Influenza virus reproduction in the MDCK cells adapted to growth in serum-free Hybris-2 medium

Whether the MDCK cell line might adapt to grow in serum-free Hybris-2 medium and influenza viruses might be reproduced in the adapted cells was studied. Seventeen passages using the Hybris-2 medium yielded cells adapted to growth in this medium (MDCK-BS). The reproduction of influenza A (H1N1 and H3N2) and B viruses versus the cells cultured in Eagle's medium was studied. The laboratory strain of influenza A/Aichi/1/68 (H3N2) and the strain B/Ohio/01/05 of influenza B in equal titers were shown to be reproduced in both control cells on Eagle's medium and MDCK-BS cells adapted to growth in the Hybris-2 medium. The reproduction of the strains A/Brisbane/10/07 (H3N2) and A/Solomon Islands/3/06 (H1N1) was less active in the MDCK cells. Each strain of influenza viruses displayed varying infective activities. The developed serum-free Hybris-2 medium may be used for cultivation of monolayer continuous MDCK cells and for their reproduction of influenza A and B viruses.     

The use of components of plant origin in the development of production technology for live cold-adapted cultural influenza vaccine

The impact of culturing conditions (multiplicity of cell culture infection with influenza virus, composition of growth and maintenance nutrient media) for the efficiency of multiplication of cold-adapted reassortant vaccine strains of influenza A and B viruses was evaluated. Soybean hydrolysate protein-based biological additive to nutrient medium provided effective reproduction of influenza A virus in MDCK cells in the presence of 2 μg/ml trypsin. The use of soybean peptone-based stabilizer provided retention of infectious titer of influenza virus grown in MDCK culture after its lyophilization to a level of 8.5 lg EID₅₀/ml. Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 3, pp. 157–160, August, 2008     

Partial change of the ts-phenotype of cold-adapted influenza virus strain grown in canine kidney cells in the presence of trypsin

The cold-adapted temperature-sensitive (ts) influenza virus strain A/Leningrad/134/17/57 (H2N2) multiplied well at 32 degrees C (optimal temperature); lower titres of infectious virus were obtained in developing chick embryos at 40 degrees C. In a canine kidney (MDCK) cell line and in primary calf kidney (CK) cells an increased reproduction of the virus was found at 40 degrees C especially in the presence of trypsin. The ratios of virus titres obtained at optimal versus higher temperatures (RCT40) were by 1,000 times lower than those found in chick embryos. Polyacrylamide gel electrophoresis revealed a comparable synthesis of the cold-adapted influenza virus strain polypeptides HA, NP, M and NS in MDCK cells, regardless whether they were incubated at optimal or non-permissive temperatures.     

Agglutination of human O erythrocytes by influenza A(H1N1) viruses freshly isolated from patients

The hemagglutinin titers of 10 influenza A (H1N1) viruses were examined using the erythrocytes of several species. Human O erythrocytes showed the highest agglutination titer to the viruses, whereas chicken erythrocytes showed a low titer. These findings were noted for at least 10 passages by serial dilutions of the viruses in Madin-Darby canine kidney (MDCK) cells. All influenza A(H1N1) viruses, plaque-cloned directly from throat-washing specimens of patients, also agglutinated human O but not chicken erythrocytes. The results of a hemadsorption test indicated that chicken erythrocytes possess less affinity to MDCK cells infected with the A/Osaka City/2/88(H1N1) stain than to those infected with the A/Yamagata/120/86(H1N1) strain which is used as an inactivated influenza vaccine in Japan. However, there were no significant differences between the A/Osaka City/2/88 and the A/Yamagata/120/86 strains in the hemagglutination inhibition test. Since human O erythrocytes have high agglutination activity to influenza A(H1N1) and also to A(H3N2) and B viruses in MDCK cells, these erythrocytes may be useful for the serological diagnosis of influenza.     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

Effect of simultaneous administration of cold-adapted and wild-type influenza A viruses on experimental wild-type influenza infection in humans.

