PR8 Strain Research Articles

Alterations of pulmonary defense mechanisms by protein depletion diet

Pulmonary defense mechanisms were quantitated in mice that were fed a protein-free diet (PFD) for periods of 2 and 3 weeks. Despite the severe weight loss and emaciation induced by the diet, the bactericidal mechanisms in their lungs were preserved against aerogenic challenges with staphylococcus aureus, Proteus mirabilis, and Listeria monocytogenes. Phagocytic assays of alveolar macrophages that were retrieved by pulmonary lavage from PFD-fed animals showed a decrease in Fc receptor-mediated binding activity but no alteration in the ingestion of sensitized erythrocytes. In contrast, the PFD induced defects in both the attachment phase and the engulfment phase of the phagocytic process when the challenge organism was Candida krusei. The PFD suppressed the pulmonary inflammatory response after mice were infected with influenza virus strain PR8; such mice also failed to eliminate infectious virus from their lungs. Virus infection in control mice suppressed pulmonary antibacterial defenses against challenges with S. aureus and P. mirabilis, and defect that was ameliorated in the lungs of PFD-fed mice with viral pneumonia. The data demonstrated that pulmonary defense mechanisms were modulated by a PFD but that the observed effect was dependent on the agent used to test host defenses.

Adherence of staphylococcus aureus to influenza A virus-infected Madin-Darby canine kidney cell cultures.

Madin-Darby canine kidney cells infected with influenza A virus (strains PR8, FM1, Jap 305, and Tex 1) were tested with nine strains of Staphylococcus aureus and group B Streptococcus type Ic to determine whether mammalian cells become susceptible to bacterial adherence as a result of virus infection. Bacterial adherence to virus-infected cells varied depending on the virus strain and on the strain of bacteria tested. A quantitative radioassay was developed to study the parameters of adherence. Attachment of 3H-labeled S. aureus grown in chemically defined or biologically complex medium to FM1 virus-infected cells was significantly increased (P less than 0.0005) compared with attachment to control cells. Adherence coincided with the appearance of hemadsorption, which is a marker of the presence of virus-induced glycoproteins on the cell surface. Adherence was temperature dependent, increased with a decrease in hydrogen ion concentration, and was not affected by the presence of K+, Mg2+, or Ca2+. Adherence was blocked when 3H-labeled S. aureus was pretreated with trypsin but not when cells were pretreated with neuraminidase.

Properties of host components of PR8 influenza virus.

The PR8 strain of influenza A virus was grown in canine kidney cell cultures and in chick and duck embryos. After purification by density gradient procedures, the virus preparations were tested for the presence of host antigens with antisera to appropriate host materials. All PR8 preparations were shown to have serologically detectable host components. These host antigens appeared not to be closely associated with the hemagglutinin or neuraminidase spikes, since virus from which essentially all of these structures were removed formed substantial precipitates with antiserum to host material. Evidence is also reported which suggests that cellular actin is an integral component of highly purified PR8 virions, though probably unrelated to host antigen.

The Influence of Defective-Interfering Particles of the PR-8 Strain of Influenza A Virus on the Pathogenesis of Pulmonary Infection in Mice

Homologous autointerference mediated by defective-interfering (DI) particles was first described with the PR-8 strain of influenza virus. However, little is actually known about the influence of DI particles of influenza virus on the pathogenesis of pulmonary infection. The present studies were designed to determine (1) the requirements for successful autointerference in vivo with DI particle-enriched PR-8 influenza virus and (2) the effects of DI particle-enriched virus on the development and progression of otherwise lethal pulmonary infection in mice. PR-8 influenza virus passaged in chicken eggs, but not that passaged in Madin-Darby bovine kidney cells, and enriched in DI particles markedly attenuated pulmonary infection in seven-week-old Swiss and four-week-old C57B16/Cr mice but not in three- to four-week-old Swiss mice. In addition, replication of influenza virus, straining of viral antigen, and numbers of infiltrates in lungs of mice infected with DI particle-enriched influenza virus were reduced in comparison with values in mice infected with comparable amounts of wild-type influenza virus. The protection mediated by DI particle-enriched virus appeared to be related to augmented humoral immune responses in infected mice rather than to autointerference with replication of wild-type virus.

Haemagglutination-inhibiting (HI) antibodies against four prototype strains of influenza A virus in different age groups.

