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Context: Preventing loss of vaccine potency during storage and handling is increasingly important as new, more expensive vaccines are introduced, in at least 1 case requiring a different approach to storage. Little information is available about the extent to which staff in private physicians' offices meet quality assurance needs for vaccines or have the necessary equipment. Although the National Immunization Program at the Centers for Disease Control and Prevention (CDC) in 1997 developed a draft manual to promote reliable vaccine storage and to supplement published information already available from the CDC and the American Academy of Pediatrics, the best ways to improve vaccine storage and handling have not been defined., Objectives: To estimate the statewide prevalence of offices with suboptimal storage and handling, to identify the risk factors for suboptimal situations in the offices of private physicians, and to evaluate whether the distribution of a new National Immunization Program draft manual improved storage and handling practices., Design: Population-based survey, including site visits to a stratified, random sample of consenting private physicians' offices. At least 2 months before the site visits, nearly half (intervention group) of the offices were randomly selected to receive a draft CDC manual entitled, "Guideline for Vaccine Storage and Handling." The remainder was considered the control group. Trained graduate students conducted site visits, all being blinded to whether offices were in the intervention or control groups. Each site visit included measurements of refrigerator and freezer temperatures with digital thermometers (Digi-thermo, Model 15-077-8B, Control Company, Friendswood, TX; specified accuracy +/- 1 degrees C). Their metal-tipped probes were left in the center shelf of cold storage compartments for at least 20 minutes to allow them to stabilize. The type of refrigerator/freezer unit, temperature-monitoring equipment, and records were noted, as were the locations of vaccines in refrigerator and freezer, and the presence of expired vaccines. Other information collected included the following: staff training, use of written guidelines, receipt of vaccine deliveries, management of problems, number of patients, type of office, type of medical specialty, and the professional educational level of the individual designated as vaccine coordinator., Participants: Two hundred twenty-one private physicians' offices known by the Georgia Immunization Program in 1997 to immunize children routinely with government-provided vaccines., Outcome Measures: Estimates (prevalence, 95% confidence interval [CI]) of immunization sites found to have a suboptimally stored vaccine at a single point in time, defined as: vaccine past expiration date, at a temperature of /=9 degrees C in a refrigerator or >/=-14 degrees C (recommended for varicella vaccine) in freezer, and odds ratios (ORs) for risk factors associated with outcomes. We performed chi(2) analysis and Student's t tests to compare the administrative characteristics and quality assurance practices of offices with optimal vaccine storage with those with suboptimal storage, and to compare the proportion of offices with suboptimal storage practices in the groups that did and did not receive the CDC manual., Results: Statewide estimates of offices with at least 1 type of suboptimal vaccine storage included: freezer temperatures measuring >/=-14 degrees C = 17% (95% CI: 10.98, 23.06); offices with refrigerator temperatures >/=9 degrees C = 4.5% (95% CI: 1.08, 7.86); offices with expired vaccines = 9% (95% CI: 4.51, 13.37); and offices with at least 1 documented storage problem, 44% (95% CI: 35.79, 51.23). Major risk factors associated with vaccine storage outside recommended temperature ranges were: lack of thermometer in freezer (OR: 7.15; 95% CI: 3.46, 14.60); use of freezer compartment in small cold storage units (OR: 5.46; 95% CI = 2.70, 10.99); lack of thermometer in refrigerator (OR: 3.07; 95% CI: 1.15,8.20); and failure to maintain temperature log of freezer (OR: 2.70; 95% CI: 1.40, 5.23). Offices that adhered to daily temperature monitoring for all vaccine cold storage compartments, compared with those that did not, were 2 to 3 times more likely to assign this task to staff with higher levels of training, have received a recent visit from the state immunization program, and be affiliated with a hospital or have Federally Qualified Health Center status. In addition, sites using >1 refrigerator/freezer for vaccine storage were more likely to have at least 1 cold storage compartment outside recommended temperature ranges. We found no significant differences in the data reported above between the intervention group (received copy of the draft manual) and the control group (did not receive copy of draft manual), even when controlling for the annual number of immunizations given or the type of office. (ABSTRACT TRUNCATED)