On the basis of the ability of the attenuated cold-adapted strain of influenza A virus to suppress disease production in ferrets simultaneously infected with epidemic influenza virus (P. Whitaker-Dowling, H.F. Maassab, and J.S. Youngner, J. Infect. Dis. 164:1200-1202, 1991), an evaluation of the ability of the cold-adapted virus to modify clinical disease in humans was made. Adult volunteers with prechallenge serum hemagglutination-inhibition titers to the influenza A/Kawasaki/86 (H1N1) virus of < or = 1:8 received either 10(7) 50% tissue culture infective doses of the wild-type A/Kawasaki virus or a mixture of 10(7) 50% tissue culture infective doses of each of the wild-type virus and a cold-adapted A/Kawasaki reassortant virus by intranasal drops in a randomized, double-blind fashion. Symptoms and wild-type virus shedding were assessed daily for 6 days following challenge. Results were compared with those derived from another group of volunteers who received only cold-adapted virus. Volunteers who received the mixed inoculum of cold-adapted and wild-type viruses had lower symptom scores than those who received wild-type virus alone, suggesting that coinfection with the cold-adapted virus may modify wild-type virus infection, but the differences were not statistically significant in this small study. The data demonstrate that administration of cold-adapted influenza A virus to humans at the time of wild-type virus infection is a safe procedure.     

MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells

The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes.     

Location of ts defects in the genome of cold-adapted recombinant influenza A virus vaccine strains

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.     

Further development (MDCK) of live cold-adapted influenza vaccine: cultivation of vaccine strains in production fermenters

Optimal conditions were developed for cultivating the cold-adapted reassortant live influenza vaccine (CARLIV) in MDCK cells, which were in their turn cultivated in fermenters with serum-free medium and microcarrier. The use of MDCK cells meets all national and WHO requirements to continuous cells used in the production of biological preparations. CARLIV cultivated under such conditions well preserve their ts-mutations and mutation, which entail substitutions of amino acids, in all CARLIV genome segments. Provided the cultivation conditions are optimal, the output of multivalent CARLIV in a 101 fermenter can reach 100000 doses.     

Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2)

For the past three decades the cold-adapted (ca) and temperature sensitive (ts) master donor strain, A/Leningrad/134/17/57 (H2N2) has been successfully used as the basis for the live attenuated reassortant influenza A vaccine. This donor strain was developed from A/Leningrad/134/57 (H2N2) wild-type (wt) virus following 17 passages in eggs at 25 degrees C. Our detailed investigation has revealed that the A/Leningrad/134/17/57 (Len/17) master donor stock is a mixed population comprised of numerous variants of the ca/ts Len/17 influenza virus. We have identified these variants to exhibit a broad range in their temperature sensitive phenotype when assayed on Madin-Darby canine kidney (MDCK) cells at 37 degrees C. A selection of these variant clones has been fully characterized by sequencing in order to understand the variability in the ts phenotype. This study has also addressed the feasibility of using cell culture technology for the propagation and subsequent manufacturing of the cold-adapted influenza vaccine (CAIV), particularly with respect to retaining the defined mutations that contribute toward the ca/ts phenotype.     

Recombinant cold-adapted attenuated influenza A vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants

A previously described cold-adapted attenuated virus, A/Leningrad/134/17/57 (H2N2), was further modified by 30 additional passages in chicken embryos at 25 degrees C. This virus had a distinct temperature-sensitive (ts) phenotype, grew well in chicken embryos at 25 degrees C, and failed to recombine with reference ts mutants of fowl plague virus containing ts lesions in five genes coding for non-glycosylated proteins (genes 1, 2, 5, 7, and 8). Recombination of A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79 (H1N1) and A/Bangkok/1/79(H3N2) yielded ts recombinants 47/25/1(H1N1) and 47/7/2 (H3N2). These recombinants inherited their ts phenotype and ability to reproduce in chicken embryos at 25 degrees C from the cold-adapted parent. Analysis of the genome composition of the recombinants obtained by recombination of the cold-adapted donor with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) showed that recombinants 47/25/1(H1N1) and 47/7/2 (H3N2) inherited five and six genes, respectively, from the cold-adapted parent, and hemagglutinin and neuraminidase genes from the wild-type strains.     

