Experimental influenza infection: influence of stress

The effect of immobilization stress on the course of various forms of influenza infection has been investigated. Influenza was produced in 10-14-week-old inbred mice by intranasal infection with pathogenic influenza virus strain A/PR/8/34 (H1N1) at different doses. Immobilization for 6 hr resulted in the appearance of virus-inhibiting activity in the serum of mice. This activity suppressed the reproduction of test-virus in tissue culture, it was resistant to acid pH 2.0 treatment and to heating at 56 degrees C. However, the high level of virus-inhibiting activity failed to protect the animals from subsequent development of lethal influenza infection. Immobilization stress caused a transient depression of virus induced interferon (IFN) production, as revealed by the use of virus inducer at early intervals after stress. Contemporarily, the stress could aggravate the course of virus infection promoting its transition from non-lethal form into a lethal one and virus penetration into brain.     

Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2)

Summary.  Immunization with recombinant adenoviral vaccine that induces potent immunity has been applied to many infectious diseases. We report here developing a recombinant adenoviral vaccine encoding the HA gene from swine H3N2 influenza virus (SIV). Two replication-defective recombinant adenoviruses were generated: (1) rAd-HA: recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus, and (2) rAd-vector: a control recombinant adenovirus containing adenovirus and transfer plasmids without a foreign HA gene. Mice given rAd-HA developed high titers of neutralizing and hemagglutination inhibition antibodies to SIV in comparison to mice inoculated with rAd-vector or PBS as early as 2 weeks after immunization, and these antibodies were substantially increased in the mice given rAd-HA within the next 3 weeks following the first dose. However, these antibodies were not able to neutralize the virus, A/HK/68 (H3N2), used for challenge. Nonetheless mice immunized with one or two doses of rAd-HA were protected from lethal challenge with heterologous virus, A/HK/1/68 (H3N2). A statistically significant (P < 0.03) difference between survival rates of rAd-HA mice vs. rAd-vector or PBS mice was observed. Received April 2, 2002; accepted June 18, 2002     

Antiviral action of monoclonal antibodies

The antiviral effect of monoclonal antibodies MAK-14-7 possessing the neutralizing activity was studied on the model of Venezuelan equine encephalomyelitis virus in cell cultures and in experimentally infected laboratory animals. Mouse monoclonal antibodies exerted a marked specific antiviral effect in tissue culture and in white mice inhibiting virus reproduction by 1-3-5 log PFU/ml. The degree of inhibition of virus reproduction is inversely proportional to the multiplicity of infection. When used in combination with remantadine, ribavirin, interferon, and its inducer poly (I) X poly (C), the monoclonal antibodies MAK-14-7 exerted additive antiviral effect.     

Interferon production by leukocytes infiltrating the lungs of mice during primary influenza virus infection.

Lung fluids and leukocytes were obtained from unprimed C3H mice by transpleural lavage at intervals after infection with influenza A/Hong Kong/68 virus and were tested for interferon activity. Lavage fluid interferon titers correlated directly with lung virus titers and with initial increases in leukocyte yields from infected lungs. In contrast to cultured lymph node cells from infected animals or leukocytes from lungs of uninfected mice, washed leukocytes obtained from the lungs of mice infected 2 to 6 days earlier produced interferon spontaneously in culture. The physiochemical, biological, and antigenic properties of both the interferon in lavage fluids and that produced by lung lavage leukocytes were similar and characteristics of alpha interferon. Fractionation studies indicated that macrophages and T lymphocytes were primarily responsible for the interferon produced in culture. The early presence and significant numbers of interferon-producing leukocytes in infected lungs suggests that these cells have an early role in defense against influenza virus infection.     

