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Composition of diet modifies toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cold-adapted rats.

The effect of a high carbohydrate, high fat or high protein diet was studied on the acute toxicity of TCDD (125 micrograms/kg) in cold-adapted (4 +/- 1 degrees C) rats. Within 10 days after dosing, TCDD-treated rats fed a high carbohydrate or a high protein diet reduced their caloric intake by 25% whereas those fed a high fat diet consumed only 15% fewer kcal/MBS (metabolic body size). TCDD-treated rats fed a high protein diet lost body weight at the same rate as their pair-fed controls, whereas body weight loss in high fat-fed rats was significantly higher than in their pair-fed controls. In contrast, TCDD-treated rats fed a high carbohydrate diet effectively maintained their body weight in the 4 days immediately after TCDD dosage, whereas their pair-fed controls lost weight. Mortality in TCDD-treated animals was 100% irrespective of the diet; all pair-fed control rats (except one fed a high protein diet) were terminated on days corresponding to the spontaneous death of their TCDD-treated pairs. Mean time to 50% mortality and mean time to death were significantly longer in TCDD-treated rats fed a high carbohydrate diet in comparison with the other two TCDD-treated groups (p less than 0.05), although caloric intake was comparable. Serum triiodothyronine (T3) was reduced in TCDD-treated animals fed a high fat or a high carbohydrate diet but not in those fed a high protein diet; serum thyroxine (T4) was reduced in all the treated groups, irrespective of diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Acclimatization↗

Cold adaptation of a psychrophilic chitinase: a mutagenesis study.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

Adaptation, Biological↗

Interference between cold-adapted (ca) influenza A and B vaccine reassortants or between ca reassortants and wild-type strains in eggs and mice.

Interference between pairs of cold-adapted (ca) influenza A and B vaccine reassortant viruses and between ca influenza B reassortant viruses and wild-type influenza strain A/PR/8/34 was studied in embryonated eggs and/or in the respiratory tract of three strains of mice. Interference in eggs between ca A and B reassortants occurred after 2 days but was masked by later cycles of growth. When doses of 10(6) EID50 of the reassortant viruses B/60/32/R and A/47/6/R were administered to outbred mice, interference with the growth of A/47/6/R occurred in the turbinates; interference with the growth of B/60/32/R did not occur. However, interference with the growth of B/60/32/R was noted in the lungs of Balb/c mice after co-infection with 10(6) EID50 of both A/PR/8/34 and B/60/32/R. Interference did not occur when the dose of B/60/32/R was increased to 10(7) EID50 per mouse. When CBA mice were co-infected with 10(6) EID50 of both B/60/32/R and the influenza A ca reassortant R/34, or 10(6) EID50 of B/60/32/R,R/34 and A/Leningrad/134/57, interference did not occur. Interference could not be detected in mice following dual infection from serum responses to individual viruses.

Animals↗

Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines.

Clinical studies previously demonstrated that live influenza A virus vaccines derived by genetic reassortment from the mating of influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus with epidemic wild-type influenza A viruses are reproducibly safe, infectious, immunogenic, and efficacious in the prevention of illness caused by challenge with virulent wild-type virus. These influenza A reassortant virus vaccines also express the ca and temperature sensitivity (ts) phenotypes in vitro, but the genes of the ca virus parent which specify the ca, ts, and attenuation (att) phenotypes have not adequately been defined. To identify the genes associated with each of these phenotypes, we isolated six single-gene substitution reassortant viruses, each of which inherited only one RNA segment from the ca parent virus and the remaining seven RNA segments from the A/Korea/1/82 (H3N2) wild-type virus parent. These were evaluated in vitro for their ca and ts phenotypes and in ferrets, hamsters, and seronegative adult volunteers for the att phenotype. We found that the polymerase PA gene of the ca parent specifies the ca phenotype and that the PB2 and PB1 genes independently specify the ts phenotype. The PA, M, PB2, and PB1 genes of the ca donor virus each contribute to the att phenotype. The finding that four genes of the ca donor virus contribute to the att phenotype provides a partial explanation for the observed phenotypic stability of ca reassortant viruses following replication in humans.

Adult↗

[Safety and high immunogenicity for children 3 to 15 of a mono- and a divaccine from recombinant influenza A/Brazil /11/78 (H1N1) and A/Bangkok/1/79 (H3N2) viruses obtained from a special donor attenuation base--the cold-adapted virus A/Leningrad/134/47/57 (H2N2)].

