Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: nonrandom reassortment of genes during preparation of live virus vaccine candidates by recombination at 25 degrees between recent H3N2 and H1N1 epidemic strains and cold-adapted A/An Arbor/6/60.

Genetic compositions of 35 recombinant cold-adapted influenza A(H3N2 and H1N1) candidate live attenuated vaccine strains have been determined. The viruses, which had been obtained by recombination (reassortment) at 25° between contemporary epidemic wild-type strains and cold-adapted A/Ann Arbor/6/60(H2N2), followed by selection for growth at 25° of virus with wild-type HA and NA, have a highly restricted genetic composition. Eighteen of the thirty-five recombinants had RNAs coding for the three polymerase (P) proteins, NP, M, and NS, from the cold-adapted mutant A/Ann Arbor/6/60 had only the HA and NA of the wild-type strains. Only 4 out of 64 theoretically possible combinations of genes coding for nonglycoprotein viral products were detected. The restricted genetic composition of cold-adapted recombinants produced at 25° supports the evaluation of this method of preparing live vaccine strains to determine whether recombinants with constant gene composition have predictable levels of attenuation for man.     

Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID₅₀) for each recombinant was approximately 10^5.0 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID₅₀ and approximately 70% of ca or ts vaccinees who received 10 to 32 HID₅₀ of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.     

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.     

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.     

Mutations in the genes coding for hemagglutinin and neuraminidase in cold-adapted variants of the influenza virus A/Leningrad/134/57 (H2N2)

An analysis of ts-mutations in the genomes of native and cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus based on the use of fowl plague virus ts mutants was carried out. The recombination test was done by the conventional method in chick embryo fibroblast culture (genes PB2, PB1, PA, NP, NA, M and NS) or cell systems permissive for reproduction of human influenza virus (gene HA). The cold-adapted strain A/Len/17 used for preparation of live influenza vaccine (LIV) for adults was shown to have 4 ts mutations: three in "internal" genes (PB2, NP, and M) and one in gene 4 coding for hemagglutinin (HA). The more attenuated cold-adapted donor A/Len/47 for preparation of similar LIV for children acquired three additional ts mutations: two (PB1 and NS) in "internal" genes and one in gene 6 coding for neuraminidase (NA). The accumulation of ts mutations in the genome of cold-adapter strains was found to be accompanied by a decrease in their pneumotropicity for mice as well as their detectability in different organs of these animals.     

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.     

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.     

Genetic recombination between natural isolates of influenza virus serotypes H1N1 and H3N2

Oligonucleotide mapping of individual genes was used for search of possible genetic recombinants between natural isolates of influenza H1N1 and H3N2 viruses isolated in the USSR in 1977-1979. No antigenic hybrids and recombinants with the antigenic structure H3N2 were found, however, it was shown that isolates of H1N1 viruses of 1979 (the A/USSR/61/79 strain) might represent genetic recombinants carrying genes P1 + P2 from H3N2 viruses, the M-gene of the USSR/61/79 virus being closest in its structure to the analogous gene of the earliest isolate of H3N2 viruses, namely A/Hong Kong/1/68. Possible selective advantages of virus recombinants having M-genes from viruses of a different serotype are discussed.     

Production of early cytokines after infection with A/Leningrad/134/57 (H2N2) wide-type virus and cold-adapted attenuated vaccine viruses

The production of proinflammatory cytokines was studied following experimental infection of BALB/c mice with influenza viruses that differed in virulence. The generation of TNF-alpha, IL-6, IL-12, and IFN-gamma was investigated in the lung homogenates in the early periods after intranasal infection of mice with A/Leningrad/134/57 (H2N2) wild-type virus and cold-adapted attenuated vaccine viruses: A/Leningrad/134/17157 (H2N2) and A/Leningrad/134/47/57 (H2N2). Wild-type virus induced substantially higher levels of proinflammatory cytokines: TNF-alpha, IL-6, IL-12, and IFN-gamma. After infection with the cold-adapted viruses, the levels of the cytokines were reduced as compared to those induced by the wild-type virus. The A/Leningrad/134/47/57 virus was marked by a noticeable production of IL-6 and IFN-gamma in the murine lung, but it was less than with wild-type virus infection. At the same time, the more attenuated strain A/Leningrad/134/47/57 induced TNF-alpha and IFN-gamma in the quantities similar to those in the control animals. Thus, a response of proinflammatory cytokines in early infection in the murine lung depended on the level of viral replication in the lower respiratory tract and on the attenuation of influenza virus strains.     

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.     

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.     

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

BackgroundDespite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny.Study designWe document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09.ResultsComplete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID₅₀ of 10⁴/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia.ConclusionsIt is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.     

High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.     

Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis

The high mutation rate of influenza virus, combined with the increasing worldwide use of influenza virus-specific drugs, allows the selection of viruses that are resistant to the currently available antiviral medications. Therefore, reliable tests for the rapid detection of drug-resistant influenza virus strains are required. We evaluated the use of a procedure involving real-time polymerase chain reaction (PCR) followed by melting point analysis (MPA) of hybrids formed between the PCR product and a specific oligonucleotide probe for the identification of point mutations in the influenza A virus neuraminidase gene (NA) that are associated with oseltamivir resistance [resulting in the amino acid change H275Y for seasonal and pandemic influenza A(H1N1) viruses and E119V for A(H3N2) viruses]. Therefore, 54 seasonal A(H1N1) (12 oseltamivir-resistant and 42 sensitive strains), 222 A(H1N1)2009 (5 resistant, 217 sensitive), and 51 A(H3N2) viruses (2 resistant, 49 sensitive) were tested by MPA, and the results were compared to those obtained by sequencing the NA gene. The results clearly indicate that the identification of drug resistance mutations by MPA is as accurate as sequencing, irrespective of whether MPA is performed using clinical material or the corresponding isolate. MPA enables a clear identification of mutations associated with antiviral resistance.     