Effects of Low- and High-Passage Influenza Virus Infection in Normal and Nude Mice

A human isolate of type A Hong Kong influenza virus (H3N2) was adapted to mice by serial passage. Lung homogenates from mice who received low passage levels contained about the same quantity of virus (10^6.2−6.95 50% tissue culture infective doses/ml) as those from mice who received high passage levels (10^5.95−6.45 50% tissue culture infective doses/ml); however, death occurred only in animals given high-passage virus. Passage 3 (P3) and passage 9 (P9) viruses were selected as representative of low-passage and high-passage viruses, respectively. Although minimal differences were detected in infectivity for rhesus monkey kidney tissue cultures and mice, P9 virus was at least 10,000 times more lethal for mice (mean lethal dose = 10^4.2). Infection with P3 virus was accompanied by minimal bronchitis and bronchiolitis only, whereas P9-infected animals exhibited marked bronchitis, bronchiolitis, and pneumonia. Striking thymic cortical atrophy was also demonstrable in the P9-infected animals and, although virus was more commonly recovered from thymuses from these animals, immunofluorescent studies revealed only a few cells containing influenza virus antigens. To further explore the participation of thymus-derived lymphocytes in influenza, athymic nude mice and furred immunocompetent littermates were given 500 50% mouse infectious doses of P9 virus. Nude mice exhibited an increased survival time and, in contrast to the extensive lung pathology seen in furred littermates, manifested minimal cellular infiltration and no tissue destruction in lungs. Brains from nude mice exhibited encephalomalacia with lymphocytic perivascular cuffing, which was not seen in furred animals. Virus was recovered from brains of 6 of 13 nude mice and 1 of 10 furred animals. The contrasting models suggest that thymus-dependent cells play a significant role in the inflammatory response to influenza virus infection and should prove useful for probing host-virus interactions which characterize influenza virus virulence.     

Effect on influenza virus infection on the production of interferon by alveolar and peritoneal cells in vitro

The investigation was carried on mice infected with influenza A/053/74/H3N2 virus. 129/Ao/Boy, inbred mice, were inoculated intranasally with influenza virus. The alveolar and peritoneal cells from infected and uninfected animals were induced in vitro with Newcastle disease virus. It was shown that the alveolar cells from infected mice produce more interferon than the cells from control mice, but the peritoneal cells from both groups of animals produced the same amount of interferon.     

Pulmonary cell susceptibility in mice and rats to influenza virus when infected in vivo and in vitro

The purpose of the case study was to evaluate comparatively the relative contribution of cell susceptibility and the inhibiting effect of factors of pulmonary epithelial lining in mice and rats to influenza virus A/Aichi/2/68 (H3N2) adapted to mice as related with the development of infection process in the lungs of experimental animals when infected in vivo and in vitro. Mice and rats were infected aerogenically with different doses of influenza virus. The primary cell-culture suspensions sampled from the lungs of mice and rats were used to study the adsorption and dynamics of influenza virus production in infection by different dose of influenza virus in vitro. The cell suspensions were shown to be able to produce the influenza virus for as long as 48 hours after infection. It was for the first time that the results denoted the identical susceptibility of primary pulmonary cells in mice and rats to influenza virus. A lower pulmonary susceptibility to influenza virus in rats versus mice could be indicative of that the surface factors of epithelial lining contribute essentially to shaping the pulmonary susceptibility to influenza virus since there is no difference of the susceptibility of pulmonary cells to influenza virus between the two above animals' species.     

Cytotoxic activity of normal killers and the lymphocytic proliferative response to specific viral antigens in influenza in mice

Inoculation of CBA mice with pathogenic influenza A/PR8/34 (H1N1) virus and non-pathogenic A/Krasnodar/101/59 (H2N2) virus demonstrated that the pathogenic strain enhanced the synthesis of serum interferon and activated the function of normal killers, but had a relatively low capacity of causing in vitro proliferative response of spleen lymphocytes of intact and in vivo primed animals. In contrast, the nonpathogenic virus had less marked interferon-inducing capacity and that of activating normal killers, but induced a very high proliferative response of lymphocytes in culture.     

Effect of cyclosporin A on murine natural killer cells.

