Mouse-adapted Influenza Virus Research Articles

Recombinant neuraminidase vaccine protects against lethal influenza

The N2 neuraminidase gene of A/Victoria/3/75 influenza virus was engineered to encode a secretable protein (NAs) by replacing the natural N-terminal membrane anchor sequence with the cleavable signal sequence of the corresponding influenza hemagglutinin gene. Soluble NAs was expressed by a baculoviruslinsect cell system and accumulated in the medium at levels between 6 and 8 μg ml −1. A combination of biochemical and standard chromatographic techniques allowed the purification of NAs to homogeneity. Crosslinking analysis indicated that NAs was partly recovered as an authentic tetrameric protein, while the remaining fraction was composed of dimeric molecules and small amounts of monomeric NAs. Purified NAs was supplemented with low-reactogenic adjuvants and used to immunize mice. After a challenge infection with a lethal dose of homologous mouse-adapted X47 influenza virus, vaccinated animals showed resistance against severe disease symptoms and were protected from lethality. Based on the results of a passive immunization experiment, it may be concluded that preformed antibody plays a central role in the mechanism by which vaccination with NAs confers viral protection.

Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,4′-trihydroxy-8-methoxyflavone, from the roots of Scutellaria baicalensis

When mouse-adapted influenza virus A/PR/8/34 ( A PR8 ) (10 PFU/cell) was adsorbed to Madin-Darby canine kidney (MDCK) cells at 4°C for 1 h and incubated at 37°C, release of the virus from the cells was detected in the medium from 4 h after incubation and reached to plateau at 8 h. However, 5,7,4′-trihydroxy-8-methoxyflavone (F36) from the roots of Scutellaria baicalensis significantly reduced this single-cycle replication of A PR8 from 4 h to 12 h after incubation by dose-dependent manner and the dose which decrease the virus titer one tenth was 11 μM. F36 (50 μM) did not inhibit the adsorption of A PR8 to MDCK cells, but reduced release of the virus in the medium, when it was added at 0 or 2 h after the incubation. The cell-associated virus determined by sialidase activity was also reduced by F36 treatment at 0 or 2 h. F36 also inhibited the fusion of A PR8 with liposomes containing bovine brain mixed gangliosides at pH 5.0. However, F36 little affected on the elongation activity of the viral RNA-dependent RNA polymerase in vitro. These results suggest that F36 reduces the replication of A PR8 by inhibiting the fusion of the virus with endosome/lysosome membrane which occurs at early stage of virus infection cycle. Whereas, when F36 was added to the MDCK cells infected with A PR8 at 3 or 4 h after incubation, release of the virus in the medium was reduced but the cell-associated virus was increased in comparison with control. Scanning and transmission immunoelectron microscopic studies revealed that F36 inhibited the budding of progeny A PR8 from the MDCK cell surface and microvilli, when it was added at 3 h after incubation. The accumulation of the A PR8 antigen was observed on the cell surface by immunofluorescence and transmission immunoelectron microscopies by the addition of F36. These results suggest that F36 also shows anti-influenza virus activity against A PR8 by inhibiting the budding of the progeny virus from the cell surface, when it was added at budding stage of virus infection cycle.

Cytokines and the acute phase response to influenza virus in mice.

This study characterized selected aspects of the acute phase response after intranasal inoculation of mice with two doses of mouse-adapted influenza virus differing in lethality. Mice given 140 plaque-forming units (PFU) of virus (58% survival) gradually decreased food and water intake to nearly zero over 6 days; survivors then slowly increased intakes. Declines in these behaviors were parallel to decreases in body temperature and general locomotor activity and were associated with elevated activities of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferons in lung lavage fluid. Circulating levels of these cytokines were not increased. After 55,000 PFU of virus (100% mortality), food and water intake fell to near zero within 48 h, temperature and locomotor activity decreased significantly, and activities of IL-1 and IL-6 were elevated in lung lavage fluid. These data show that cytokine activities in the lungs are elevated in a time frame that supports the hypothesis that cytokines could mediate behavioral and physiological changes in mice during acute influenza infections.

Antiviral activity of plant flavonoid, 5,7,4'-trihydroxy-8-methoxyflavone, from the roots of Scutellaria baicalensis against influenza A (H3N2) and B viruses.

