Search

Persons attending for routine influenza vaccination in an urban practice each provided three specimens of blood for evaluating their immunological response. 138 (67%) of the 206 persons were defined as "at risk" by reason of morbidity as given in the guidelines published by the Chief Medical Officer. The mean age was 67 yr and 65% were aged 65 yr or more. By day 7, 71% of 31 persons had protective H(1)N(1) titres, 61% H(3)N(2) and 42% B. These proportions were similar to those found at day 14 and at day 21 based on 159 persons. These findings suggest that an effective immune response is mounted within seven days of vaccination indicating that the vaccination of persons "at risk" is worthwhile even after an epidemic has established itself. This is not a reason to modify present policy of routine vaccination in early winter well before epidemics are likely to occur.

Zanamivir, a neuraminidase inhibitor under license from Biota, has been developed and launched by Glaxo Wellcome for the treatment of influenza virus infection [277127]. While registered as drugs, neuraminidase inhibitors are technically vaccines as they are believed to work by provoking an immune response, thus preventing the spread of infection from one cell to another within the respiratory tract [314356]. It is administered, using an inhaler, directly to where the virus replicates, minimizing the potential for side effects. Zanamivir has been launched, under the name Relenza, for the treatment of influenza in Australia [325148], New Zealand [335591] and the UK [341871]. It is registered in Mexico and Armenia [346474], and approved in Japan [351580]. In October 1999, it was launched in the US [352392] and Glaxo confirmed in January 2000, that zanamivir has been launched all over Europe, with the exception of Greece and Spain, where launch is imminent [352513]. In October 1999, the British Pharma Group (BPG) wrote to the UK Prime Minister to seek a discussion on the implications for the British-based pharmaceutical industry of the recommendation from NICE's Raised Appraisal Committee that Relenza should be banned from the NHS [342597]. An FDA submission was made in October 1998 [302732,304779]; the FDA advisory committee met on February 24 1999 to discuss the application [313583]. The Antivirals Drugs Advisory Committee voted against recommending zanamivir for FDA approval [316196]. Members suggested that additional studies using a different endpoint or targeting specific high-risk populations would be more likely to garner support [317454]. Following a discussion with Glaxo Wellcome in April 1999, the FDA indicated that it would continue to review the NDA for zanamivir [320565]. In July 1999, the company received final FDA approval for the drug for the treatment of both influenza A and B [334127], and aimed to initiate a phase IV trial program for the detection of consumer problems with the product, following launch of the product in time for the 1999-2000 flu season [352392,335048]. In September 1998, Glaxo applied to the Canadian Health Protection Branch (HPB) for approval to market zanamivir for the treatment and prevention of influenza. A separate application for Fast Track approval was also made with the HPB [299375,299472]. In November 1999, zanamivir received approval in Canada [346474]. Glaxo expects to make MAA and NDA filings for the use of zanamivir as a prophylactic in 1999 [333494]. In July 1998, Merrill Lynch predicted that as zanamivir offered only modest benefits gained from a narrow treatment window, potential would be limited and forecast annual sales of US dollar 30 million [302048]. In January 1999, Paribas predicted sales of pounds 75 million in 1999, rising to pounds 280 million in 2003 [317650]. In April 1999, ABN Amro predicted sales of pounds 23 million in 1999, rising to pounds 326 million in 2003 [328676]. In August 1999, Lehman Brothers estimated that sales of zanamivir will reach US dollar 400 million by 2003, and that it is an obvious candidate for an OTC switch, although this is not likely until 5 years after launch [336356].

Influenza A virus initiated worldwide epidemics (pandemics) in 1918, 1957, 1968 and 1977. A revised calculation of the 1918-1919 pandemic estimates that 40 million persons died and 500 million were infected. The mortalities in 1957 and 1968 were nearly 6 million. Biological and genetic characteristics of the causative agents of the more recent pandemics, have been well studied but little is known about the causative agent of the Great Pandemic in 1918. Genetic characterisation of the 1918 virus has been achieved by sourcing virus RNA from formalin fixed lung samples or by exhuming frozen victims of the outbreak from Arctic regions. Initial analysis of the HA gene from two USA sources indicates a virus related to swine and human influenza with no base insertion at the HA1-HA2 cleavage junction which, at least in avian influenza A, characterises high virulence. Important unanswered questions are whether the 1918 virus spread pantropically perhaps to include the brain and hence cause encephalitis including the later lethargic forms, or whether infection was confined to the respiratory tract. Re-examination of reports of respiratory disease in England and France in 1916-1917 may indicate a non-Spanish origin of the pandemic and a period of 2 years for the virus to be seeded worldwide. In contrast the other two pandemic viruses in 1957 and 1968 appeared to originate in Asia. New anti-neuraminidase drugs in conjunction with amantadine and novel developments with influenza vaccines would be expected to ameliorate the disease in a future pandemic., (Copyright 2000 John Wiley & Sons, Ltd.)