In mice, cyclosporin A decreased the natural killer cell-enhancing effect of two interferon inducers, infective murine cytomegalovirus and nonreplicating Newcastle disease virus. It also inhibited murine cytomegalovirus replication at doses greater than 20 mg/kg, but it did not significantly inhibit interferon induction by Newcastle disease virus. In cell culture, cyclosporin A had no direct effect on the natural killer activity of spleen mononuclear cells derived from normal or murine cytomegalovirus-infected animals. However, at 50 micrograms/ml it significantly reduced the ability of interferon to enhance the natural killer activity of normal spleen cell suspensions. The inhibitory effect of cyclosporin A on natural killer cell activity in infected mice may be partly explained by its ability to block the action of interferon.     

The sensitization of mice with a wild-type and cold-adapted variant of influenza A virus. II. Secondary cytotoxic T cell responses.

Reductions in virus titres and the generation of enhanced cytotoxic T cell (Tc) activity in the lungs of mice primed either with a wild-type, parental (H2N2) influenza virus, A/AA/6/60, or a cold-adapted variant A/AA/6/60-ca and challenged 6 weeks later with a H1N1 A/WSN virus showed that both H2N2 viruses could sensitize the mice. A comparison of graded sensitizing doses of each virus showed that inocula of 10(6) tissue culture infective doses (TCID50) of the ca-variant or 10(3) TCID50 of the wild-type virus gave similar results. The spleens and lungs of normal mice were found to contain similar levels (circa 1/10(5) cells) of precursor Tc cells and the level in the lung did not increase 2 days after intranasal (i.n.) inoculation of A/WSN virus. Two and 6 weeks after priming mice with 10(5) TCID50 of either virus, the lungs contained about a 20-fold increase in the precursor Tc cell frequency. In contrast, sensitization with a sub-lethal dose of a mouse-adapted A/WSN virus caused a 100-fold or greater increase. Sensitization of mice with the parental but not the ca-variant virus caused an increase in frequency of precursor Tc cells in the spleens of the sensitized mice and this might reflect the very low level of replication of the ca-variant virus in the mouse lung.     

An early ‘classical’ swine H1N1 influenza virus shows similar pathogenicity to the 1918 pandemic virus in ferrets and mice

The 1918 pandemic influenza virus has demonstrated significant pathogenicity in animal models and is the progenitor of ‘classical’ swine and modern seasonal human H1N1 lineages. Here we characterize the pathogenicity of an early ‘classical’ swine H1N1 influenza A virus isolated in 1931 compared to the pathogenicity of the 1918 pandemic virus and a seasonal H1N1 virus in mice and ferrets. A/Swine/Iowa/31 (Sw31) and the 1918 influenza viruses were uniformly lethal in mice at low doses and produced severe lung pathology. In ferrets, Sw31 and 1918 influenza viruses caused severe clinical disease and lung pathology with necrotizing bronchiolitis and alveolitis. The modern H1N1 virus caused little disease in either animal model. These findings revealed that in these models the virulence factors of the 1918 influenza virus are likely preserved in the Sw31 virus and suggest that early swine viruses may be a good surrogate model to study 1918 virulence and pathogenesis.     

Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens.

The immunogenicity of several cold-adapted (ca) viruses was compared in CSL mice with that of wild-type parental viruses with similar surface antigens, according to the vaccinating dose required to clear a challenge consisting of 10(4.5) 50% tissue culture infective doses of the wild-type virus. All ca viruses were less immunogenic than their wild-type parental strains by a factor of 10(1.3) to 10(3.4), probably due to the restricted capacity of ca viruses to replicate in the respiratory tracts of mice. However, their immunogenicity was considerably enhanced when two quite small doses were administered 3 weeks apart. The immunogenicity of ca viruses when administered in two doses and wild-type viruses when administered as a single dose varied according to their surface antigens. It was highest for viruses with the H2N2 A/Ann Arbor/6/60 and H3N2 A/Queensland/6/72 surface antigens and lowest for those with H1N1 A/HK/123/77 surface antigens. When two doses consisting of 10(5.0) 50% tissue culture infective doses of A/Ann Arbor/6/60-ca were administered at an interval of 3 weeks, solid immunity was induced against the wild-type A/Ann Arbor/6/60 parental virus, two heterologous H3N2 strains, and an H1N1 strain.     

