Respiratory virus surveillance. FluWatch project, 2000-2001. End of season update.

[Article in English, French]

Macey JF, Winchester B, Squires SG, Tam T, Zabchuk P, Li Y.

Field Epidemiology Training Program, Centre for Surveillance Coordination and Division of Respiratory Diseases, Bureau of Infectious Diseases, Ottawa, Ontario.

The 2000-2001 season was a relatively mild season worldwide. In Canada, lower than usual activity was reported for all national indicators of influenza activity, including the rate of influenza-like illness (ILI), the percentage of laboratory-confirmed cases of influenza and provincial/territorial influenza activity levels. However, there were a number of interesting characteristics of this year's influenza season. In contrast to the predominance of influenza A, and in particular the A/Sydney/5/97 (H3N2)-like virus over the past 3 years, influenza B predominated overall this season. Influenza A (H3N2) accounted for < 1% of all characterized isolates (H1N1 accounted for 49% of isolates). Increased laboratory-confirmed influenza activity began in the West (Yukon, prairie provinces and British Columbia) in mid-December, followed by the Atlantic provinces in mid- to late January and Ontario and Quebec in mid- February and March.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11416943&dopt=Abstract flu, influenza



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IL-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection.

van der Sluijs KF, van Elden LJ, Nijhuis M, Schuurman R, Pater JM, Florquin S, Goldman M, Jansen HM, Lutter R, van der Poll T.

Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands.

Secondary pneumococcal pneumonia is a serious complication during and shortly after influenza infection. We established a mouse model to study postinfluenza pneumococcal pneumonia and evaluated the role of IL-10 in host defense against Streptococcus pneumoniae after recovery from influenza infection. C57BL/6 mice were intranasally inoculated with 10 median tissue culture infective doses of influenza A (A/PR/8/34) or PBS (control) on day 0. By day 14 mice had regained their normal body weight and had cleared influenza virus from the lungs, as determined by real-time quantitative PCR. On day 14 after viral infection, mice received 10(4) CFU of S. pneumoniae (serotype 3) intranasally. Mice recovered from influenza infection were highly susceptible to subsequent pneumococcal pneumonia, as reflected by a 100% lethality on day 3 after bacterial infection, whereas control mice showed 17% lethality on day 3 and 83% lethality on day 6 after pneumococcal infection. Furthermore, 1000-fold higher bacterial counts at 48 h after infection with S. pneumoniae and, particularly, 50-fold higher pulmonary levels of IL-10 were observed in influenza-recovered mice than in control mice. Treatment with an anti-IL-10 mAb 1 h before bacterial inoculation resulted in reduced bacterial outgrowth and markedly reduced lethality during secondary bacterial pneumonia compared with those in IgG1 control mice. In conclusion, mild self-limiting influenza A infection renders normal immunocompetent mice highly susceptible to pneumococcal pneumonia. This increased susceptibility to secondary bacterial pneumonia is at least in part caused by excessive IL-10 production and reduced neutrophil function in the lungs.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15187140&dopt=Abstract flu, influenza