The data on complete safety and high immunogenic potency of intranasal live influenza vaccine from the recombinant A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) strains prepared with a special donor of attenuation, a cold-adapted A/Leningrad/134/47/57 (H2N2) virus, have been obtained. The possibility of using a bivaccine from the recombinant strains constructed on the basis of a single donor of attenuation and inheriting from it most of the genes coding for nonglycosylated proteins was demonstrated. Administration of this bivaccine to children was not accompanied by any increase in the rate of postvaccination febrile reactions, but produced in the vaccinees an intensive immune response to the antigenic components of the vaccine which was in agreement with the data obtained after separate administration of monovaccines.

Adaptation, Physiological↗

A mutation in the PA protein gene of cold-adapted B/Ann Arbor/1/66 influenza virus associated with reversion of temperature sensitivity and attenuated virulence.

Reassortant SG3 inherits only the acidic polymerase (PA) protein gene from the cold-adapted B/AA/1/66 influenza virus (ca B/AA/1/66) and all remaining genes from a virulent, wild-type virus. This reassortant demonstrates attenuated virulence in ferrets and expresses a ts phenotype characteristic of the ca parent. During virulence evaluation of SG3, a virulent, non-ts revertant virus (designated SG3rFL) was isolated from the lungs of one ferret. In order to determine whether the reversion of SG3 resulted from mutation of the PA gene and/or as the result of extragenic supressor mutations, the revertant PA gene of SG3rFL was transferred to a reassortant (SG3r) inheriting only the revertant PA gene from SG3rFL and all remaining genes from SG3. Reassortant SG3r was non-ts and virulent, indicating that mutation of the PA gene was sufficient for the reversion of the ts and attenuation phenotypes expressed by SG3rFL. The nucleotide and predicted amino acid sequences of the SG3rFL PA gene were determined and compared to those of wt and ca B/AA/1/66. The predicted PA proteins of wt and ca B/AA/1/66 are known to differ by six amino acid substitutions including a valine to methionine substitution at residue 431. The PA proteins of ca B/AA/1/66 and SG3rFL were distinguished by only the single amino acid substitution of methionine to isoluecine also occurring at residue 431. Thus, the methionine residue was implicated in the attenuation of ca B/AA/1/66 and its reassortants. The hydropathic properties of valine, isoleucine, and methionine suggested that reversion involved the restoration of hydrophobic character at this site.

Amino Acid Sequence↗

[The selection of cold-adapted variants of the influenza viruses H1N1 and H3N2 and their antigenic and genetic characteristics].

As a result of serial passages (42 passages) at low temperatures (26 degrees--28 degrees C) of two influenza H1N1 and H3N2 virus strains stable cold-adapted (ca) variants were produced. Investigations of them showed the ca A/USSR/03/84 (H1N1) variant to have ts-mutations in genes 1, 2, 4, 5, 7, and 8 and the ca A/USSR/215/79 (H3N2) to have ts-mutations in genes 1, 3, 4, 5, 7 and 8. These ca-variants may be recommended as attenuation donors to be used in recombination experiments with epidemic influenza viruses in order to obtain attenuated reassortant candidate vaccine strains.

Adaptation, Physiological↗

Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides.

In order to improve the thermal stability (t(1/2)) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane-pyridine complex as reducing agent, increased thermal stability (t(1/2), 168 min at 70 degrees C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t(1/2), 18 min at 70 degrees C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane-pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.

Candida↗

Efficacy of a cold-adapted, intranasal, equine influenza vaccine: challenge trials.

A randomised, controlled, double-blind, influenza virus, aerosol challenge of horses was undertaken to determine the efficacy of a cold-adapted, temperature sensitive, modified-live virus, intranasal, equine influenza vaccine. Ninety 11-month-old influenza-naïve foals were assigned randomly to 3 groups (20 vaccinates and 10 controls per group) and challenged 5 weeks, 6 and 12 months after a single vaccination. Challenges were performed on Day 0 in a plastic-lined chamber. Between Days 1 and 10, animals were examined daily for evidence of clinical signs of influenza. Nasal swabs for virus isolation were obtained on Day 1 and Days 1 to 8 and blood samples for serology were collected on Days 1, 7 and 14. There was no adverse response to vaccination in any animal. Following challenge at 5 weeks and 6 months, vaccinates had significantly lower clinical scores (P = 0.0001 and 0.005, respectively), experienced smaller increases in rectal temperature (P = 0.0008 and 0.0007, respectively) and shed less virus (P<0.0001 and P = 0.03, respectively) over fewer days (P<0.0001 and P = 0.002, respectively) than did the controls. After the 12 month challenge, rectal temperatures (P = 0.006) as well as the duration (P = 0.03) and concentration of virus shed (P = 0.04) were significantly reduced among vaccinated animals. The results of this study showed that 6 months after a single dose of vaccine the duration and severity of clinical signs were markedly reduced amongst vaccinated animals exposed to a severe live-virus challenge. Appropriate use of this vaccine should lead to a marked reduction in the frequency, severity and duration of outbreaks of equine influenza in North America.