Interferon in experimental viral infections in mice: tissue interferon levels resulting from the virus infection and from exogenous interferon therapy.

In mice given single intraperitoneal doses of interferon, serum interferon levels peaked at 1 h postinjection and were reduced to zero at about 8 h. The interferon concentrations in spleen, liver, and lungs were about 100-fold higher than could be expected from the amount of serum contained in these organs. In the brain only low levels of antiviral activity were detected. In mice infected intraperitoneally with Mengo virus, viral replication in the brain occurred around day 4 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). This was preceded, however, by viral replication in the spleen and by the appearance of modest amounts of interferon in spleen and serum. In these mice protection could be obtained with relatively small doses of interferon, provided protection could be obtained with relatively small doses of interferon, provided they were given before the time of maximal levels of endogenous serum interferon. In mice infected intranasally with vesicular stomatitis virus, virus replication in the brain started within 24 to 48 h and increased with time; also, small amounts of interferon (10(2) to 10(2.5) U/g) were already detectable on days 1 and 2. The major peak of virus replication in the brain occurred on days 5 to 6 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). In this model early treatment with interferon also provided protection, but only if given in larger doses than in the Mengo virus system. Athymic (nu/nu) mice developed a chronic systemic infection when inoculated with a demotropic strain of vaccinia virus. No interferon was detected in sera, livers, spleens, or lungs of these animals; some mice had low levels of interferon-like antiviral activity in the brain, but no attempt was made to characterize this material. Daily administration of large doses of interferon failed to exert an effect on the development of this chronic disease. Yet, normal (NMRI) mice were protected against acute infection with dermotropic or neurotropic strains of vaccinia virus, and athymic mice were partially protected against acute lethal infection with neurotropic vaccinia virus.     

Characterization of a 1980-swine recombinant influenza virus possessing H1 hemagglutinin and N2 neuraminidase similar to that of the earliest Hong Kong (H3N2) virus

A recombinant (H1N2, formerly Hsw 1N2), A/swine/Ehime/1/80 was found to possess antigenic, biological and genomic characteristics different from those of a previous A/swine/Kanagawa/2/78 (H1N2) strain. Five monoclonal antibodies to A/NJ/8/76 definitely differentiated the hemagglutinin molecules of the former virus from the latter, showing that these viruses differed, at least, at two antigenic determinants. Neuraminidase-inhibition tests with monoclonal antibodies to different H2N2 and H3N2 viruses revealed that the A/swine/Ehime/1/80 strain contained a neuraminidase very similar to that of the late human Asian (H2N2) and the earliest Hong Kong (H3N2) viruses. Growth comparison of swine and human isolates indicated that A/swine/Ehime/1/80 and A/swine/Shizuoka/1/78 (H1N1) failed to grow at 42 degrees C, while A/swine/Kanagawa/2/78 and its possible parental virus, A/swine/Kanagawa/4/78 (H1N1) replicated efficiently at this stringent temperature. These results revealed that the viruses having growth characteristics similar to those of avian influenza virus were present in the swine population. RNA analysis by oligonucleotide mapping suggested that A/swine/Ehime/1/80 may be a recombinant between A/swine/Shizuoka/1/78-like and A/Aichi/2/68 (H3N2)-like viruses. To further determine the gene constellation of this recombinant virus, DNA-RNA hybridization was performed by using DNA segments complementary for swine (H1N1) virus RNA and the entire RNAs of three viruses. The molecular hybridization could define the genomic composition of the recombinant, indicating that only the neuraminidase gene of this virus is derived from the earliest Hong Kong (H3N2)-like virus and remaining seven genes from swine (H1N1) virus.     