The 1997 Hong Kong outbreak of an avian influenzalike virus, with 18 proven human cases, many severe or fatal, highlighted the challenges of novel influenza viruses. Lessons from this episode can improve international and national planning for influenza pandemics in seven areas: expanded international commitment to first responses to pandemic threats; surveillance for influenza in key densely populated areas with large live-animal markets; new, economical diagnostic tests not based on eggs; contingency procedures for diagnostic work with highly pathogenic viruses where biocontainment laboratories do not exist; ability of health facilities in developing nations to communicate electronically, nationally and internationally; licenses for new vaccine production methods; and improved equity in supply of pharmaceutical products, as well as availability of basic health services, during a global influenza crisis. The Hong Kong epidemic also underscores the need for national committees and country-specific pandemic plans.

To enhance quality assurance of vaccine distribution by public health programs in the US, various methods for packing vaccines were validated. Validation involved both tests in an environmental chamber and actual shipping of packages by commercial overnight delivery service. Dry ice was used with vaccines needing to be kept at temperatures lower than -14 degrees C, and water-based cold packs with other vaccines. The latter could be used in two ways. When frozen, and placed over two or three faces of well-insulated boxes, assortments of vaccines were kept cold but not frozen for 2 days or more. However, packages with -15 degrees C cold packs may reach < 0 degree C. When cold packs at refrigerator temperature cover four to six faces of well insulated boxes, vaccine freezing in winter conditions or warming in temperate conditions was slowed considerably. These approaches, which require materials costing less than approximately 1% of the cost of the vaccines they protect, provide examples of packaging suitable for overnight delivery of vaccines in the US in different seasons.

It is now more than 30 years since the first cold-adapted influenza viruses were developed in Russia as potential live, attenuated vaccines. In the past 15-20 years considerable experience has been gained from Russian and joint Russian-US laboratory and clinical studies with type A monovalent and bivalent vaccines prepared with genetic reassortant viruses derived from one of these cold-adapted viruses in particular. A/Leningrad/134/57. More recent experiences include use of trivalent cold-adapted vaccines with a type B component. The overall high level of safety of individual and combined vaccines in pre-school and school-aged children, with illness reductions in open field trials equivalent to that seen with inactivated vaccines, is such as to suggest that practical measures might now be justified to facilitate expansion of the use of these vaccines to other countries. It is proposed that further experimentation with the Russian cold-adapted live attenuated vaccines should be focused on issues that will relate to the public health perspective, i.e. selection of the single best candidate type A and B vaccines for intense study using as criteria their potential for meeting licensing requirements outside Russia, and documenting the clinical protective efficacy of a single vaccine dose compared to two doses as studied until now. Resolution of these issues is important to ensure that costs for future live vaccine production, control, and utilization will be kept at lowest levels so that expanded use of live vaccines will have maximum cost-benefit and afford-ability. To guide those interested in these issues, examples are given of populations for whom a licensed live cold-adapted vaccine might be considered, together with indications of extra data needed to fully validate each suggested use.

Increases in infectiousness, neurotropism and virulence were found in a laboratory variant of influenza A/Seal/Massachussets/1/80 (H7N7) virus having a highly cleavable hemagglutinin. Sequential passage from host to host further increased pathogenicity of the H7N7 virus in mice, ferrets and rats.

Schoolchildren of 30 to 34 schools of Novgorod were vaccinated over a three-year period with Russian live cold-adapted attenuated vaccine for children and whole-virus inactivated vaccines and placebo for comparative field study of the vaccines properties and efficacy. In control trials both bi- and trivalent live attenuated vaccines were well tolerated and areactogenic. A whole-virus inactivated trivalent vaccine induced mild and moderate fever and local reactions in 2-4% of the vaccinees. Special observations are necessary to establish the possibility of use and to determine a dose of this inactivated vaccine for immunization of children, especially those of 7-10 years of age. All the vaccines induced HI antibody production in 50-80% and antineuraminidase in 50-70% of seronegative children. The pattern of the results was similar to that in revaccinated children with preexisting antibody at a level of 1:20, but much lower in children with the initial titre above 1:20. After the 3rd year of vaccination the immune response of the vaccinees was similar, most of the results depending on the initial antibody titre and also on the change of vaccine strains. This raises a question of the expediency of annual influenza revaccination of the same person after 2 years of successful immunization and of the necessity of vaccine strains replacement after 2-3 years of use.