We investigated effects of isoscutellarein-8-methylether (5,7,4'-trihydroxy-8-methoxyflavone, F36) from the roots of Scutellaria baicalensis on the single-cycle replication of mouse-adapted influenza viruses A/Guizhou/54/89 (H3N2 subtype) and B/Ibaraki/2/85 in Madin-Darby canine kidney (MDCK) cells. The agent suppressed replication of these viruses from 6 to 12 h after incubation in a dose-dependent manner by 50% at 20 microM and 90% at 40 microM, respectively. F36 (50 microM) reduced the release of B/Ibaraki virus in the medium by 90-93% when it was added to the MDCK cells at 0 to 4 h after incubation. The cell-associated virus determined by sialidase activity was also reduced by the treatment at 0 to 4 h. F36 (120 microM) inhibited the low pH-dependent membrane fusion of both the viruses with the liposome containing mixed gangliosides from bovine brain. However, the agent little affected the hemagglutination and RNA-dependent RNA polymerase activities of these viruses in vitro. These results suggest that F36 inhibits the replication of A/Guizhou and B/Ibaraki viruses at least partly by inhibiting the fusion of viral envelopes with the endosome/lysosome membrane which occurs at the early stage of the virus infection cycle. F36 (0.5 mg/kg) showed no antiviral activity against A/Guizhou and B/Ibaraki viruses in mice when administered intranasally 5 min prior to virus inoculation, whereas it significantly inhibited their proliferation in the mouse lung when administered intranasally 7 times (total 3.5 mg/kg) from 18 h before to 54 h after virus infection.

In vivo anti-influenza virus activity of Kampo (Japanese herbal) medicine “Sho-seiryu-to” and its mode of action

When BALB/c mice were treated with a Kampo (Japasese herbal) medicine “Sho-seiryu-to” (SST) (2 g/kg, 10 times) orally from 7 days before to 4 days after the infection and infected with mouse-adapted influenza virus A/PR/8/34 by nasal site-restricted infection, replication of the virus in the nasal cavity and spread of the virus to the lung were efficiently inhibited at 5 days after infection in comparison with water-treated mice. However, another Kampo medicine “Kakkon-to” showed no anti-influenza virus activity in the same condition. The antiviral IgA antibody in the nasal and broncho-alveolar washes of the SST treated mice increased significantly in comparison with that of water-treated control. Oral administration of SST (2 g/kg, 18 times) from 7 days before to 13 days after vaccination also significantly augmented serum hemagglutination-inhibiting antibody by nasal inoculation of influenza HA vaccine (5 μg/mouse) that was insufficient to induce antiviral antibody. SST did not inhibit the replication of mouse-adapted influenza virus A/PR/8/34 in Madin-Darby canine kidney cells. SST also did not inhibit the influenza virus sialidase activity against sodium p-nitrophenyl- N-acetyl- α-D-neuraminate and hemagglutination by mouse-adapted influenza virus A/PR/8/34. SST showed no influence on interferon production in nasal wash of mice at 5 days after the virus infection. These results suggest that SST confers better protection against influenza virus infection through augmentation of production of antiviral IgA antibody but not direct action to the virus, and can be used as an adjuvant to nasally inoculated influenza HA vaccine.

The influenza virus variant A/FM/1/47-MA possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth.

Genetic analysis of mouse-adapted influenza virus variant A/FM/1/47 (FM) MA has previously identified four genome segments, 4, 5, 7, and 8, that are statistically associated with virulence. On sequencing these genome segments, we found single amino acid replacements at amino acid 47 of the HA2 subunit of the hemagglutinin and at amino acid 139 of the matrix protein. Mutation was not detected in segments 5 and 8, obviating a role for these genes in FM-MA virulence. FM-MA replicates to higher titer than FM in MDCK cells and in mouse lung. FM X FM-MA reassortants were used to show that the M1 gene controlled replication in MDCK cells as well as in mouse lung.

Behavioral thermoregulation in mice inoculated with influenza virus

Mice housed at 30°C and inoculated with a mouse-adapted influenza virus show a fall in body temperature ( T b) and a decrease in food intake to almost 0 grams per day. This study tested whether the fall in T b could be accounted for by the decreased food intake and whether the fall in T b was due to a decrease of thermoregulatory set point or to an inability to maintain T b at set point level. The fall in T b of influenza-infected mice was greater than that of food-deprived mice. When food deprived, mice given access to a thermal gradient increased their preference for warmer areas in the gradient and, as a result, T b did not fall as much as T b of starved mice not given access to a thermal gradient. When infected with influenza virus, mice given a thermal gradient decreased T b less and at a slower rate than mice not given a gradient. However, this fall in T b of influenza-infected mice was greater than that of food-deprived mice given a thermal gradient. Mice given a thermal gradient increased their preference for the warmer temperatures after inoculation; this returned to preinoculation preference for cooler temperatures during the later days of infection despite a continuous fall in T b. Influenza-infected mice given a thermal gradient survived significantly fewer days than infected mice not given a thermal gradient. We conclude that the influenza-induced fall of T b in mice cannot be explained solely by the decrease in food intake, and is partially due to a decrease in thermoregulatory set point.