深圳地区人和鸡群中H9N2亚型流感病毒病原学和血清流行病学调查

目的：了解甲型流感病毒N9N2亚型毒株在深圳地区鸡群和人群中的分布。方法：采用常规的鸡胚双腔法来分离病毒。抗体测定，采用红细胞凝集抑制（HI）试验和中和试验测定法。结果：从深圳地区农贸市场鸡群中分离到27株H9N2亚型流感病毒，但未能从人群中分离到H9N2病毒。约有26％人血清中检测到H9亚型毒株的抗体，（HI滴度≥20），同时还发现抗体阳性率和几何均数随人群年龄增长而增高，同时与职业有关。然而，在鸡群中H9毒株的抗体阳性率仅为7％。结论：禽H9N2毒株不仅能感染人，而且在深圳地区人群和禽类中较为广泛的分布。人H9N2很大可能来源于鸡的H9N2毒株。     

Safety of and serum antibody response to cold-recombinant influenza A and inactivated trivalent influenza virus vaccines in older adults with chronic diseases.

Forty older adults with chronic diseases were vaccinated intranasally with either influenza A/California/10/78 (H1N1) (CR37) or influenza A/Washington/897/80 (H3N2) (CR48) virus. No clinically significant morbidity or decrement in pulmonary function occurred postvaccination. Two (15%) recipients of CR37 virus and twelve (44%) recipients of CR48 virus became infected with vaccine virus, as indicated by a fourfold rise in serum hemagglutination inhibition antibody titer; a fourfold rise in serum immunoglobulin G (IgG) or IgA antibody titer, indicated by enzyme-linked immunosorbent assay; isolation of vaccine virus from nasal washings; or all of these. Within 1 year after cold-recombinant vaccine virus vaccination, 18 vaccines received inactivated trivalent influenza virus vaccine parenterally. Of the vaccinees, 13 (72%) developed a fourfold rise in serum antibody titer to H1N1 antigen and 16 (89%) developed a fourfold rise in serum antibody titer to H3N2 antigen. We conclude that administration of these cold-recombinant vaccine viruses to older adults with chronic diseases was safe, but that serum antibody response rates were lower than those achieved with subsequently administered inactivated influenza virus vaccine given parenterally. However, the higher seroconversion rates attained by using the inactivated trivalent influenza virus vaccine do not necessarily mean that it is more efficacious in preventing infection or severe illness or both due to natural wild-type influenza A virus.     

Use of live cold-adapted influenza A H1N1 and H3N2 virus vaccines in seropositive adults.

To investigate the immunogenicity and protective efficacy of cold-adapted influenza vaccine in individuals with underlying immunity to influenza A virus, we administered cold-adapted H1N1 and H3N2 vaccines to adults with prevaccination serum hemagglutination inhibition antibody titers of 1:16 or more and challenged them 1 month afterwards with homologous wild-type influenza A virus. Both cold-adapted vaccines were immunogenic in seropositive adults. In addition, individuals receiving cold-adapted vaccines had lower rates of virus shedding and illness following challenge with wild-type influenza virus than did unvaccinated seropositive volunteers.     

Growth characteristics of Japanese encephalitis virus in chick embryo cells with special reference to the applicability of the culture to the production of inactivated vaccine

Summary The mouse-adapted Nakayama strain of JE virus was adapted by serial passage to growth in primary chick embryo (CE) cells. It was clone-purified three times. The resulting virus was able to induce a clear CPE in CE cells. It was inoculated to CE cells cultured in bottles and in a spinner culture system and the optimal conditions to give the highest yield were examined. Under the conditions adopted, a basal medium containing no serum or serumalbumin could be used as maintenance medium without lowering the efficiency for yielding a high titer of virus. Also, virus concentrations in culture fluids obtained in the bottle and spinner cultures were about equal. Virus obtained from the spinner culture was concentrated by ethanol precipitation and inactivated by beta-propiolactone treatment in order to produce a vaccine. Three lots of vaccine so prepared were tested for their potencies by the mouse protection test with promising results.     

Cold-Adapted Variants of Influenza A Virus: Evaluation in Adult Seronegative Volunteers of A/Scotland/840/74 and A/Victoria/3/75 Cold-Adapted Recombinants Derived from the Cold-Adapted A/Ann Arbor/6/60 Strain

Influenza A/Scotland/74 (H3N2) and A/Victoria/75 (H3N2) cold-adapted (ca) recombinant viruses, prepared by mating the A/Ann Arbor/6/60 (H2N2) ca donor virus and influenza A wild-type virus, were evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer, 相似文献   

Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine.