Four influenza type B viruses isolated in Russia during periods of relatively low (1987-8) or high (1990-1) influenza B activity were characterized antigenically using a microneutralization assay. These isolates were antigenically similar to contemporary reference strains from either of two separate lineages represented by B/Victoria/2/87 and B/Yamagata/16/88. The evolutionary relationships of the variable portion of the haemagglutinin (HA1) genes of these viruses were determined by comparison with influenza B HA1 sequences previously obtained. The Isolate B/USSR/2/87, collected during the 1987-8 influenza season, was found to be closely related to viruses on the B/Victoria/2/87 lineage that circulated during the 1988-9 influenza season in the United States. Sequence analysis of the isolates from the 1990-1 influenza season demonstrated cocirculation of viruses from both the B/Victoria/2/87 and B/Yamagata/16/88 lineages in Russia, confirming the antigenic analysis.

Children aged 7-14 years in Novgorod, Russia, were given Russian live cold-adapted or inactivated influenza vaccines or placebo over a 2-year period. Schools were randomly assigned as a whole to one of the preparations. In the first year, the vaccines were bivalent, containing types A (H3N2) and A (H1N1) components. In the second year, the vaccines also contained a type B component. In the first year, all viruses isolated were type A (H3N2); in the second, about three-quarters of the isolates were type B and the rest type A (H1N1). During both years, the vaccines protected the vaccinated children. Where significant differences existed, the live attenuated vaccine was more protective than the inactivated. Vaccination rates in schools in which live attenuated vaccines had been used were inversely related to illness rates of staff and unvaccinated children, suggesting that viral transmission had been reduced by the vaccine.

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 microM AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 microM monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25 degrees, but were very prominent at 45 degrees. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.

Recombinant baculoviruses that express the M2 protein from the genes of either the amantadine-sensitive, influenza A/Ann Arbor/6/60 virus or a laboratory-derived, amantadine-resistant mutant of this virus were constructed. Addition of amantadine or rimantadine at 2 micrograms/ml to cultures of Sf9 cells infected with the recombinant baculoviruses increased the yield of the M2 protein from the amantadine-sensitive virus approximately 10-fold, but did not increase the yield of the M2 protein from the amantadine-resistant virus. Flow cytometry demonstrated that the increased production of M2 in the presence of amantadine resulted in increased cell surface expression of the M2 protein. Pulse-chase experiments indicated that whereas the rate of synthesis of the M2 protein increased in the presence of amantadine, the M2 protein was stable in both the presence and absence of amantadine. Addition of amantadine to Sf9 cells as late as 72 h after infection with the recombinant virus increased the production of M2 protein. These data suggest that the M2 protein exerts some biological activity in Sf9 cells.

Objective: To determine whether volunteer family physician reports of the frequency of influenza-like illness (ILI) usefully supplement information from other influenza surveillance systems conducted by the Centers for Disease Control and Prevention., Design: Evaluation of physician reports from five influenza surveillance seasons (1987-88 through 1991-92)., Setting: Family physician office practices in all regions of the United States., Participants: An average of 140 physicians during each of five influenza seasons., Interventions: None., Outcome Measures: An office visit or hospitalization of a patient for ILI, defined as presence of fever (temperature > or = 37.8 degrees C) and cough, sore throat, or myalgia, along with the physician's clinical judgment of influenza. A subset of physicians collected specimens for confirmation of influenza virus by culture., Results: Physicians attributed 81,408 (5%) of 1,672,542 office visits to ILI; 2754 (3%) patients with ILI were hospitalized. Persons 65 years of age and older accounted for 11% of visits for ILI and 43% of hospitalizations for ILI. In three of five seasons, physicians obtained influenza virus isolates from a greater proportion of specimens compared with those processed by World Health Organization laboratories (36% vs 12%). Influenza virus isolates from sentinel physicians peaked from 1 to 4 weeks earlier than those reported by World Health Organization laboratories. Physicians reported peak morbidity 1 to 4 weeks earlier than state and territorial health departments in four of five seasons and 2 to 5 weeks earlier than peak mortality reported by 121 cities during seasons with excess mortality associated with pneumonia and influenza., Conclusions: Family physicians provide sensitive, timely, and accurate community influenza morbidity data that complement data from other surveillance systems. This information enables monitoring of the type, timing, and intensity of influenza activity and can help health care workers implement prevention or control measures.