In vivo anti-influenza virus activity of plant flavonoids possessing inhibitory activity for influenza virus sialidase

Isoscutellarein (5,7,8,4′-tetrahydroxyflavone) from the leaf of Scutellaria baicalensis non-competitively inhibited (IC 50, 20 μM) the hydrolysis of sodium p- nitrophenyl-N- acetyl-α- D - neuraminate by influenza virus sialidase with an apparent K i value of 41 μM. Negligible inhibitory activity was observed for mouse liver sialidase at a concentration of 79 μM. Isoscutellarein also inhibited the replication of influenza virus A/WSN/33 in Madin-Darby bovine kidney cells with 50% virus inhibitory dose at 16 nmol/well and influenza virus A/PR/8/34 in the allantoic sac of embryonated egg with little toxic effects. The flavone showed significant anti-influenza virus activity in vitro similar to isoscutellarein-8-methylether (F36) (Nagai, T., Miyaichi, Y., Tomimori, T., Suzuki, Y. and Yamada H., 1990, Chem. Pharm. Bull. 38, 1329–1332), and more potent virucidal activity in ovo than F36. However, F36 completely prevented proliferation of mouse-adapted influenza virus A/PR/8/34 in mouse lung by the intranasal (0.5 mg/kg) and intraperitoneal (4 mg/kg) administrations, and it was more potent than the known anti-influenza virus substance, amantadine. Intranasal administration of F36 (0.5 mg/kg) also protected mice against a lethal. influenza virus A/PR/8/34 infection. Isoscutellarein significantly inhibited lung virus proliferation when administered intranasally or orally to mice. F36 and isoscutellarein showed negligible toxic effect against mice. These results suggested that flavones, which have potent influenza virus sialidase inhibitory activity, have anti-influenza virus activity in vivo.

Anti-influenza response achieved by immunization with a synthetic conjugate.

The peptide corresponding to sequence 91--108 of the hemagglutinin of type A H3N2 influenza virus has been synthesized by the solid-phase peptide synthesis method and covalently attached to several macromolecular carriers. The conjugate with tetanus toxoid was used for immunization of rabbits and mice. The immunoglobulin fraction of the rabbit antiserum showed the presence and antipeptide antibodies by both agar gel diffusion and radioimmunoassay. In the latter assay, the antibodies showed marked crossreactivity with the intact virus of the A/Texas/77 strain. The antibodies were also capable of inhibiting the hemagglutination of chicken erythrocytes by the virus; the highest hemagglutination inhibition titer (1:32) was achieved with a serum-resistant strain of A/Texas/77. When the in vitro virus plaque formation assay was used with monolayers of Madin--Darby canine kidney (MDCK) cells, the number of plaques was reduced on interaction with the immunoglobulin fraction of the antiserum, which was effective up to a dilution of 1:32. Preliminary results indicate that C3H/DiSn mice immunized with the peptide--tetanus toxoid conjugate are partially protected against a further challenge with A/Texas mouse-adapted influenza virus. The results are thus indicative of the efficacy of the synthetic material in eliciting anti-influenza immune response.

From synthetic antigens to synthetic vaccines

The availability of synthetic antigens permitted a systematic elucidation of the molecular basis of antigenicity, as well as of other phenomena, and to establish the genetic control of immune response and its link to the major histocompatibility region of the species. Moreover, it allowed a conceptual approach to production of vaccines made up of synthetic fragments of relevant virus antigens. This was first demonstrated with a bacteriophage, MS-2, that can be efficiently inactivated with antibodies obtained with an immunogen in which a synthetic peptide corresponding to a sequence of 20 amino acid residues within its coat protein was covalently linked to a polymeric synthetic carrier. When the synthetic adjuvant N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) was chemically linked to the same molecule, the resulting immunogen was effective in aqueous solution. This approach has now been used for diphtheria: a synthetic peptide from the A-chain of the toxin, covalently attached together with MDP to the synthetic carrier multichain poly(DL-alanine) induced antibodies cross-reactive with the diphtheria toxin as well as protection against the dermonecrotic activity of the toxin. A similar approach has been described also by R. Arnon and her colleagues in our laboratory for influenza virus, making use of a peptide with sequence 91–108 of the hemagglutinin of type A H3N2 strains attached to tetanus toxoid. The results indicated that the conjugate led to partial protection against A/Texas mouse-adapted influenza virus.

Caractères biologiques de souches du virus de l'influenza adaptées à 29 °C et à 41 °C

Two mouse-adapted influenza virus strains were adapted to growth at 29 °C by a gradual lowering of the temperature of incubation in embryonated eggs. The growth of these two cold variants was completely inhibited at 41 °C. The cold and hot variants showed no significant difference in rise in the infectious titer in the mouse lung but a marked reduction in mortality and pathological response was observed with the cold variants, while good antibody response was stimulated in both cases. The cold variants were better interferon inducers in chick embryo. However, neither cold nor hot variants induced more than detectable amounts of interferon in blood, spleen, or lungs of infected mice. These cold variants possess many characteristics suitable for an effective live virus vaccine.