Human immunodeficiency virus (HIV-1) associated immune deficiency has the characteristics of chronic graft versus host disease (GVHD) caused by human leukocyte antigen (HLA) class 2 incompatibility. The envelope glycoprotein fragment TKAKRRVVEREKR mimics HLA class 1 C molecules serologically, and also mimics an immune regulatory T cell epitope, in the region of amino acids 67 to 71, within the HLA DR beta chain. This beta chain alloepitopic region (between amino acids 67 to 80) furnishes peptides predicted to bind optimally to HLA class 1 B alleles. The hypothesis predicts that viral parameters, such as viral load, and clinical parameters, such as rate of progress to acquired immune deficiency syndrome (AIDS) and severity of the associated immune deficient state, are linked to the HLA B and HLA DR beta chain haplotype in infected patients. Immune suppression is caused by HLA class 1 B restricted CD8+ T cells which normally regulate HLA class 2 DR restricted antigen specific responses. The hypothesis further predicts the severity of immune deficiency to be linked to those HLA DR beta chain allotypes which express the amino-acid glutamine (Q) in position 70.

We provide data on the prevalence of SRH antibody to influenza A/Singapore/1/57 (H2N2). Approximately 10.3% of sera had antibody to the influenza A (H2N2) subtype virus in comparison to the 36.9% of positive sera to a representative influenza A (H3N2) and 31.5% to influenza A (H1N1) viruses. The percentage of subjects with antibody constantly decreased from the older to the younger age groups. Persons born after 1968 were essentially seronegative, whereas subjects born before 1900, and in the decade 1950-1959, showed the highest antibody levels to influenza A (H2N2) viruses. These age groups also appeared to have 'protective' levels of anti-HA antibody to influenza A (H2N2) virus. An antigenic variant of A/Singapore/1/57 virus was selected in the laboratory using a monoclonal antibody to HA. Serological comparison of the new in vitro variant with the parental virus and two naturally occurring viruses, namely A/England/12/64 and Tokyo/3/67, showed that certain human sera were able to distinguish the variant, indicating a restricted antibody repertoire in these adult and children's sera, providing an explanation of how such variants could actually arise in nature.

We report the isolation of two influenza A(H3N2) virus strains which were unable, in the first passages in MDCK cell culture, to agglutinate chicken erythrocytes, though reacting with guinea pig and turkey red blood cells. This observation demonstrates that the occurrence of this phenomenon is not exclusive to influenza A(H1N1) viruses, as previously reported. In order to investigate the molecular basis of this phenomenon, we analysed the nucleotide sequence of the HA-1 region, presumed to be involved in the switch of haemagglutination properties, from the virus present in the original samples and in the corresponding strains isolated and cultivated in MDCK cells and in embryonated eggs. The substitution of amino acid 138 (Ala-->Ser) in MDCK cells could be related to the change in haemagglutination characteristics.