Sera of 197 apparently well persons were tested for residual haemagglutination-inhibiting antibodies against live Hong Kong/68, A/FM/47 and A/PR/34 strains. Sera of 62 well persons, regularly exposed to contacts with swine, were tested against an inactivated A/New Jersey/76 antigen. Those born some time before and during a certain influenza era showed a significantly greater proportion of homologous residual titres against the subtype prevailing in that influenza era, than those born after the termination of the same era. In each of the seven age groups tested both the percentage of positives and the geometric mean titres were usually highest against the Hong Kong strain (representing the most recent era); the next highest were those against the FM1 strain and the lowest were those against the PR8 strain (representing the most distant of these three influenza eras). The serological involvement of donors exposed to regular contacts with swine was relatively stronger against the New Jersey antigen than the response of other serum donors shown against the other three, more recent, prototypes of influenza virus A. The oldest age groups showed significantly lower antibody response against the PR8, FM1 and Hong Kong strains (but not against the New Jersey antigen) than the next one or two of the younger age groups.

Individual antigen-specific T lymphocytes: helper function in enabling the expression of multiple antibody isotypes.

In recent years antigen-specific T cells have been shown to be capable of mediating a number of diverse functions in collaboration with B cells in humoral immune responses. One of the more intriguing roles attributed to helper T cells is the promotion of the synthesis of multiple immunoglobulin isotypes by B cells in T-dependent antibody responses. The experiments presented in this report were carried out to determine if an individual antigen-specific T lymphocyte has the capability to enable the production of antibodies of multiple immunoglobulin heavy chain isotypes. We describe an experimental system which allows for the isolation and antigenic stimulation of individual helper T cells in a splenic environment which provides an excess of primary B cells for collaboration with isolated T lymphocytes. Employing this system we have demonstrated that an individual antigen-specific T lymphocyte, specific for the PR8 strain of influenza virus, has the capacity to enable primary B-cell PR8-specific antibody responses of more than a single immunoglobulin isotype. The implications made by these studies regarding the problem of genetic restrictions regulating T-cell-B-cell interaction is discussed.

Influenza virus antigen in postencephalitic parkinsonism brain. Detection by immunofluorescence.

Brains of six patients with postencephalitic parkinsonism were examined for influenza virus antigen by direct immunofluorescence. Tissue sections were exposed to fluorescein-conjugated globulin directed against the NWS, WSN, WS, and PR8 strains of influenza A 0 , the ribonucleoprotein of the WSN strain, the swine strain of influenza A, the Japan strain of influenza A 2 , the Lee strain of influenza B, measles, and herpes simplex virus. Intranuclear fluorescent antigen was detected in hypothalamic and midbrain sections prepared from all six cases after staining with antibody to the two neurotropic influenza A 0 strains (NWS and WSN) and to the ribonucleoprotein of the WSN strain. No fluorescence was observed after staining of similar sections prepared from the brains of five patients with idiopathic Parkinson disease with labeled influenza NSW antiserum.

Further observations on the effect of air ions on influenza in the mouse.

In earlier papers we reported that the course of respiratory infections in mice produced by intranasal instillation of measured amounts of: (1) a fungus, COCCIDIOIDES IMMITIS, (2) a bacterium, KLEBSIELLA PNEUMONIAE and (3) a virus, PR8 strain of influenza virus was materially affected by the air ion environment. When the challenge dose of influenza virus was administered as an aerosol, as described here, the cumulative mortality rate was completely uninfluenced by shifts in the concentration of positive and negative air ions in the ambient atmosphere and by the accompanying electrical fields. A hypothetical mechanism accounting for the different results obtained with intranasal and aerosol challenge is presented.

Ascending influenza spread in mice by intraperitoneal inoculation determined by immunofluorescence.

The sequence of antigen accumulation in the lung of mice infected intraperitoneally with PR8 strain of influenza virus was pursued by means of immunofluorescent (IF) staining. Influenza infection following intraperitoneal inoculation will be characterized by the fact that only when a large inoculum of virus was injected, around 50% of mice succumbed to generalized pneumonitis 6 days after infection. In such infections, virus growth in the lung reached a maximum at 3 days following virus inoculation, and at this stage, pathological features of mouse lung were comparable to those after intranasal instillation. However, initial spread of virus in the lung was found to be different from that after intranasal inoculation. IF was detected as early as 30 min in reticular cells locating along the lymphsinus of parabronchial lymph nodes, and this IF diminished at 24 hr. Since then, the infection spread through alveolar sac and IF was detectable in both type A and B alveolar cells. Ascending mode of infection through bronchioles was then detected from 24 hr and the intensity of IF in the epithelial cells along bronchial lining reached maximum at 120 hr.

Presença do vírus da gripe no sangue e órgãos de animais inoculados por via intracardíaca

Executou-se o presente trabalho com a finalidade de isolar o vírus da gripe do sangue e de órgãos de camundongos inoculados por via intracardíaca. A observação foi feita com intervalos de 3 a 120 dias após à inoculação. O isolamento do vírus apresentou algumas variações, conforme o tempo decorrido e o material examinado. A presente publicação foi feita em prosseguimento ás anteriores (1 e 2) que tratam da persistência do vírus da gripe em animais inoculados. Neste caso, inocularam-se camundongos brancos por via intracardíaca, ainda não experimentada por nós.

