Studies on the elimination of non-specific inhibitors in sera against influenza viruses with the aid of filtrates ofVibrio cholerae

Summary In the sera of humans and various animals there are two different non-specific inhibitors (α-and β-inhibitors) which may be specifically abolished by two substances (α-and β-“enzymes”) both present in filtrates ofV. cholerae. This neutralization is necessary for the rapid classifying of influenza virus strains with the aid of the haemagglutination inhibition test. The egg line strains of the A- and A′-groups are only sensitive to β-inhibitor, while the mouse line strains of the same groups are only sensitive to the α-inhibitor. This does not apply to strains of the B group, where mouse as well as egg lines are sensitive to both inhibitors. With the aid of isolated β-inhibitor (easily to be prepared from cattle serum), it is possible to decide in a quick manner whether or not a strain from the A- or A′-group has previously been adapted to the mouse. With the technical assistance of Miss I.de Nooyer and with the financial support provided by the Institute for Preventive Medicine, Leyden; N.V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Curacao Fund for Preventive Medicine.     

Antibody response against strains of influenza-A virus in ferrets with basic immunity

Summary In Holland ferrets are offered for sale with basic antibodies against one or more sub-groups of strains of influenza-A virus. With the haemagglutination inhibition test low basic antibody titers can only be detected by neutralising the non-specific inhibitor in the ferret sera. When such a ferret is infected with a strain of a heterologous sub-group, a considerable rise in antibodies is obtained against the homologous and heterologous sub-groups. It is very probable that the same holds for human beings. Some examples are given of haemagglutination-inhibition tests with serum pairs from adults who suffered from influenza in the epidemic of the winter of 1949 in Holland from which the non-specific inhibitor had been eliminated.Sub-unit of the Influenza Research Team of the Institute of Preventive Medicine, Leyden. With the financial support of the Institute of Preventive Medicine, the Department of Medical Research Philips Roxane (Weesp), the State Department of Science, and the Jan Dekker Fund.     

Studies on the increase of antibodies in nasal secretions of human beings after intracutaneous vaccination with influenza virus,with the aid of the haemagglutination inhibition technique

Summary With the aid of the haemagglutination inhibition test in nasal secretions of human beings it is possible to demonstrate an increase of antibodies against influenza virus after pararespiratory vaccination, provided the non-specific inhibition of the secretions is climinated by means of enzymes ofVibrio cholerae. The antititre of the nasal secretions fluctuated greatly. After intracutancous vaccination of 0.3 ml in the skin of the back with concentrated vaccine it amounted on an average to 3% of that of the blood titre. The possible importance of this simplified technique for further investigations is pointed out. Financial support was provided by the Institute of Preventive Medicine, Leyden; N. V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Cura?ao Fund for Preventive Medicine.     

The influence of treating ferret influenza antisera with enzymes of crude filtrate ofVibrio cholerae on the titre of the antibodies

Summary After treating influenza antisera from ferrets with enzymes of a crude filtrate ofVibrio cholerac (pH 7.6) for the purpose of eliminating the non-specific inhibitors it is not possible to detect a significant fall in the titre of the antibodies with the aid of the egg protection test. Financial support was provided by the Institute for Preventive Medicine, Leyden; N. V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Cura?ao Fund the Preventive Medicine.     

Some aspects of the adaptation of influenza virus onto mice

Summary The author describes the adaptation onto mice of an influenza A strain, isolated byMulder from the tracheal mucosa of a man, who died from an acute fulminant staphylococcal-pneumonia and discusses the nature of the adaptation phenomenon.Communication of the Workteam of Acute Respiratory Diseases of the Institute for Preventive Medicine.     

Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli

Vilcek, Jan (New York University School of Medicine, New York, N.Y.), and John H. Freer. Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli. J. Bacteriol. 92:1716-1722. 1966.-Extracts prepared from washed cells of Escherichia coli B by sonic treatment and subsequent filtration through a 0.45-mu membrane filter significantly inhibited plaque formation with Sindbis virus in cultures or primary chick embryo cells up to a dilution of 1:20,000. The inhibitor acted on the cells rather than directly on the virus. The inhibiting substance was nondialyzable. Treatment of crude extracts with nucleases, trypsin, chymotrypsin, pepsin, or ether had no effect on the activity. Treatment with pronase destroyed the virus-inhibiting effect. Extracts prepared from two strains of E. coli B and one strain of E. coli K-12 all showed inhibitory activity against Sindbis virus. The inhibitor was present in the cytoplasmic fraction of bacteria. It was also active against Sindbis virus in human cells and showed some activity against vesicular stomatitis and vaccinia viruses in different types of cells. Interferon was not shown to be involved in the inhibition, although actinomycin D partially reversed the inhibitory activity of the extracts.     

Characterization of the Noncoding Regions of the 1918 Influenza A H1N1 Virus

The terminal noncoding region (NCR) sequences of the eight gene segments of the influenza A/Brevig Mission/1/1918 (H1N1) virus were determined by rapid amplification of cDNA ends (RACE). Chimeric viruses encoding the open reading frames of the 1918 virus but flanked by either the wild-type 1918 NCR sequences or the NCR sequences of two other H1N1 virus strains, A/WSN/1933 and A/New York/312/2001, were produced. No growth differences between the NCR variant 1918 influenza viruses were noted.     