Administration, Intranasal↗

Direct and total effectiveness of the intranasal, live-attenuated, trivalent cold-adapted influenza virus vaccine against the 2000-2001 influenza A(H1N1) and B epidemic in healthy children.

BACKGROUND: The efficacy of the intranasal, live-attenuated, trivalent cold-adapted influenza virus vaccine (CAIV-T) against influenza A(H3N2) and B infections in healthy persons is established, but its effectiveness against natural influenza A(H1N1) infection is unknown. OBJECTIVE: To assess the effectiveness of CAIV-T in healthy children during the 2000-2001 influenza A(H1N1) and B epidemic. DESIGN: Community-based, nonrandomized, open-label trial from August 1998 through April 2001. SETTING: Intervention and comparison communities in central Texas. PARTICIPANTS: Healthy children, aged 1.5 to 18 years, from the intervention communities received a single dose of CAIV-T at least 1 time or more in 1998, 1999, and/or 2000. MAIN OUTCOME MEASURES: The incidence of medically attended acute respiratory illnesses during the 2000-2001 influenza epidemic was compared in 3794 health plan CAIV-T recipients with age-eligible, health plan nonrecipients in the intervention communities for direct effectiveness (n = 9325), and with those in the 2 comparison communities for total effectiveness (n = 16,264). RESULTS: The 2281 CAIV-T recipients in 2000 had significant direct protection against medically attended acute respiratory illness of 18% to 20% during the biphasic influenza A(H1N1) and B epidemic, and 17% to 26% during influenza A(H1N1) predominance. The 931 recipients of CAIV-T in 1999 containing influenza A/Beijing/262/95(H1N1) and B/Beijing/184/93-like viruses had persistent heterovariant protection against the 2000-2001 influenza A/New Caledonia/20/99(H1N1) and B/Sichuan/379/99 variants. The 616 recipients of a single CAIV-T dose in 1999 only, including those younger than 5 years with no prior natural exposure to influenza A(H1N1) viruses, showed persistent protection. CONCLUSION: Healthy children who received CAIV-T in 2000 or 1999 were protected against new variants of influenza A(H1N1) and B in the 2000-2001 influenza epidemic.

Acute Disease↗

Nucleotide sequences of the neuraminidase genes of influenza A/Leningrad/134/57 (H2N2) virus and two of its live, attenuated, cold-adapted variants.

The nucleotide sequences of the neuraminidase (NA) genes of the A/Leningrad/134/57 (H2N2) wild-type (Len/wt) virus as well as two of its live attenuated, cold-adapted (ca) variants, A/Leningrad/134/17/57 (Len/17) and A/Leningrad/134/47/57 (Len/47), were determined. In comparison with Len/wt, one nucleotide change (C-225 to A) was found in the NA gene of Len/17. This change codes for a Thr-to-Asn substitution at position 69 of NA. The NA gene of the more attenuated Len/47 ca virus has one silent (T-814 to C) and two coding nucleotide substitutions, C-78 to T (Ala-20 to Val) and C-225 to A (Thr-69 to Asn). These sequence data were used to design a PCR-restriction technique to determine the origin of the NA gene in candidate live, attenuated vaccine reassortants made by reassorting these ca strains with current field viruses.

Amino Acid Sequence↗

Infectivity and reactogenicity of reassortant cold-adapted influenza A/Korea/1/82 vaccines obtained from the USA and USSR.

The safety and immunogenicity of two live influenza A virus vaccine strains, the CR 59 and 17/25/1 cold-adapted (ca) reassortants, were evaluated in 170 healthy young adult volunteers. The vaccines were produced by recombining A/Korea/1/82 (H3N2) wild-type virus with either A/Ann Arbor/6/60 (H2N2) or A/Leningrad/134/17/57 (H2N2) ca donors of attenuation. Both vaccines were well tolerated in volunteers. The 17/25/1 strain, prepared from A/Leningrad, infected at least 70% of seronegative volunteers after the first dose and 84% after the second; the CR 59 strain infected 62% and 72% of volunteers after first and second doses, respectively. Among the vaccinees who were initially seropositive, 17/25/1 infected 66% after one dose and 85% after two, while CR 59 infected 62% and 71%, respectively. Despite differences in temperature sensitivity, genetic composition, and serological reactivity to monoclonal antibodies, both vaccines behaved almost identically in animal models and man. We conclude that both donors of attenuation may be of great potential value.