It has been proposed that the highly conserved human immunodeficiency virus type 1 (HIV-1) envelope gp120 carboxy-terminal sequence, TKAKRRVVEREKR (CT120), may represent a functional mimic of the human leukocyte antigen (HLA) class II DR beta-chain third hypervariable region (HVR3) sequence motif located at position 69-81. Presentation of this potentially pathogenic fragment by HLA class I and/or II molecules, in a manner analogous to the indirect pathway of allorecognition, may induce both widespread cellular activation and also break self-tolerance, resulting in the selective and progressive anti-self HLA class II-directed immune suppression, which is a central feature of HIV-1 infection and the associated acquired immune deficiency syndrome (AIDS). To investigate the functional role of the HIV-1 gp120 C-terminal fragment T cell lines (TCLs) were raised from three healthy HIV-1-seronegative subjects at low risk of HIV-1 exposure, by repeated stimulation with a short synthetic 13-mer CT120 peptide in vitro. Graded concentrations (10[3] to 5 x 10[4]) of CT120 TCLs suppressed the primary 6-day proliferation of autologous PBMCs in response to the soluble antigens tetanus toxoid (TT) and purified protein derivative (PPD). In contrast, CT120 TCLs demonstrated no suppressive effect on 3-day phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen (PWM) mitogenic responses. Fractionation of CT120 TCLs into highly purified CD4+ and CD8+ T cell subsets demonstrated that the CD8+ T cell fraction mediated the suppressor effector function. HLA restriction analysis revealed a complex pattern as both anti-HLA class II DR and anti-HLA class I (A, B, C) MAbs inhibited proliferation of oligoclonal CD8+ CT120 TCLs. Strategies aimed at specifically inhibiting such putative immunopathogenic HIV-1-encoded T cell epitopes may be an important consideration for development of future HIV-1 immunotherapy.

The kinetics of inactivation of four different strains of HIV-1 (RF, MN, SF2 and IIIB) by beta-propiolactone (BPL) and binary ethylenimine (BEI) were studied under various conditions. The conditions that would be required for the reduction of virus infectivity by at least 10(20) TCID50 ml-1 were estimated on the basis of the experimental rates of inactivation obtained. A multiple step procedure including treatment with 0.2% BPL, 0.05% sodium cholate, 10 mM BEI and 0.02% formaldehyde was designed to inactivate HIV-1 for use as an experimental vaccine. Complete inactivation of virus infectivity was confirmed by prolonged cell culture. The experimental vaccine preparation was analysed for the presence of HIV-1 proviral DNA utilizing the polymerase chain reaction. After treatment with both BPL and BEI proviral DNA was detected in one of four samples using primers encoding a 244 bp segment of the pol region of the viral genome. Proviral DNA could not be detected in any of the four samples using primers encoding segments of > 400 bp in the gag and reverse transcriptase region.

We have developed a unique multiple step procedure to inactivate human immunodeficiency virus chemically with a very high safety margin while retaining antigenically active structural virion proteins, including gp120, in the final immunogen. The whole virus preparation (1-10 micrograms per dose) was highly immunogenic in a variety of small mammals and induced antibodies that recognized homologous and heterologous strains of HIV-1. Sera from immunized animals bound to peptides representing the entire sequence of the external glycoprotein gp120. Neutralizing antibodies active against the homologous immunizing strain and against heterologous HIV-1 strains were also elicited. Sera with virus neutralizing activity did not bind to MHC class I proteins derived from the human cell line used to grow the virus.

To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the National Academy of Sciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV-1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (Ile), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild-type "AZT-sensitive" RTs co-existed with mutated "AZT-resistant" RTs in the same patient sample.

The enzyme-linked immunospot assay was used to examine the humoral immune response in 15 healthy volunteers immunized with either split or subunit inactivated trivalent influenza vaccine containing A/Beijing/353/89 (H3N2), A/Taiwan/1/86 (H1N1) and B/Yamagata/16/88. The rapidity of the individual B-cell and serum antibody response was examined in lymphocyte and serum samples collected at various time intervals after vaccination. A rapid serological response was detected with increases in antibody titre detected in the majority of volunteers by 7-8 days postvaccination. Influenza-specific plasma cells were detected as early as 4 days postvaccination, higher numbers of IgA and IgG antibody-secreting cells (ASC) were observed which peaked at 7-8 days postvaccination. The number of ASCs then declined, with low numbers of cells detected at 11 days postvaccination. Influenza-specific IgA ASCs were predominantly of the IgA1 subclass. This rapid immune response may have a bearing on future vaccination policies of unimmunized 'at risk groups' in times of high influenza activity.

