IMMUNIZATION EXPERIMENTS WITH SWINE INFLUENZA VIRUS

1. Swine influenza virus obtained from the lungs of infected ferrets or mice, when administered intramuscularly or subcutaneously, immunizes swine to swine influenza. 2. Ferrets, which have received subcutaneous injections of swine influenza virus obtained from the lungs of infected ferrets, are immune to intranasal infection with this virus. Similar injections with virus from the lungs of infected mice or swine do not immunize. 3. Mice can be immunized to intranasal infection with swine influenza virus by the subcutaneous injection of virus obtained from the lungs of infected mice, but not by similar injection with virus from the lungs of infected ferrets or swine. Repeated injections induce greater immunity than a single one. 4. Intraperitoneal inoculation of both mice and ferrets with swine influenza virus immunizes them to intranasal infection and it appears to make little or no difference whether the virus used as vaccine is obtained from the lungs of infected mice, ferrets, or swine. 5. Field experiments in which swine influenza followed the intramuscular administration of virus are cited as examples of the hazard involved in the use of this means of immunization in a densely crowded susceptible population.     

NEUTRALIZATION TESTS WITH SERA OF CONVALESCENT OR IMMUNIZED ANIMALS AND THE VIRUSES OF SWINE AND HUMAN INFLUENZA

Human and swine influenza viruses were regularly neutralized by their homologous immune sera. However, the sera of animals convalescent from infection with either the swine or human influenza virus possessed little, if any, neutralizing capacity for the heterologous virus. Hyperimmunization of animals against swine influenza virus tended to increase the neutralizing capacity of their sera for human influenza virus, but in an inconstant fashion, whereas repeated inoculations with human influenza virus frequently resulted in sera with strong neutralizing activities against swine influenza virus. These observations serve to emphasize both the immunological distinctiveness and the interrelationships of swine and human influenza viruses.     

Is rinderpest virus the archevirus of the Morbillivirus genus?

Groups of 6-39 monoclonal antibodies identifying 3-18 distinct epitopes on the nucleoprotein (NP), polymerase (P), hemagglutinin (H; equivalent in canine distemper and rinderpest viruses), and fusion (F) components of measles and canine distemper viruses were characterized in immunofluorescence tests with fixed Vero cell cultures infected with measles, canine distemper and rinderpest viruses. The majority of NP-specific monoclonal antibodies reacted with all three viruses, but one-third of the antibodies only reacted with the homologous virus. A few antibodies detected epitopes uniquely shared between either measles and rinderpest viruses or canine distemper and rinderpest viruses. Of the P-specific antibodies, two-thirds only reacted with the homologous virus, one antibody detected an epitope shared between canine distemper and rinderpest viruses, and the rest reacted with all three viruses. Also, the majority of antibodies against the H component were type-specific, but four antibodies reacted both with measles and rinderpest viruses. In contrast, the F component was antigenically highly conserved. 17 of 21 antibodies against this component reacted with all three viruses; one antibody reacted only with measles and rinderpest virus F components, and three antibodies reacted only with the homologous virus. No monoclonal antibody of any specificity selectively reacted with only measles and canine distemper viruses. Furthermore, the measles virus H component appeared to be more closely related to the equivalent rinderpest virus component than to the canine distemper virus component. Thus, it is proposed that rinderpest virus is the archevirus of the morbillivirus group from which canine distemper virus was first to evolve and, more recently (perhaps about 5,000 years ago), measles virus.     

INTERFERENCE BETWEEN VIRUSES IN TISSUE CULTURE

The influence of one virus on the growth of another in tissue culture was investigated. The 17DD High strain of yellow fever virus was found capable of completely suppressing the growth of both the Asibi strain of the same virus and of the heterologous West Nile virus, even when these were added to the cultures in large amounts. The 17DD High strain of yellow fever virus and the West Nile virus produced either partial or complete suppression of growth of the Venezuelan equine encephalomyelitis virus, depending upon the quantity of the latter inoculated into the cultures. Owing to lack of methods for the detection of interference except in a single direction, reciprocal interference with these viruses could not be investigated. The 17DD High strain of yellow fever virus and the West Nile virus were able to suppress completely, or almost completely, the growth of influenza A virus added to the infected cultures in maximal amounts. Interference in the reverse direction, even with the use of small amounts of the neurotropic viruses, was not demonstrable. Cultures infected with the 17DD High strain of yellow fever virus were examined for the presence of neutralizing antibodies and non-specific antiviral substances; neither was found present.     