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen (HA) from the strains A/New Caledonia/20/99 (H1N1) (pV1A5) and A/California/04/2009 (H1N1) (pVEH1), respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD(50) of mouse-adapted A/New Caledonia/20/99 (H1N1). Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses (P<0.05) than the pVAX1 control group. Additionally, the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group (P<0.05). Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus (H1N1), while the pandemic influenza DNA vaccine only partially protects against this virus.     

Stochastic processes are key determinants of short-term evolution in influenza a virus

Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.     

Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.     

Phylogenetic analysis reveals the global migration of seasonal influenza A viruses

The winter seasonality of influenza A virus in temperate climates is one of the most widely recognized, yet least understood, epidemiological patterns in infectious disease. Central to understanding what drives the seasonal emergence of this important human pathogen is determining what becomes of the virus during the non-epidemic summer months. Herein, we take a step towards elucidating the seasonal emergence of influenza virus by determining the evolutionary relationship between populations of influenza A virus sampled from opposite hemispheres. We conducted a phylogenetic analysis of 487 complete genomes of human influenza A/H3N2 viruses collected between 1999 and 2005 from Australia and New Zealand in the southern hemisphere, and a representative sub-sample of viral genome sequences from 413 isolates collected in New York state, United States, representing the northern hemisphere. We show that even in areas as relatively geographically isolated as New Zealand's South Island and Western Australia, global viral migration contributes significantly to the seasonal emergence of influenza A epidemics, and that this migration has no clear directional pattern. These observations run counter to suggestions that local epidemics are triggered by the climate-driven reactivation of influenza viruses that remain latent within hosts between seasons or transmit at low efficiency between seasons. However, a complete understanding of the seasonal movements of influenza A virus will require greatly expanded global surveillance, particularly of tropical regions where the virus circulates year-round, and during non-epidemic periods in temperate climate areas.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Revelation of Antiviral Activities by Artificial Sulfation of a Glycosaminoglycan from a Marine Pseudomonas

Sulfated derivatives of a glycosaminoglycan containing l-glutamic acid produced by a marine Pseudomonas species, No. 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction. Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were investigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells. Both preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and 5.2 μg/ml, respectively, whereas they had no antiviral activity against FluV-B. No cytotoxicity of either product was noted against MDCK cells at the 50% cytotoxic concentration of 100 μg/ml. Received April 4, 1998; accepted July 24, 1998.     

The question of the presence of influenza virus in the faeces of mice after infection and feeding with the virus

Summary Repeated attempts to duplicateSarracino andSoule's experiments to identify influenza virus in the faeces of infected mice proved to be a failure. Other, more sensitive methods for extracting minute amounts of influenza virus from contaminated material are described, but they also proved incapable of extracting influenza virus from the faeces of infected mice. Attempts to infect animals by the intestinal route produced no result and at the same time we failed to prove that the influenza virus can pass the alimentary canal without being destroyed.     

Neurosecretion in the distal stump of the sectioned hypothalamic-hypophyseal tract in the toad

Summary After section of the hypothalamic-hypophyseal neurosecretory pathway of the toad accumulation of neurosecretory material both proximally and distally to the plane of section was observed. When, in other animals, a second section close to the neurohypophysis was performed, neurosecretory material was absent in the distal stump resulting from the first section. The pile-up of neurosecretion in the distal stump of the unisectioned tract is attributable to a backward flow from the neurohypophysis to the lesion rather than to local synthesis of neurosecretion.The present investigation was carried out as a consequence of a discussion by the senior author with ProfessorWilliam Etkin, Department of Anatomy, Albert Einstein College of Medicine, New York (USA).     

A universal influenza A vaccine based on the extracellular domain of the M2 protein.

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.     

Induction of homotypic and heterotypic T- and B-cell immunity with influenza A virus in mice

Infection of mice with live influenza A virus induces cytolytic T lymphocytes (CTL) as well as B cells capable of reacting with target cells infected with the appropriate virus subtypes. In Balb/c mice CTL reveal a broad cross-reactivity against all influenza A substrains known. In contrast B-cell responses are restricted to virus subtypes which are identical in regard to the hemagglutinin (HA) of the sensitizing virus. Reinfection with homologous live influenza virus within 6–7 months results in no or in a drastically diminished B-cell response as compared to a priming situation and fails to induce CTL. Inability to induce secondary immunity to homologous influenza virus was correlated with the presence of circulating antibodies specific for the sensitizing virus subtype. Cross-boosting with heterologous live influenza A virus induces homotypic and heterotypic CTL and B-cell immunity with characteristics of secondary responses. Preparations of inactivated intact influenza virus are unable to reactivate CTL memory in vivo but induce B-cell activity. B-cell responses stimulated by this procedure are restricted to the boosting virus. Attenuated viruses, which are produced by recombination of wild strains with cold-adapted strains, are also efficient in stimulating in vivo CTL memory if used for cross-boosting.     

The interrelationships of disease and culture in a primitive New Guinea community

This article explores links between disease and social standing in a primitive New Guinea community. Social and cultural events have modified the incidence of certain diseases. Furthermore, the changing patterns of disease may have influenced the development and form of social distinctions.This work is based on data collected as a predoctoral fellow in the Department of Anthropology at Columbia University. Fieldwork in New Guinea was done with NIH support under Training Grant 1 T01-MH11775-01 (related to 2 F1 MH301640-02), with Dr. Margaret Mead as the sponsor. Additional funding for analysis of data was obtained from the Harvard School of Public Health, the Department of Preventive Medicine of the Harvard Medical School, and the Institute for Transcultural Studies in New York.