Live influenza A/Victoria/75 (H3N2) virus vaccines: reactogenicity, immunogenicity, and protection against wild-type virus challenge.

Four live influenza A/Victoria/75 (H3N2) recombinant virus vaccines were administered intranasally to a total of 50 volunteers who had little or no detectable serum neutralizing antibody. A recombinant with ts-1[E] having a 38 degrees C shut-off temperature caused febrile reactions or systemic reactions or both in 21% of the volunteers, but one with ts-1A2 having a 37 degrees C shut-off temperature caused no illness. Two recombinants prepared with cold-adapted A/Ann Arbor/6/60 caused 9% febrile reactions or systemic reactions or both. Virus shedding occurred in a minority of the 50 volunteers, but 90% developed a serum neutralizing antibody response. Wild-type A/Victoria/75 virus challenge of 34 of the vaccinated volunteers and 12 others who had had prior natural A/Victoria/75 virus infection revealed similar and significant protection when compared with the 96% infection and 68% febrile illness or systemic illness or both observed in 25 unvaccinated volunteers with little or no serum antibody. These results encourage continued efforts toward development of live influenza virus vaccines.     

Evaluation of live avian-human reassortant influenza A H3N2 and H1N1 virus vaccines in seronegative adult volunteers.

An avian-human reassortant influenza A virus deriving its genes coding for the hemagglutinin and neuraminidase from the human influenza A/Washington/897/80 (H3N2) virus and its six "internal" genes from the avian influenza A/Mallard/NY/6750/78 (H2N2) virus (i.e., a six-gene reassortant) was previously shown to be safe, infectious, nontransmissible, and immunogenic as a live virus vaccine in adult humans. Two additional six-gene avian-human reassortant influenza viruses derived from the mating of wild-type human influenza A/California/10/78 (H1N1) and A/Korea/1/82 (H3N2) viruses with the avian influenza A/Mallard/NY/78 virus were evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:8) adult volunteers for safety, infectivity, and immunogenicity to determine whether human influenza A viruses can be reproducibly attenuated by the transfer of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The 50% human infectious dose was 10(4.9) 50% tissue culture infectious doses for the H1N1 reassortant virus and 10(5.4) 50% tissue culture infectious doses for the H3N2 reassortant virus. Both reassortants were satisfactorily attenuated with only 5% (H1N1) and 2% (H3N2) of infected vaccines receiving less than 400 50% human infectious doses developing illness. Consistent with this level of attenuation, the magnitude of viral shedding after inoculation was reduced 100-fold (H1N1) to 10,000-fold (H3N2) compared with that produced by wild-type virus. The duration of virus shedding by vaccines was one-third that of controls receiving wild-type virus. At 40 to 100 50% human infectious doses, virus-specific immune responses were seen in 77 to 93% of volunteers. When vaccinees who has received 10(7.5) 50% tissue culture infectious doses of the H3N2 vaccine were experimentally challenged with a homologous wild-type human virus only 2 of 19 (11%) vaccinees became ill compared with 7 of 14 (50%) unvaccinated seronegative controls ( P < 0.025; protective efficacy, 79%). Thus, three different virulent human influenza A viruses have been satisfactorily attenuated by the acquisition of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The observation that this donor virus can reproducibly attenuate human influenza A viruses indicates that avian-human influenza A reassortants should be further studied as potential live influenza A virus vaccines.     

Characterization of epidemic influenza virus A(H3N2) strains circulating in Russia in the 2003-2004 epidemic season

Studies indicated that the epidemic rise in the incidence of influenza was caused by its virus A (H3N2) circulation in Russia in the 2003-2004 season. The Center of Influenza Ecology and Epidemiology investigated 101 epidemic strains isolated the MDCK culture. Antigenic analysis showed that all viruses A(H3N2) were similar to the reference virus A/Fujian/411/02(H3N2) and only 5 strains slightly differed from the latter. Twelve (14%) strains resistant to rimantadine at a concentration of 0.5 mg/ml were identified. Investigation of paired sera from the patients demonstrated a rise of antibodies to the references of influenza virus A(H3N2) in 68.7% of cases and a less increase in those to influenza viruses A(H1N1) and B. The active circulation of A(H3N2) viruses was due not only to changes in their antigenic structure, but also to the low level of antibodies to these viruses, as shown by the analysis of donor sera.     

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.     

Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.     

两株京科猪H1N1亚型流感病毒内部基因来源的研究

目的通过内部基因研究了解两株猪(H1N1)亚型流感病毒内部基因是否含有禽流感病毒基因节段及是否与猪群中H9N2亚型毒株发生了基因重配。方法病毒在鸡胚中传代,从收获的尿囊液中提取RNA,通过逆转录合成cDNA,cDNA用PCR扩增。PCR产物用纯化试剂盒纯化,接着进行核苷酸序列测定,然后用MegAlign(Version1.03)和Editseq(Version3.69)软件进行基因进化树分析。结果两株京科猪H1N1病毒内部基因除PB2基因节段有所不同外,其余5个基因节段均相同,但与猪H1N1流感病毒内部基因相近,然而,与古典型猪H1N1毒株有差异。结论两株京科猪H1N1病毒不是基因重配株,它们的内部基因均属猪H1N1流感病毒基因系。