Antibody to influenza virus matrix protein detects a common antigen on the surface of cells infected with type A influenza viruses

Antisera to the type-specific internal influenza virus matrix (M) protein of a type A influenza virus were produced in goats. In the presence of complement, anti-M serum was cytotoxic for target cells which were infected with a variety of serologically distinct type A influenza viruses, but did not react with type B influenza virus-infected cells. Absorption experiments indicated that anti-M serum detected a common antigen(s) on the surface of type A-infected cells. This serological cross-reactivity parallels the cross-reactivity observed for the cytotoxic T-cell response to type A viruses.     

Antigen-activated dendritic cells ameliorate influenza A infections

Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections.     

Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus

Highly pathogenic H5N1 influenza viruses have infected an increasing number of humans in Asia, with high mortality rates and the emergence of multiple distinguishable clades. It is not known whether antiviral drugs that are effective against contemporary human influenza viruses will be effective against systemically replicating viruses, such as these pathogens. Therefore, we evaluated the use of the neuraminidase (NA) inhibitor oseltamivir for early postexposure prophylaxis and for treatment in ferrets exposed to representatives of two clades of H5N1 virus with markedly different pathogenicities in ferrets. Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir (5 mg/kg of body weight/day) given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when it was initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were rechallenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and the higher dose may be needed for the treatment of more virulent viruses.     

Biological properties of an influenza A virus-specific killer T cell clone. Inhibition of virus replication in vivo and induction of delayed- type hypersensitivity reactions

We tested two biological properties of a continuously growing mouse cytotoxic T cell line, L4, which is specific for influenza A virus and has been cloned and recloned many times. We previously reported that L4 cells are H-2 restricted and cross-reactive for all type A influenza viruses, whereas they do not recognize type B influenza viruses. They bear Thy-1 and Lyt-2 markers. In the present study, we show that L4 cytotoxic T cells protect mice against a lethal influenza infection on transfer to syngeneic recipients, and reduce virus titers in the lungs of mice challenged with a heterologous type A influenza virus. This provides further support for the active role of cytotoxic T cells in limiting virus replication in influenza infection. We could also demonstrate that the cloned cytotoxic T cells induce a delayed-type hypersensitivity skin reaction in the footpads of mice challenged with live or inactivated influenza virus. This reaction can be observed at 24 h, but has declined by 48 h. A clone of cells derived from L4 that has lost its cytotoxic potential and its ability to recognize infected cells did not induce a delayed-type hypersensitivity reaction in the presence of virus. Thus, cytotoxic T cells actively killing influenza virus-infected cells are able to induce a delayed-type hypersensitivity skin reaction to homologous and heterologous type A influenza viruses.     

Efficacy of 2'-deoxy-2'-fluororibosides against influenza A and B viruses in ferrets.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.     

A COMPLEX VACCINE AGAINST INFLUENZA A VIRUS : QUANTITATIVE ANALYSIS OF THE ANTIBODY RESPONSE PRODUCED IN MAN

A quantitative study of the antigenicity of various vaccines containing influenza A virus has been made in human beings. A complex vaccine prepared from chick embryos inoculated with both influenza A virus and the X strain of canine distemper virus was found to be more effective than other vaccines in stimulating the production of neutralizing antibodies against the former virus. The increased antibody levels which resulted from the administration of this vaccine remained almost unaltered for at least 5 months.     

Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals

In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P3) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3) and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 microM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.     

EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : II. SOME FACTORS AFFECTING THE INCIDENCE OF TRANSMITTED INFECTION

Evidence has been presented that with the experimental model described, infected mice vary in their ability to transmit influenza virus infection. This variation is not explained by differences in titers of influenza virus in the nose, throat, trachea, or lungs of good transmitters. Older mice acquire transmitted influenza virus infection more readily than younger mice. Seasonal variations in the incidence of transmitted influenza virus infection occur.     