Delay of Fatal Pneumonia in X-Irradiated Mice Inoculated with Mouse-Adapted Influenza Virus, PR8 Strain

Adult male albino mice were infected by intranasal and airborne routes with highly adapted Type A influenza virus, PR8 strain, one day after whole-body X-irradiation. Graded amounts of X-rays had little effect, if any, on susceptibility to this virus as measured by infectivity and mortality. However, pretreatment with 350 R reduced the frequency of early deaths due to influenzal pneumonia but did not prevent later deaths. The findings demonstrate that the number of days to death in irradiated mice inoculated with influenza virus is independent of the mortality rate and should not be ignored in evaluating radiation effects in infectious processes.

Effect of an Exogenous Interferon on Experimental Influenza Virus Infection and Interferon Production in White Mice

WE have investigated the effect of an exogenous interferon obtained from inbred albino mice (asw strain) after infection with a mouse-adapted influenza virus (strain Em2, type A1) on the dynamics of the influenza virus infection, in parallel with interferon production.

Multiplication of mouse-adapted influenza virus in mouse lungs after infection with very low doses.

WE studied the dynamics of the infection with influenza virus in mice after intra-nasal administration of extremely low doses. White mice were infected with the mouse-adapted Em2 strain of the A1 type influenza virus. The virus, used in the form of infected allantoic fluid, showed LD50 titres of 105.1 and 105.45 0.1 ml., respectively, as estimated by intra-nasal inoculation of white mice, and a TCID50 titre of 104.88 and 105.52 0.1 ml., respectively, as determined in suspended chlorio-allantoic membrane tissue cultures1—herein designated as Horvath's TC. Mice weighing 16–18 gm. were inoculated under light ether anaesthesia intra-nasally in individual experiments (altogether 6 experiments in 3 independent series) with 0.05 ml. of infected allantoic fluid, containing 10−2.12–100.88 TCID50 0.1 ml.; in other words, in each case with a lower dose than 1 mouse LD50. Starting with the first day after inoculation, 3 mice were killed daily and from their lungs 10 per cent suspensions in Horvath's1 nutrient medium were prepared. The suspensions were clarified by centrifugation at 2,500 r.p.m. for 10 min. and serial 10-fold dilutions of them inoculated into Horvath's TC. The cultures were incubated at 35° C. for 72 hr. and the reproduced virus detected by haemagglutination test on plastic plates2. The results are shown in Fig. 1. It can be seen that the multiplication of the virus in the lungs depends on the dose of the virus administered and shows a cyclic character. After administration of the lowest dose (0.0038 TCID50 in 0.05 ml.), virus was first detected three days after infection. It is still present on the fourth day, but thereafter disappears and is not detectable up to day 12. After administration of a 10-times higher dose the virus appears in the lung also on day 3, it is not detectable for the next five days and appears repeatedly on day 9 or 10 after infection. The higher the dose administered, the shorter the time intervals of the cyclic appearance and disappearance of virus in the lungs. After infection with 2.6 TCID50 and 3.8 TCID50 in 0.05 ml., respectively, a complete disappearance of virus from lungs was not detected during the whole observation period, although even in these cases an appreciable variation in infectivity titres was seen. It is to be noted that the first disappearance of virus and/or a marked lowering of its titre occurred in all experiments 4–5 days after infection. Virus-neutralizing antibodies were not detected in sera of mice for 10–14 days after infection.

Comparison of quantity of egg and mouse-adapted influenza viruses required to infect each host.

SummaryParallel titrations to determine the dilution of virus essential to initiate infection in the allantoic membrane of the embryonated egg and the mouse lung were carried out. In every instance with mouse-adapted viruses, the relative quantity essential to infect the mouse and the chick embryo was approximately the same; with egg-adapted viruses approximately 2 logs more virus was required to initiate multiplication in the mouse lung than in the allantoic sac. These data describe another property of mouse-adapted influenza viruses which distinguish them from unadapted strains.

Nontoxic Influenza Virus

Summary The production of nontoxic influenza A virus has been described. This was accomplished by inoculating undiluted, non-mouse-adapted, allantoic fluid virus into the allantoic sac of the developing chick embryo. Mouse-adapted influenza virus would not cause nontoxic virus to be produced. Exactly why the virus grows as a nontoxic suspension is not understood. The virus particle, however, appears to be responsible in part, although a soluble factor has not been excluded. The nontoxic virus appears to be as good an antigen as the toxic. Although most of the attempts to produce nontoxic virus meet with success some failures are encountered. Some of the problems for further study have been set forth.