To investigate the possible mechanism of maintenance of old human influenza A (H3N2) viruses in pigs, the haemagglutinins (HAs) of seven isolates from swine were studied by analysis of nucleotide and deduced primary amino acid sequences, as well as reactivity of the HA molecule to chicken antisera and monoclonal antibodies. The swine HAs were closely similar to the HA of the A/Victoria/3/75 human variant as regards antigenic and molecular characteristics. These findings are consistent with the hypothesis that the swine HA genes were transmitted from an early human H3 virus to pigs, where they survived with limited mutations over a period of 5 years. The sequence data were also compared with swine H3 sequences to investigate genetic relationships between the H3 genes from swine viruses isolated in different geographical areas. An evolutionary tree, constructed from the nucleotide sequences of viruses isolated from pigs in China and in Italy, illustrated that, depending on the country of their isolation, the HA genes of swine influenza A (H3N2) viruses have different origins, e.g. human and avian, and evolved independently in different lineages. The study provides direct support for the hypothesis that pigs might serve as a 'mixing vessel' for the generation of pandemic strains of human influenza viruses.

A study was performed with volunteers in order to determine whether the virulence of an influenza B viral infection could be attenuated. Of a total of 62 persons, 26 received intranasal inoculations of an unpassaged influenza B virus isolate [virus U], while 26 received the same virus isolated passaged in cell culture and then in special pathogen-free embryonated hens' eggs [virus P]. The remaining 10 persons received uninoculated cell culture medium. Daily nasal wash samples were collected post-infection and scores for illness in the volunteers were evaluated. Viruses were isolated in cell culture and in eggs. Isolates were analysed by means of monoclonal antibodies (MAbs) raised against the influenza B viral haemagglutinin (HA) glycoprotein. One-step and nested polymerase chain reactions as well as direct nucleotide sequence analysis of part of the HA gene of the unpassaged specimens, together with the influenza B viruses isolated from those specimens, were performed. Nine of 26 volunteers who received the unpassaged virus became ill (illness scores from 13-84/100) whereas 7/26 of the volunteers who received the passaged virus had very mild illness (illness scores from 3-7/100). All the other volunteers remained well. Results of analysing the clinical specimens collected from the two groups of volunteers were compared. A difference in MAb reactivity, together with an aspartate for asparagine amino acid substitution at position 196 in a 432 base pair region of the viral HA gene, was observed. The loss of a potential glycosylation site at amino acid position 196-198 in the viral HA was associated with attenuation of virulence. This finding may have implications for the formulation of influenza vaccines.

When clinical isolates of influenza A and B viruses are propagated in embryonated hens' eggs or tissue culture cells, different selective pressures in vitro result in specific amino acid substitutions in the haemagglutinin (HA) gene. A proportion of such viruses which lose a potential glycosylation site near the receptor binding region of the HA at amino acid positions 196-198 appear to have reduced virulence. Direct polymerase chain reaction (PCR) amplifications of cDNA and subsequent nucleotide sequence analysis of part of the HA-1 gene of the original infecting influenza B strain and the nasal wash material from an infected volunteer were performed. The nucleotide sequences of the viral HA-1 from the nasopharynx of the infected volunteer were the same as that of the original infecting strain. Antigenic analysis of both the original infecting virus and the viruses isolated from sequential samples collected from the volunteer, all of which were cultivated on Madin-Darby Canine Kidney (MDCK) cells and in embryonated hens' eggs, revealed variation in the HA of viruses only after egg adaptation. In particular, we describe the use of direct nucleotide sequencing techniques without the use of cloning strategies in order to determine the sequence of the HA-1 gene after direct PCR amplification of clinical nasal wash material.

Influenza A (H1N1) viruses when initially isolated in mammalian cell cultures (MDCK cells) had different agglutination reactions with chicken and guinea-pig erythrocytes compared to the same viruses after passage. On first isolation the virus HA resembled the 'O' phase viruses described originally by Burnet and Bull and agglutinated mammalian but not avian erythrocytes. After passage, the virus HA resembled a classical 'D' phase virus and agglutinated both avian and mammalian erythrocytes. Monoclonal and polyclonal antisera detected antigenic differences between the HAs of the viruses in the 'O' and 'D' phases. The 'O' phase virus HA reacted preferentially with antibodies in post infection human antisera. Viruses in the 'O' phase replicated poorly in the allantoic cavity of embryonated hens' eggs whilst 'D' phase virus replicated in both MDCK cells and in embryonated hens' eggs. At least three distinguishable subpopulations of influenza A (H1N1) viruses may co-exist in clinical throat swab material, including viruses possessing HAs in the 'O' and 'D' phases and other 'D' phase viruses cultivable in embryonated hens' eggs but antigenically distinguishable from the corresponding 'D' phase virus in MDCK cells.