Eighteen strains of human influenza A (H1N1) viruses isolated between August 1986 and January 1991 were analyzed in this study. Examination of the total viral genome of 12 strains by T1 mapping revealed that considerable genetic heterogeneity exists among these viruses. Partial sequencing of each of the non-HA RNA segments of 4 viruses having divergent T1 oligonucleotide maps indicated that only one was a reassortant virus that had genes from both the influenza A (H1N1) and (H3N2) subtypes. This reassortant obtained its PB2 gene from a virus of the H3N2 subtype and the other 7 RNA segments from an H1N1 parent. Sequencing studies of the HA1 domains of the hemagglutinin (HA) genes of these 18 strains revealed that although these viruses are antigenically similar to the reference strains A/Taiwan/1/86 and A/Singapore/6/86, 7 conserved amino acid substitutions that are shared by recently isolated H1N1 viruses have occurred in the main stream of evolution of the H1N1 subtype. Our data indicate that: (1) Genetic reassortment continues to contribute to genetic variability of H1N1 viruses. (2) Genetic variants of non-reassortant H1N1 viruses are co-circulating in the world. (3) The HA's of recent H1N1 viruses are related to those of the 1986 reference strains. (4) Although there has been little detectable antigenic variability, the HA genes of human epidemic influenza A (H1N1) viruses have continued to evolve at an evolutionary rate similar to that for the H1N1 and H3N2 viruses analyzed previously.

Problem/condition: CDC monitors the emergence and spread of new influenza virus variants and the impact of influenza on morbidity and mortality annually from October through May., Reporting Period Covered: This report covers United States influenza surveillance conducted from October 1988 through May 1989., Description of System: Weekly reports from the vital statistics offices of 121 cities provided an index of influenza's impact on mortality; 58 WHO collaborating laboratories reported weekly identification of influenza viruses; weekly morbidity reports were received both from the state and territorial epidemiologists and from 153 sentinel family practice physicians. Nonsystematic reports of outbreaks and unusual illnesses were received throughout the year., Results: During the 1988-89 influenza season, influenza A (H1N1) and B viruses were identified in the United States with essentially equal frequency overall, although both regional and temporal patterns of predominance shifted over the course of the season. Throughout the season increases in the indices of influenza morbidity in regions where influenza B predominated. Only 7% of identified viruses were influenza A (H3N2), but not isolations of this subtype increased as the season waned and it subsequently predominated during the 1989-90 season. During the 1988-89 season outbreaks in nursing homes were reported in association with influenza B and A (H3N2), but not influenza A (H1N1)., Interpretation: The alternating temporal and geographic predominance of influenza strains A (H1N1) and B during the 1988-89 season emphasizes the importance of continual attention to regional viral strain surveillance, since amantadine is effective only for treatment and prophylaxis of influenza A., Actions Taken: Weekly interim analyses of surveillance data produced throughout the season allow physicians and public health officials to make informed choices regarding appropriate use of amantadine. CDC's annual surveillance allows the observed viral variants to be assessed as candidates for inclusion as components in vaccines used in subsequent influenza seasons.

A recombinant baculovirus expressing the M2 protein from influenza A/Ann Arbor/6/60 (H2N2) virus (AA60 virus) was constructed. The expressed M2 protein was recognized by a monoclonal antibody specific for the M2 protein and comigrated with the M2 protein from cells infected with AA60 virus on SDS-polyacrylamide gels. Immunofluorescence studies indicated that the expressed M2 protein was present on the surface of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus. Immunoassays using the expressed M2 protein were able to detect antibodies to the M2 protein in serum samples from humans and ferrets infected with influenza A viruses.