ANTIBODY RESPONSE OF HUMAN BEINGS FOLLOWING VACCINATION WITH INFLUENZA VIRUSES

Eleven different preparations of influenza virus were used to vaccinate large groups of human beings. The antibody response to these vaccines was measured by means of the in vitro agglutination inhibition test, and the geometric mean titers of sera taken 2 weeks after vaccination were compared. From these comparisons the following conclusions were drawn: 1. There was a wide individual variation in the antibody response of human beings to the same preparation of influenza virus administrated subcutaneously. The amount of antibody produced by a group with a low prevaccination antibody level was very nearly the same as the amount produced by groups that had higher initial levels. 2. The use of the X strain of distemper virus in the preparation of an influenza vaccine did not enhance the antigenicity of the influenza virus present. 3. Within certain limits the mean antibody response of human beings increased as the amount of virus injected was increased. When large amounts of influenza A virus were given, the antibody response was of the same order of magnitude as that which occurred following actual infection by this virus. 4. When the vaccine was prepared from allantoic fluid, there was no significant difference in the antibody response of human beings given active virus, formalin-inactivated virus, heat-inactivated virus, or virus inactivated by the drying process. 5. Ground infected chick embryos, when diluted with infected allantoic fluid, gave a greater antibody response than allantoic fluid alone (when the virus remained active). The antigenicity of such a preparation was diminished when the virus was inactivated by formalin. 6. Antibody levels 6 and 9 weeks after vaccination showed a marked drop from the 2-week postvaccination levels. In a small group the antibody levels at 5 months were still further reduced. Those individuals who possessed the higher titers tended to lose their antibodies faster than did those at a lower level.     

Review of the clinical effectiveness of the neuraminidase inhibitors against influenza B viruses

Influenza A and B viruses cause significant morbidity and mortality worldwide each year. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients. In vitro studies have shown that influenza B viruses are significantly less susceptible to oseltamivir and other neuraminidase inhibitors compared with influenza A viruses. Following analysis of published clinical studies, we show that oseltamivir does appear to have lower effectiveness in patients infected with influenza B virus compared with influenza A infected patients, but due to insufficient studies on zanamivir, laninamivir or peramivir, it was not possible to conclude the relative effectiveness of these drugs against influenza A virus compared with B virus.     

EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : IV. RELATIONSHIP OF TRANSMISSIBILITY OF DIFFERENT STRAINS OF VIRUS AND RECOVERY OF AIRBORNE VIRUS IN THE ENVIRONMENT OF INFECTOR MICE

A mouse-adapted Jap. 305 strain of influenza A₂ virus was found to be much more readily transmitted from one mouse to another than the NWS strain of influenza A₀ virus although the two viruses were equally pathogenic for mice as judged by pulmonary virus titers and lung lesions. The survival of artificially created aerosols of virus and the quantity of airborne virus required to initiate infection in mice were identical for the two viruses. The difference in transmissibility was associated with the recovery of infectious airborne virus in the environment of mice infected with the Jap. 305 strain during the period of their maximum infectiousness, but not in the environment of mice infected with the NWS strain.     

Pathogenesis of and immunity to avian influenza A H5 viruses.

In 1997 in Hong Kong, 18 human cases of respiratory illness were caused by an avian influenza A H5N1 virus. Although avian influenza viruses had not previously been known to cause respiratory illness in humans, the H5N1 viruses caused severe illness and death, primarily in individuals aged > 12 years. The introduction of H5N1 viruses into humans raised concerns about the potential of these viruses to cause a pandemic. We have used the BALB/c mouse to better understand the pathogenesis of and immunity to the H5N1 viruses in a mammalian model. Previously, we demonstrated that H5N1 viruses isolated from humans replicated efficiently in the lungs of mice without prior adaptation to this host. Two general phenotypes of pathogenicity of H5N1 viruses, based on high and low lethality for mice, were observed. We now demonstrate that in addition to a lethal outcome, H5N1 viruses with a high pathogenicity phenotype exhibit additional features that include rapid and uncontrolled replication in the lungs of infected mice, dissemination and replication of the virus in other organs, and depletion of peripheral blood leukocytes. The BALB/c mouse model was also used to better understand the parameters of protective immunity to the H5N1 viruses. Prior infection with H5N1 viruses of low pathogenicity or an antigenically related non-pathogenic H5N3 virus protected mice from death by infection with a highly pathogenic HK/483 virus. Serum hemagglutination-inhibition antibody titers of 40 or greater were associated with protection of mice from death. Immunization of mice with baculovirus-expressed recombinant H5 hemagglutinin protein or a previously defined HS-specific synthetic peptide induced MHC class II restricted CTL activity. Mice that had CTL activity but no serum hemagglutination-inhibition antibody were not protected from a lethal challenge with H5N1 virus. These results suggest that antibody is required for protection of mice against lethal challenge with H5N1 viruses of the high pathogenicity phenotype.     