Laboratory investigations of virus isolation and serum antibodies in a Mediterranean country (Italy) demonstrated that influenza A and B viruses, and often both, circulated every winter in Italy. The winter 1987/88 was characterized by a low level of influenza activity, as shown by the limited number (47) of influenza virus isolates, the majority of which (61%) belonged to the influenza B type. In contrast, the 1988/89 influenza season was exclusively associated with the circulation of influenza type A viruses. The A(H1H1) subtype was largely predominant (97%), as compared to the low incidence of the A(H3N2) subtype (3%). During the 1989/90 winter a co-circulation of A and B influenza viruses was observed, A(H3N2) strains being responsible for 96% of the virologically confirmed cases. Antigenic analysis of the virus isolates showed some antigenic variation in influenza A viruses of both H1N1 and H3N2 subtypes, whilst antigenic stability was found among the influenza B virus isolates. Overall, the above virological findings correlate with the data concerning the pattern of influenza virus circulation in Northern Europe and the UK during the three years surveyed. The results of serum antibody surveys conducted in each post-epidemic period are also reported.

Electron microscopy (EM) of cell sections showed cell associated virus-like particles (VLP), 50-60 nm in diameter, budding from the membrane of human lymphoid cells in culture. The particles had an envelope continuous with the cell membrane and a dense core that almost filled the particle. Particles 70-80 nm in diameter with prominent external spikes were found in the culture medium by negative staining (medium-associated VLP). Cell-associated VLP were also present in cord lymphocytes, both on initial separation and after culture with or without foetal calf serum, and therefore were considered to be endogenous to the cells and were not bovine diarrhoea virus. VLP were observed in most of the lymphoid cell lines examined. VLP were also found less frequently in established human tumour and nontumour cell lines. Both cell-associated and medium-associated VLP were also present in HIV infected cell cultures, and they could be distinguished from HIV by their characteristic morphology and smaller size. It was not determined whether the 2 types of particle represented the same entity, or whether they were defective virus particles or a cellular secretory product.

The antibody response of mice and adult humans to immunization with subunit vaccines derived from a pair of antigenically distinct influenza A H1N1 viruses isolated in eggs was investigated. Although the haemagglutinin molecule of each virus differed by only three amino acid residues, highly specific antibody responses were elicited in mice as determined by haemagglutination inhibition and radioimmunoprecipitation assays. Results from competitive radioimmunoassays using monoclonal antibodies of known specificity and a study of the reactivity of mouse antisera with H1N1 field strains indicated that the marked differences in the antibody responses to the two vaccines was due to an amino acid substitution in the distal tip of the haemagglutinin molecule. In contrast, crossreactive antibody responses were elicited in humans presumably due to exposure to viruses related to the candidate vaccine prior to vaccination. Although immunogenic differences are apparent in this pair of antigenically distinct viruses in naive laboratory animals, these differences are not apparent following vaccination of humans that had prior exposure to related viruses.

Multiple mutations in the reverse transcriptase (RT) gene were observed in a drug-resistant isolate of human immunodeficiency virus type 1 (HIV1) from an individual having prolonged (greater than 2 years) zidovudine (AZT) therapy. The virus replicated in PBMC's in the presence of very high concentrations of AZT (125 microM). Drug-sensitive strains were curtailed by 0.01 microM AZT. Eleven defined mutations were observed as compared with published sequences of RT for eight strains of HIV1. Eight of these mutations were found in the domain involved in nucleotide recognition and enzyme function. Only one of the mutations, giving a Thr--Tyr change at amino acid 215, matched those previously ascribed (67, 70, 215, and 219) to the generation of high-level resistance to AZT. Therefore additional amino acid changes may have significance in the emergence of super-resistant viruses.

The nucleotide sequences of ten haemagglutinin genes of representative H7N7 equine influenza viruses isolated between 1956 and 1977 have been determined by primer extension sequencing. Their nucleotide and deduced amino acid sequences demonstrate a high degree of homology. These equine viruses can be divided into two distinct subgroups, the prototype-like, and a group comprising the early American isolates and the remaining equine viruses. The equine H7 haemagglutinins form a quite distinct group compared to H7 haemagglutinins isolated from other species. Each of these equine H7 haemagglutinins possess a tetrabasic amino acid cleavage site separating the HA1 and HA2 domains but, in addition, all ten contain a nine amino acid insertion prior to the tetrabasic sequence. The haemagglutinin glycoproteins of all ten viruses are capable of cleavage activation in virus infected primary chicken embryo fibroblast cells.