Prolongada persistência do vírus da gripe em hamsters inoculados por vias parenterais e nasal

As experiências relatadas no presente trabalho visaram observar a persistência do vírus da gripe em hamsters inoculados pelas vias subcutânea, peritoneal e nasal. Usaram-se 53 hamsters na verificação da persistência do vírus nos seus organismos a qual atingiu até um ano e cinco meses, pelo menos, numa das séries de experiências. A outra parte do trabalho consistiu em verificar a passagem do vírus, em série, de hamster a hamster. Observamo-la até o máximo de nove vezes, o que foi excepcional, notando-se passagens, na série, com resultados negativos intercalados. Para esta última parte forma usados 91 hamsters, perfazendo o total de 144 em todas as experiências realizadas.

The antiviral activity of isoprinosine.

A hypothesis predicting that increase in the rate of synthesis of host messenger RNA would be associated with antiviral activity was examined. Biochemically, Isoprinosine or NPT-10381 (methisoprinol) increased the rate of rapid labeling of polyribosomal RNA and protein in uninfected monkey kidney cells in tissue culture. In tissue-culture systems, Isoprinosine exerted antiviral effects against influenza virus, PR-8 and A2 strains; herpes virus, LU strain; polio virus 3; and adenovirus 10. In in vivo mortality studies, Isoprinosine was observed to exert therapeutic antiviral effects against the A2 strain of influenza and, as well, against a herpes infection of newborn mice. Experiments were carried out which demonstrated a correlation between the in vivo anti-influenza (PR-8) effects of Isoprinosine and reduction in the virus titer in lungs of infected mice.

Respiratory Diseases in Cyclophosphamide-Treated Mice II. Decreased Virulence of PR8 Influenza Virus

Mice infected intranasally with the PR8 strain of influenza virus were treated with cyclophosphamide, a potent immunologic suppressor. During the first week of infection, mortality in the unmodified influenza infection averaged 65%, whereas in those animals also treated with cyclophosphamide it averaged 22.5%. After the first week, the mortality rate in the infected cyclophosphamide-treated animals rose to that seen during the first week in the animals only infected. This decreased mortality in the first week was found despite the fact that the cyclophosphamide-treated and infected animals had higher virus titers which persisted longer, decreased circulating antibody, and a decreased interferon response. This delayed mortality appeared to be related to the finding of decreased cellular infiltration in the lungs of infected cyclophosphamide-treated animals.

Inactivation of the hemagglutinins of type A influenza viruses by physical and chemical means: an aid to classification.

The effects of temperature and treatment with sodium dodecyl sulfate, Tween 20, dithiothreitol, trypsin, or guanidine on the hemagglutinating capacity of six strains of type A influenza virus (A(0)/PR8/34, A(1)/CAME/46, A(2)/J305/57, A(2)/Bethesda/63, A(2)/HK/Aichi/68, and A(2)/HK/80/68), one strain of swine virus (A/Swine/76/?), and one equine strain (A/Equi-2/63) were determined. The two Hong Kong strains could be readily distinguished from the earlier A(2) strains by the resistance of their hemagglutinins to trypsin treatment and their inability to recover hemagglutinating capacity after removal of dithiothreitol from treated virus preparations. In these respects, the equine strain most closely resembled the Hong Kong variants. The pattern of hemagglutination inactivation also set the swine, PR8, and CAME strains apart from each other as well as from the other five strains. The results suggest that separation of type A viruses into groups by the pattern of inactivation of their hemagglutinins may be a valuable adjunct to standard serology for a more definite classification of these viruses.

Transmissão congênita do vírus da gripe inoculado em hamsters

Relatam-se, no presente trabalho, experiências feitas em hamsters visando observar a transmissão congênita do vírus da gripe. inocularam-se 151 hamsters prenhes ou acasaladas, usando-se a via parenteral ou a nasal. o vírus foi isolado em percentagens variáveis, conforme o caso, sempre acima de 50%, quer das hamsters mães, quer dos filhotes, fetos ou embriões. Observaram-se 15,2% de perturbações embrionárias ou fetais. Processou-se, infecção latente ou inaparente nos animais e os órgãos dos que foram sacrificados não revelaram alterações comuns, macroscópicas ou microscópicas.

Antiviral immune cytolysis at an early stage of paramyxovirus infection.