The search for preparations suppressing the reproduction of the AIDS virus

Trials of clinically advantageous national inducers, thymus hormones, as well as human recombinant alpha 2-interferon were carried out in cultures of continuous lymphoblastoid cells H9/IIIB infected with HIV virus. The virus-inhibiting effect for HIV was observed with interferon in doses of 10-100 IU/ml. At a concentration of interferon of 1000 IU/ml, virus replication was inhibited completely, the interferon doses used exerting no marked toxic or antiproliferative effect on the cells. Human interferon inducers, poly(G).poly(C), PXL-6, dsRNA in concentrations of 50-100 micrograms/ml inhibited virus reproduction significantly. The highest antiviral effect was achieved with yeast dsRNA. The preparations of immunomodulators, thymarin, the 5th and 7th fractions of thymosin, noticeably stimulated proliferation of infected T-lymphocytes, reducing the relative number of cells carrying the virus-specific antigen. Combined use of preparations with different mechanisms of the antiviral effect may be advantageous in prevention and treatment of AIDS.     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.     

A novel intranasal virus-like particle (VLP) vaccine designed to protect against the pandemic 1918 influenza A virus (H1N1)

We have prepared a virus-like particle (VLP) vaccine bearing the surface glycoproteins HA and NA of the 1918 influenza A virus by infecting Sf9 cells with a quadruple recombinant baculovirus that expresses the four influenza proteins (HA, NA, M1, and M2) required for the assembly and budding of the VLPs. The presence of HA and M1 in the purified VLPs was confirmed by Western blot, and that of NA by a neuraminidase enzymatic assay. For in vivo studies, the 1918 VLP vaccine was formulated with or without an oligonucleotide containing two CpG motifs and administered in two doses 2 wk apart via the intranasal route. The antibody titers in mice immunized with VLP vaccines were higher than in mice vaccinated with an inactivated swine virus (H1N1) control, when CHO cells expressing 1918 HA were used as antigen. The opposite result was obtained when disrupted swine virus was the antigen for the ELISA test. Vaccine efficacy was evaluated by challenging immunized mice with the 1918 antigenically related influenza virus A/swine/Iowa/15/30 (H1N1) and measuring viral titers in the upper and lower respiratory tract. Mice immunized with VLP vaccine plus CpG demonstrated significantly lower viral titers in the nose and lungs than did the control on days 2 and 4 postchallenge and completely cleared the virus by day 6. Furthermore, they did not show symptoms of disease although there was a minor decrease in body weight. Mice vaccinated with VLP alone also demonstrated significantly lower viral titers in the nose and lungs than did the placebo group as well as the inactivated virus group on days 4 and 6 postchallenge. These results suggest that it is feasible to make a safe and immunogenic vaccine to protect against the extremely virulent 1918 virus, using a novel and safe cell-based technology.     

Identification and characterization of a highly virulent triple reassortant H1N1 swine influenza virus in the United States

A highly virulent H1N1 influenza A virus, A/Swine/Kansas/77778/2007 (KS07), which caused approximately 10% mortality in finishing pigs, was isolated from herds in the Midwestern United States. Molecular and phylogenic analysis revealed this swine isolate was a triple reassortant virus, similar to an H1N1 virus that infected humans and pigs at an Ohio county fair in August 2007. A pig challenge model was developed to evaluate the pathogenicity and transmission capacity of the KS07 virus. The results confirmed that the KS07 virus is highly virulent in pigs and easily transmitted to sentinel animals. The KS07 virus failed to cross-react with a panel of H1-specific swine sera. Interestingly, the KS07 virus shed for a prolonged period up to 7 days in infected pigs, indicating that this virus can spread efficiently between animals. The highly virulent H1N1 swine influenza virus is further evidence of reassortment among avian, human and swine influenza viruses and justifies the need for continued surveillance of influenza viruses in swine.     

Factors influencing helper-independent adeno-associated virus replication

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.