Serological analysis of a group of 63 influenza H1N1 viruses isolated from pigs in Italy in the period 1976-1988 revealed the presence of two distinct antigenic subpopulations: some viruses possessed a haemagglutinin indistinguishable from that of viruses typically associated with pigs, i.e., A/New Jersey/8/76 (H1N1), whereas others showed a close antigenic relatedness with the haemagglutinin of avian-like H1 viruses. These findings represent further evidence that influenza A viruses from avian species may be transmitted to mammals. The surface and internal proteins of some of these viruses were also analyzed biochemically to evaluate the molecular relatedness among viruses circulating in non-human hosts.

A study of the immunogenicity of the inactivated trivalent subunit influenza vaccine for the 1989/90 season was performed in what proved to be an influenza epidemic year. One hundred student volunteers at The London Hospital Medical College participated in the study and the findings indicated that there was an excellent serological match between the epidemic strain of influenza A (H3N2) and the vaccine strain. Before vaccination, the geometric mean titre (GMT) to A/England/308/89, a representative H3N2 epidemic strain in the United Kingdom from the 1989/90 season, was 46. Post-vaccination the antibody levels rose and 99% of vaccinees had HI titres of greater than or equal to 40, the GMT being 131. The serological responses were also investigated against other circulating influenza A (H3N2 and H1N1) and B strains. Preliminary results of an evaluation of the rapidity of the immune response showed that in three of six subjects rises in HI antibody appeared within two days of vaccination.

Influenza A (H1N1) and influenza B viruses from clinical samples were isolated in the amniotic cavity of embryonated hens' eggs by classical techniques and propagated in the allantoic cavity. Virus progeny from different eggs which had been inoculated with virus material from the same clinical sample possessed antigenically distinguishable haemagglutinins (HAs). Virus progeny of some eggs possessed HAs which were serologically identical to those of virus isolated in parallel in mammalian (MDCK) cells. These egg-grown viruses possessing HAs with the antigenic phenotype of mammalian cell-grown viruses appeared to be antigenically related to epidemic influenza virus because post-infection human sera reacted to high titre with the virus HA. Specific nucleotide changes were detected in the HAs of the viruses isolated directly in eggs at positions 163 and 189 for influenza A (H1N1) viruses or positions 141 and 196 to 198 for influenza B viruses. Egg-isolated viruses which possessed the antigenic phenotype of mammalian cell-grown viruses retained glycosylation sites at positions 163 and 196. The viruses isolated directly in embryonated hens' eggs which possessed the HA antigenic phenotype and glycosylation sites of MDCK cell-grown virus can, unlike the latter viruses themselves, be used as candidate influenza vaccine viruses.

Eighteen influenza A (H3N2) viruses were isolated during a single outbreak in a closed community of Carmelite nuns. Serological analysis of the virus haemagglutinin (HAs), using a panel of monoclonal antibodies, demonstrated antigenic microheterogeneity. In contrast, no significant biochemical differences were detected in viral genes by RNA:RNA hybridisation or in structural or nonstructural polypeptides analysed by high-resolution polyacrylamide gel electrophoresis (PAGE) or by limited proteolysis followed by PAGE. Influenza A (H3N2) viruses isolated in the vicinity of the convent were distinguishable from each other and from the epidemic viruses isolated in the convent both antigenically and biochemically.

An influenza B virus was passaged in man (virus A) and then in human embryo trachea (C) and into embryonated eggs (D) or directly into eggs (B). Virus A, B, and C had the same (cell-like) haemagglutinin phenotype on reaction with selected monoclonal antibodies while D had an "egg-like" phenotype. The viruses were administered at a dose of 1,000 TCD50 (for MDCK cells) by intranasal inoculation to groups of 27 or 28 volunteers. Viruses A, B, and C all produced disease in six to eight volunteers, whereas D produced no illness and only four volunteers were infected. The viruses shed by the volunteers were indistinguishable from those with which they were inoculated. The haemagglutinin genes of the viruses were sequenced and changes were detected indicating amino acid substitutions at position 196-198 in the attenuated egg-grown virus D whereby a potential glycosylation site present in the other viruses was lost.