Antiviral antibody and rabbit complement added as early as 5 min after infection, and with relatively low virus/cell multiplicity, lysed mouse ascites lymphoma cells infected with Sendai or Newcastle disease virus. Inactive Sendai virus at much higher input also sensitized ascites cells and mouse fibroblast monolayers to early antiviral immune cytolysis. At 4 C where adsorption but no penetration occurred, antibody removed virus from the cell membrane and little cytolysis was observed. The ascites cells were sensitive to antibody and complement at all times after the start of penetration and uncoating, indicating that viral envelope antigen is constantly present on the cell membrane. Significant cross-reactions by immune cytolysis between Newcastle disease virus- and Sendai virus-infected cells suggested possible participation of host antigens of the viral envelope. No comparable antiviral immune cytolysis was observed with influenza strains PR8 and WSN. Cell viability was estimated by dye exclusion and the ability to form acid from glucose as indicated by colorimetric pH of the medium. The relation of antiviral immune cytolysis to changes in the membrane resulting in cell fusion is considered.

Attachment of myxoviruses to artificial membranes: Electron microscopic studies

An artificial lipoprotein membrane consisting of phosphatidylcholine and the glycoprotein fetuin, carrying the specific sialic acid receptor for myxoviruses, has been used in electron microscopic investigations of viral attachment. Techniques of formation and preparation of the membrane for electron microscopy are described. The membrane produced is visualized as a coherent structure approximately 70 Å thick, but with no discernible substructure. Myxoviruses (the PR8 strain of influenza A 0 and the Sato strain of Newcastle disease virus) were found to become attached to the membrane, whereas viruses for which the membrane has no specific receptor (adenovirus types 2 and 5, and herpesvirus) were not attached. Membranes formed of sialic acid-free fetuin showed almost complete absence of viral attachment and appeared much thinner. Attachment was also blocked by pretreatment of the membrane with antibody directed against fetuin. Antibody binding studies were made using rabbit antiviral and antifetuin sera, and a goat antirabbit globulin conjugated with ferritin. Binding of antifetuin antibody showed that the membrane had a one-sided structure with virus bound only to the side which accepted antibody, and that soluble fetuin released from the membrane was not responsible for binding of virus aggregates. Ferritin labeling of attached virus showed that the viral envelope remained intact and was not disrupted on contact or incorporated into the membrane. The system offers a means of investigating some current problems involving viruscell interactions.

Aerosol Inoculator for Exposure of Human Volunteers

The performance of an aerosol inoculator for human volunteers is described in tests that used the PR8 strain of type A influenza virus and sodium fluorescein as a physical tracer. Virus recovery from the aerosols was approximately 1% and was unaffected by such variables as prolonged aerosolization, total airflow, relative humidity, or method of sampling. The recovery of sodium fluorescein from the aerosol was approximately 12% and was influenced by total airflow rates and relative humidity. With this apparatus, it should be possible to deliver reasonably predictable and measurable doses of respiratory viruses to human subjects. The design makes it possible to dismantle the inoculator into its component parts to facilitate portability.

RNA and protein synthesis in chick embryo lung cell monolayer cultures infected with influenza virus.

When monolayer cultures of 15 day chick embryo lung cells were infected with PR8 strain influenza virus, newly-synthesised virus was first detected in the medium 4 hours post-infection, and continued to be released from the cells for at least 3 days. No obvious cytopathic changes were observed during this period. Synthesis of RNA and protein was measured in infected cells over a period of 13 hours post-infection by pulse-labelling with3H-uridine and14C-valine for 15 minutes following 30 minutes exposure to actinomycin D (1.5 μg/ml) at hourly intervals. The rate of incorporation of uridine into RNA of infected cells increased and decreased, relative to that in uninfected cells, three times during the 13 hour period, giving peaks of RNA synthesis in infected cells at intervals of approximately 4 hours. At its maximum rate, PR8 virus-induced RNA synthesis was less than 5% of total cellular RNA synthesis measured in the absence of actinomycin D. Labelled RNA extracted from actinomycin D-treated infected cells sedimented in the 16S–18S region on sucrose density gradients, and no such species of labelled RNA was found in uninfected cells. The rate of incorporation of valine into protein of infected cells varied by little more than 10% from that in uninfected cells during the period of study.

The effect of air ions on bacterial and viral pneumonia in mice.

Ambient air untreated for removal of ordinary pollutants and ionized with tritium powered generators to contain 1–2 × 105 small positive ions/cm3 accelerated the rate of death of mice challenged intranasally with measured doses of KLEBSIELLA PNEUMONIAE or of the PR8 strain of influenza virus. The differences between the cumulative mortality rates of controls and ion-treated animals were significant (p < 0.05 to p < 0.001) for several days of the period of observation. Exposure of infected mice to an electrical field of the same strength as that used for ion-treated mice showed no statistically significant field effect. Repetition of the influenza virus experiments using pollutant-free air and ion densities averaging 4.1 × 105 small positive ions/cm3 produced essentially the same results. The observation that high concentrations of positive ions accelerate the rate of death in pulmonary infections with KLEBSIELLA PNEUMONIAE and with influenza virus conforms to the pattern of earlier work with COCCIDIOIDES IMMITIS.