Adolescent↗

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate.

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.

Adaptation, Physiological↗

Sensitization of mice with wild-type and cold-adapted influenza virus variants: immune response to two H1N1 and H3N2 viruses.

Two A strain influenza viruses, A/Hong Kong/123/77 (A/HK/123/77) (H1N1) and A/Queensland/6/72 (A/Qld/6/72) (H3N2), and the two cold-adapted reassortants which possess the surface antigens of these strains (CR35 and CR6, respectively) were tested for their ability both to induce primary cytotoxic T-cell (Tc cell) responses in mice and to sensitize mice for a second Tc cell response when challenged with a distantly related A strain virus, A/Shearwater/72 (H6N5). After intranasal inoculation, A/Qld/6/72 replicated to higher titers in the lung (1 to 2 log10 50% egg infective doses) than did A/HK/123/77 or either of the reassortants. A/Qld/6/72 induced higher Tc cell responses in the lung than did CR6, and both were more effective than either A/HK/123/77 or CR35 in this respect. When similar doses (10 or 10(3) hemagglutinin units) of each virus were injected intravenously into mice and the spleens were tested for Tc cell activity 6 days later, both A/Qld/6/72 and CR6 were ca. 100-fold better at inducing a primary Tc cell response than A/HK/123/77 or CR35. In contrast, the H1N1 and H3N2 viruses gave rather similar anti-hemagglutinin antibody titers (after intravenous injection) and delayed-type hypersensitivity reactions (after subcutaneous injection). If mice were primed with a low dose of these viruses (10(4) 50% egg infective doses intranasally), A/Qld/6/72 and CR6 were more effective than A/HK/123/77 or CR35 at sensitizing for a secondary Tc cell response when challenged with A/Shearwater/72, but if larger doses were given either intranasally (10(6) 50% egg infective doses) or intravenously (10 to 10(3) hemagglutinin units), all viruses sensitized the mice equally well, despite the fact the A/Shearwater/72 gives a poor primary Tc cell response in mice. Thus, the viral glycoprotein antigens can be important in determining the immunogenicity of the virus and, particularly, the class I antigen-restricted Tc cell response of the host.

Animals↗

The HA1 of cold-adapted influenza B vaccine is not altered during replication in human vaccinees.

Influenza viruses recovered from 14 children 2-10 days after vaccination with an egg-grown, cold-adapted influenza B vaccine (B/AA/1/86) were analyzed. Hemagglutination-inhibition (HI) assays using monoclonal antibodies did not detect antigenic differences between the vaccine strain and the viruses recovered from the vaccinees. Furthermore, nucleotide sequence analysis of the HA1 region did not reveal any changes compared to the sequence of the vaccine strain. These findings indicate that influenza B vaccine hemagglutinin is genetically stable during replication in human vaccinees.

Animals↗

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus.

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2 +/- 0.2 microM and 11.2 +/- 0.2 microM at 5 degrees C and 20 degrees C respectively.

Bacillus↗

Class-specific antibody responses in school children vaccinated with an A/Brazil/11/78 (H1N1)-like recombinant influenza virus prepared from the A/Leningrad/134/57 paediatric cold-adapted donor strain.

Forty-three school children from 8 to 11 years old were vaccinated intranasally with two doses of a paediatric attenuated influenza vaccine developed by reassortment between cold-adapted A/Leningrad/134/57(H2N2) and an A/Brazil/11/78(H1N1)-like strain. Two vaccine doses were administered 1 month apart in a randomized, blind, placebo-controlled study. Although the first vaccine dose had a low infectivity titre, overall 65% of children who received two doses of vaccine showed serological evidence of infection by HI tests. Serum IgA antibody responses against the vaccine strain were detected in nearly 50% of the vaccines and serum IgG antibody responses were detected in approximately equal to 40% by an enzyme immunoassay.

Antibodies, Viral↗

Developments with antarctic microorganisms: culture collections, bioactivity screening, taxonomy, PUFA production and cold-adapted enzymes

There have been recent research developments with Antarctic prokaryotes in the areas of isolations of novel bacterial, culture collections, bioactivity screening, taxonomy, production of polyunsaturated fatty acids (PUFAs), cold-adapted enzymes and bioremediation. Research to date confirms the novelty of bacteria isolated from this extreme environment. Opportunities now exist to exploit these and other findings to develop possible new biotechnological products from Antarctic microorganisms.

Journal Article↗