An enzyme-linked immunosorbent assay was used to measure nasal-wash and serum isotype-specific hemagglutinin antibody responses in 109 seronegative (hemagglutination-inhibiting titer less than or equal to 1:8) adults vaccinated intranasally with live attenuated A/Washington/897/80 (H3N2) or A/California/10/78 (H1N1) cold-adapted (ca) virus or with licensed subvirion vaccine subcutaneously. Live and inactivated virus elicited serum immunoglobulin A (IgA) responses in 83 and 96% of vaccinees, respectively, and elicited serum IgG responses in 72 and 100% of vaccinees. Inactivated virus induced higher titers of serum antibodies than did live virus and stimulated a nasal-wash IgG response more often than did live virus (94 versus 59%, P less than 0.01). In contrast, only 38% of inactivated virus vaccinees had local IgA responses compared with 83% of live virus vaccinees. Serum IgA and IgG and nasal IgG antibody titers remained elevated above prevaccination levels for at least 6 months in most of the live and inactivated vaccine responders, but the mean level of local IgA antibody induced by infection with live virus vaccine, in particular, decreased substantially. Considered in the context of previous work, the finding that live virus vaccine induced relatively long-lasting antibody in both local and serum compartments suggested that this vaccine may be a suitable alternative to inactivated vaccine for use in healthy persons.     

Resistance of adults to challenge with influenza A wild-type virus after receiving live or inactivated virus vaccine.

The efficacy of live attenuated cold-adapted (ca) reassortant influenza A H3N2 and H1N1 virus vaccines against experimental challenge with homologous wild-type virus 7 months after vaccination was compared with that of licensed inactivated virus vaccine in 106 seronegative (hemagglutination-inhibiting antibody titer less than or equal to 1:8) college students. The live attenuated virus vaccines induced as much resistance against illness as did the inactivated vaccine. Vaccine efficacy, measured by reduction in febrile or systemic illness in vaccines, compared with that in controls was 100% for ca H3N2 vaccine, 84% for inactivated H3N2 vaccine, 79% for ca H1N1 vaccine, and 67% for inactivated H1N1 vaccine. Less protection was conferred against upper respiratory tract illness; there was 50 and 77% protection in ca and inactivated H3N2 vaccines, respectively, but there was no protection in ca or inactivated H1N1 vaccinees. The duration, but not the magnitude, of H1N1 wild-type virus shedding in both ca and inactivated vaccinees was significantly reduced compared with controls. In contrast, a significant reduction in the duration and magnitude of H3N2 virus shedding was observed in ca vaccinees but not in inactivated vaccines. After wild-type virus challenge, live ca virus vaccinees demonstrated resistance at least as great 7 months postvaccination as did inactivated virus vaccinees. These observations indicate that live virus vaccines may be a satisfactory alternative to inactivated vaccines for healthy persons.     

The genes associated with trans-dominance of the influenza A cold-adapted live virus vaccine

Segment 7 (M) of the cold-adapted live influenza A virus vaccine plays a primary role in the ability of this virus to interfere with the replication of wild-type influenza A viruses. This conclusion is based on several lines of evidence. Single gene reassortant viruses derived by crossing influenza A/Ann Arbor/6/60 (H2N2) cold-adapted donor virus with an epidemic wild-type strain, A/Korea/1/82 (H3N2), were tested for their ability to interfere with wild-type parental virus in the Madin-Darby line of canine kidney cells and embryonated eggs. It was apparent in both hosts that the single gene reassortant carrying segment 7 (M) derived from the cold-adapted virus was dominant over wild-type virus. Additional confirmation of the role of segment 7 (M) in trans-dominance of the cold-adapted vaccine virus was derived from the analysis of reassortants produced by mixed infection by a wild-type virus and its cold-adapted reassortant vaccine strain. After three serial passages, the virus yield contained a high proportion of reassortants carrying segment 7 (M) of the cold-adapted parental strain. When used in mixed infections, these reassortants were dominant over the replication of the parental wild-type virus.