Efficient expression of the tumor-associated antigen MAGE-3 in human dendritic cells, using an avian influenza virus vector

Dendritic cells (DCs) are the most potent inducers of immune reactions. Genetically modified DCs, which express tumor-associated antigens (TAA), can efficiently induce antitumor immunity and thus have a high potential as tools in cancer therapy. The gene delivery is most efficiently achieved by viral vectors. Here, we explored the capacity of influenza virus vectors to transduce TAA genes. These viruses abortively infect DCs without interfering with their antigen-presenting capacity. In contrast to other viruses used for DC transduction, influenza viruses can be efficiently controlled by antiviral pharmaceuticals, lack the ability to integrate into host chromosomes, and fail to establish persistent infections. Genes encoding a melanoma-derived TAA (MAGE-3), or the green fluorescence protein (GFP), were introduced into a high-expression avian influenza virus vector. Monocyte-derived mature DCs infected by these recombinants efficiently produced GFP or MAGE-3. More than 90% of the infected DCs can express a transduced gene. Importantly, these transduced DCs retained their characteristic phenotype and their potent allogeneic T cell stimulatory capacity, and were able to stimulate MAGE-3-specific CD8(+) cytotoxic T cells. Thus influenza virus vectors provide a highly efficient gene delivery system in order to transduce human DCs with TAA, which consequently stimulate TAA-specific T cells.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

建立微量空斑减少方法测定人血清H7N9禽流感病毒中和抗体水平

目的 建立微量空斑减少的方法测定人血清中H7N9禽流感病毒中和抗体水平。方法 确定H7N9禽流感病毒感染MDCK细胞后形成空斑数目,进行病毒定量,系列稀释待测抗体,加入已定量病毒,计算病毒被抗体中和后空斑减少数目,确定中和抗体的滴度。实验中以H7N9病例血清测定该方法的灵敏度,以其他亚型流感病毒抗体阳性人群血清评估微量空斑减少方法的特异性,同时将该方法与微量中和实验进行比较分析。结果 微量空斑减少方法不仅能够检测H7N9禽流感病例中和抗体水平,而且该方法检测季节性流感病毒抗体、H5N1和H9N2禽流感病毒抗体与H7N9禽流感病毒抗体之间均无交叉反应,与微量中和实验检测结果相一致。结论 建立的微量空斑减少方法具有较好的灵敏度和特异性,能够用于人血清H7N9禽流感病毒中和抗体的检测。     

Encephalitis induced in non-human primates by canine distemper virus adapted to human neural cells

The Onderstepoort strain of canine distemper virus (CDV) adapted to human oligodendroglioma, neuroblastoma and glioblastoma cells, was intracerebrally inoculated into cynomolgus monkeys. All the three viruses caused periventricular encephalitis involving the brain stem. When the neurovirulence of these viruses were compared in terms of clinical signs and histopathological changes, the oligodendroglioma-adapted virus showed the neurovirulence of the highest degree inducing degeneration of axons and glial cells. Chronic encephalitis was also observed. The neuroblastoma-adapted virus induced predominantly nerve-cell degeneration although clinically this virus showed slightly lower degree of neurovirulence than the oligodendroglioma-adapted viruses. The glioblastoma-adapted virus showed clinically much lower neurovirulence than the other two viruses; all monkeys infected with this virus survived and produced high level of antibody in most cases. Histopathologically degeneration of axons and glial cells was characteristics although the incidence was less frequent than the oligodendroglioma-adapted virus. Predominant involvement of nerve cells by neuroblastoma-adapted virus and predominant involvement of axon and glial cells by oligodendroglioma-adapted virus and by glioblastoma-adapted virus suggest that in vitro tropism of the virus to neural cells is partially reflected on tropism of the virus in the CNS.