The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991, B/Hong Kong/22/89- or B/Yamagata/16/88-like viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA1 domains of representative viruses confirmed the continued existence of two main lineages among recent strains of influenza B virus and identified unique amino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.

During the 1989-90 influenza season, 98% of all influenza viruses isolated in the United States and reported to CDC were influenza A. Almost all those that were antigenically characterized were similar to influenza A/Shanghai/11/87(H3N2), a component of the 1989-90 influenza vaccine. Regional and widespread influenza activity began to be reported in late December 1989, peaked in mid-January 1990, and declined rapidly through early April 1990. Most of the outbreaks reported to CDC were among nursing-home residents. Considerable influenza-associated mortality was reflected in the percentage of deaths due to pneumonia and influenza (P&I) reported through the CDC 121 Cities Surveillance System from early January through early April. More than 80% of all reported P&I deaths were among persons greater than or equal to 65 years. In contrast to the predominance of influenza A during 1989-90, during the 1990-91 influenza season 86% of all influenza virus isolations reported were influenza B. Widespread influenza activity was reported from mid-January through April 1991, with regional activity extending into May. Outbreaks were reported primarily among schoolchildren, and no evidence of excess influenza-associated mortality was found. Almost all the influenza B isolates tested were related to influenza B/Yamagata/16/88, a component of the 1990-91 influenza vaccine, but were antigenically closer to B/Panama/45/90, a minor variant.

Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins.

Outbreaks of influenza A (H3N2, A/Shanghai/11/87-like) occurred in two partially (60% and 79%) vaccinated nursing home populations in January 1988. A retrospective cohort study using chart review was designed to assess the effectiveness of influenza vaccination and amantadine prophylaxis (100 mg per day) in controlling the outbreaks and to determine the amantadine susceptibility of influenza viruses isolated from case-patients. The point estimate of vaccine efficacy in preventing influenza-like illness was -33% (95% confidence interval -115% to 18%). However, 9% of vaccinated case-patients died within 14 days after onset of influenza-like illness compared with 26% of unvaccinated case-patients (relative risk = 0.4, 95% confidence interval 0.1-1.0). There was no significant difference in illness severity among case-patients who became ill before amantadine prophylaxis was started (n = 84) compared with those who became ill while taking amantadine (n = 34). Four virus isolates obtained before amantadine prophylaxis was started demonstrated 52-68% inhibition by 1 microgram/ml of amantadine; by comparison, six isolates (resistant viruses) obtained from residents who became ill while taking amantadine demonstrated 1-18% inhibition. The resistant viruses had four different RNA sequences in the gene coding for the M2 protein transmembrane region. Three resistant viruses with identical RNA sequences were isolated from residents living in contiguous rooms who had onset of signs and symptoms during a 6-day interval. Further studies are needed to determine how frequently and under what circumstances resistant viruses occur when antiviral agents are used to control institutional influenza A outbreaks. Strategies for antiviral agent administration that limit the emergence and transmission of resistant virus strains may be needed.

Antigenic and genetic variations have been analyzed in eight consecutive isolates recovered from a child with severe combined immunodeficiency syndrome persistently infected with naturally acquired type A (H1N1) influenza virus over a 10-month period. Hemagglutination inhibition reactions and T1 oligonucleotide fingerprinting demonstrated that these viruses were related to strains causing outbreaks in the United States at that time (1983 to 1984) but that antigenic and genetic differences between consecutive isolates could be detected. This variation between isolates was examined further by sequencing the RNAs encoding the HA1 region of the hemagglutinin (HA) and the nucleoprotein (NP) in five of the consecutive isolates. Multiple point mutations were detected in both genes, and a deletion of one amino acid was detected in the HA. Depending on the isolates compared, 5.8 x 10(-3) to 17 x 10(-3) substitutions per nucleotide site per year were detected in the RNAs encoding the HA1, and 3.5 x 10(-3) to 24 x 10(-3) substitutions per nucleotide site per year were detected in the NP gene. Fifty-four percent of the base changes in the HA1 and 73% in the NP led to amino acid substitutions. A progressive accumulation of mutations over time was not observed, suggesting that the genetic diversity of these viruses may best be interpreted as the result of shifts in the population equilibrium (quasi-species) of replicating variant genomes.

Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.

Analysis of the effects of amantadine during a single cycle of replication of A/FPV/Rostock virus in vitro showed that, as with other influenza A viruses, an M2 protein-dependent step early in infection was inhibited. No effect was observed on later steps in replication under the conditions used.

Full-length cDNA clones of the nucleoprotein (NP) genes of influenza A/Ann Arbor/6/60 and B/Ann Arbor/1/86 viruses were constructed from virion RNA and subsequently expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector, Autographa californica nuclear polyhedrosis virus. Western blot analysis of lysates prepared from Sf9 cells infected with the recombinant viruses confirmed that the baculovirus-expressed NP antigens were reactive with monoclonal antibodies specific for either type A or B NP and with anti-NP antibodies in human serum samples. Electrophoretic analysis indicated that the expressed NP antigens comigrated with NP purified from influenza A or B virions and that the recombinant NP antigens represented greater than 10% of total protein in infected cells. Dilutions of clarified Sf9 cell lysates were used as antigens in a standard enzyme immunoassay to detect serum antibody specific for influenza A or B viruses. The results from assays using the baculovirus-expressed NP antigens showed good correlation with the results obtained using bacterially expressed NP antigen as well as complement fixation. Therefore, baculovirus-expressed NP antigens have the potential to be used to develop reproducible and routine assays for the serodiagnosis of influenza virus infections as an alternative to the complement fixation or haemagglutination inhibition tests.

The protective efficacy of influenza hemagglutinin expressed from recombinant vaccinia virus was compared with that induced by inactivated or infectious influenza vaccines. Intraperitoneal and intranasal routes of vaccination were compared. All the vaccines except the intranasally administered, inactivated vaccine induced detectable levels of neutralizing and hemagglutination-inhibiting antibodies in the serum of mice at 28 days postvaccination. Immunization with any of the intranasally administered vaccines reduced the amount of influenza virus nucleoprotein antigen in lungs after challenge with a homologous, mouse-adapted strain of influenza virus. Intraperitoneally administered vaccines failed to provide such protection. These results indicated that the route of vaccine administration may be the most critical factor for inducing protective immunity. The results also showed that in this mouse model a recombinant DNA-based vaccine could provide protection equivalent to that provided by conventional attenuated and inactivated influenza vaccines.

During 1988-1989 two highly distinct antigenic variants of influenza type B were recognized in hemagglutination-inhibition tests with postinfection ferret serum. These viruses were antigenically related to either B/Victoria/2/87, the most recent reference strain, or B/Yamagata/16/88, a variant that was isolated in Japan in May 1988. All influenza B viruses isolated in the United States during an epidemic in the winter of 1988-1989 were antigenically related to B/Victoria/2/87. However, in several countries in Asia, both B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses were isolated. Sequence analysis of the hemagglutinin (HA) genes of several influenza B isolates from 1987 to 1988 indicated that the HA1 domains of the B/Yamagata/16/88-like viruses and B/VI/87-like viruses isolated in 1988 differed by 27 amino acids. Evolutionary relationships based on this sequence data indicated that the B/Yamagata/16/88-like viruses were more closely related to epidemic viruses from 1983 (B/USSR/100/83-like viruses) than to more recent reference strains such as B/Victoria/2/87. All other Asian strains, as well as selected isolates from the United States in 1988, were confirmed by sequence analysis as being genetically related to B/Victoria/2/87. These data provide clear evidence that two parallel evolutionary pathways of influenza type B have existed since at least 1983 and that viruses from each of the separate lineages were isolated from cases of influenza B in 1988. This finding is similar to earlier observations for type A H1N1 and H3N2 influenza viruses.