A synthetic nucleoside analogue 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboamide (ribavirin or RTCA) inhibits the replication of tissue culture of influenza B virus and also a wide range of influenza A viruses of human, animal and avian origin. The synthesis of influenza virus-induced antigens and also structural and non-structural polypeptides is inhibited by RTCA as detected by immunofluorescence and by pulse labelling experiments with [35S]-methionine. The inhibitory effects of RTCA on influenza A virus replication in tissue culture is reversed by a molar excess of guanosine or zanthosine which suggests that the compound acts at an early stage of virus RNA synthesis prior to the utilisation of the latter nucleosides. A possible inhibitory effect of RTCA on cellular DNA replication is not excluded.

Antigenic relationships among the matrix proteins of influenza A viruses were analysed using a competitive radioimmunoassay technique. There were at least two antigenic determinants on the M protein of A/PR/8/34 virus. One antigenic determinant was shared by several influenza A viruses of human, avian, swine or equine origin, while the second determinant was not necessarily shared by all influenza viruses since it was not detected on the M protein of A/chicken/Germany/N/49 virus. In addition, the cross-reactive determinant(s) of the M protein of N virus was not equally represented in other influenza A virus subtypes.

The major influenza A virus structural antigens, matrix protein, nucleoprotein, haemagglutinin and neuraminidase were measured rapidly and accurately using a rocket immunoelectrophoresis technique. Virus was disrupted with 1% (w/v) sodium sarcosyl and electrophoresed into agarose containing specific antiserum to the individual virus structural proteins in 0.05 M-barbitone buffer, pH 8.6, for 1 to 4 h. For haemagglutinin antigen assays statistical analysis indicated that the coefficient of variation within an immunoelectrophoresis plate was 8.0% for antigen concentrations in the range 15 to 40 microgram/ml protein. For haemagglutinin and matrix protein the method was sufficiently sensitive to measure concentrations of antigens as low as 1.5 and 2.0 microgram/ml respectively. By incorporation in the agarose of mixtures of antisera against specific antigens of the virus, haemagglutinin, matrix or nucleoprotein could be assayed simultaneously.

Intratypic electrophoretic mobility differences in high resolution SDS-polyacrylamide gels were detected between corresponding matrix (M) proteins, nucleoproteins (NP), haemagglutinin (HA) and the non-structural polypeptides NS1 and NS2 induced in Vero cells by human influenza A viruses of the antigenic subtypes H1N1 and H3N2. Such phenotypic differences were distinguishable in both H1N1 and H3N2 viruses isolated in single school and city outbreaks. Additional intratypic variation was detected in the biological property of virus plaquing in MDCK cells. Although the biochemical basis is not established, phenotypic variation could represent an additional factor influencing the epidemiology of influenza A viruses.

Viral recognition of specific receptors in the host cell plasma membrane is the first step in virus infection. Attachment is followed by a redistribution or capping of virus particles on the cell surface which may play a role in the uptake process. Certain viruses penetrate the plasma membrane directly but many, both enveloped and non-enveloped viruses, are endocytosed at coated pits and subsequently pass into endosomes. The low pH environment of the endosome facilitates passage of the viral genome into the cytoplasm. For some viruses the mechanism of membrane penetration is now known to be linked to a pH-mediated conformational change in external virion proteins. As a consequence of infection there are alterations in the permeability of the plasma membrane which may contribute to cellular damage. Recent advances in the understanding of these processes are reviewed and their relevance to the development of new strategies for vaccines emphasised.

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.

Antigenic variants of A/Texas/77 (H3N2) virus were selected in vitro using monoclonal antibody to virus haemagglutinin (HA). The antigenic variants and parental A/Texas/77 viruses were used to to evaluate the frequency of anti-HA antibodies in the sera of children and adults using single-radial-haemolysis (SRH) tests. Twenty to 41% of selected sera from adults, which contained antibody to the parental A/Texas/77 virus, failed to react with the different antigenic mutant viruses. A higher proportion of sera from children (37-58%) failed to react with the antigenic variants. Certain human sera and particularly those of children would appear to possess a more limited antibody repertoire to influenza HA, potentially allowing new antigenic variants to escape neutralization and spread in the community.