A reassortant cold-adapted (ca) influenza B experimental live attenuated intranasal vaccine was evaluated for safety and immunogenicity in children by means of a blind, placebo controlled study. The vaccine contained the haemagglutinin and neuraminidase genes, and the gene for its non-structural proteins from wild-type (wt) B/Ann Arbor/1/86 virus, the contemporary strain at the time of the study. Other genes were derived from ca B/Leningrad/14/55 virus. No increase in illness rates was seen in the children from ages 3-15 years given vaccine at maximum potency (a one in two dilution of infectious allantoic fluid, having a titre of 10(7.0) EID50) compared to children given placebo. About 60% of seronegative children, ages 3-7 years, exhibited a detectible antibody response following one dose of intranasal vaccine, with the seroresponse rate rising to greater than 70% after two doses of vaccine. Immunogenicity was lowest in seropositive children age 8-15 years, reaching a maximum of 36% after two doses. Results indicated that the vaccine was highly attenuated, and probably of adequate immunogenicity for kindergarten age children. The lower immunogenicity in older children suggests the vaccine might be overly attenuated for use in school-age children who are more likely to have a history of prior natural infection with influenza B virus. Further clinical and epidemiological studies of protection are needed to fully assess this.

The adaptation of two influenza B strains (B/Leningrad/14/55 and B/Ann Arbor/1/66) to replication at 25 degrees C is described. Comparison of the two viruses indicates that both also exhibit temperature sensitive phenotypes, although that of the virus B/Leningrad/14/55 is less pronounced. When inoculated into ferrets both viruses replicate well in the trachea, but only the B/Leningrad/14/55 cold-adapted virus replicates in the lungs. This virus exhibited a moderate level of attenuation in the animals, in contrast to the B/Ann Arbor/1/66 cold-adapted virus, which was fully attenuated. Reassortant viruses deriving the surface antigens of the contemporary wild type virus B/Ann Arbor/1/86 and most or all of their other genes, from one or other cold-adapted parent, were virtually indistinguishable from their respective cold-adapted parents. The B/Leningrad/14/55 reassortant was slightly more attenuated than its cold-adapted parent in ferrets. These studies extend knowledge of the properties of viruses used to prepare experimental live influenza B human vaccines.

Mice and guinea pigs were immunized with the haemagglutinin (HA) of influenza B-USSR/100 virus, either in Syntex Adjuvant Formulation-1 (SAF-1) or in saline. Antibody titres were determined by ELISA, haemagglutination inhibition and virus neutralization. Animals immunized with HA in SAF-1 had significantly higher antibody titres than did animals immunized with HA in saline. Both 3-week-old and 13 1/2-month-old mice had greater and more uniform antibody responses to HA in SAF-1 than to HA in saline.

Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the influenza B antibodies detected influenza B reference viruses collected between 1940 and 1984. Pools of either influenza A or influenza B monoclonal antibodies were used to detect influenza viruses reisolated from clinical specimens in tissue culture. At 48 h after inoculation, the influenza A monoclonal antibodies detected 64% of H1N1 and 94% of H3N2 influenza A specimens, and the influenza B monoclonal antibodies detected 79% of the influenza B specimens. The results of this study suggest that the monoclonal antibodies described should provide useful diagnostic reagents for workers in virology laboratories who wish to isolate and identify influenza virus but have been unable to obtain consistent supplies of animal sera specific for influenza A or B viruses.

Monoclonal antibodies that are broadly reactive with either influenza A or influenza B viruses were used to develop a 2- to 3-h antigen capture time-resolved fluoroimmunoassay (TR FIA) for detecting influenza viral antigens in both original nasopharyngeal aspirate specimens and in tissue cultures inoculated with nose or throat swab specimens. The lower limit of sensitivity of the assay was about 10 pg of protein as determined with purified influenza A nucleoprotein expressed by recombinant DNA. When the TR FIA was performed with 96 nasopharyngeal aspirate specimens collected during outbreaks of influenza A (H3N2) virus and the results were compared with serodiagnosis results with paired sera, the specificity and sensitivity of TR FIA for the demonstration of influenza A infections were 95 and 85%, respectively. In culture confirmation assays, more than 80% of the swab specimens that grew influenza A or B virus within 7 days could be identified by the TR FIA within 48 h of the inoculation of cells. The results are consistent with those previously reported for respiratory syncytial virus and extend the applicability of monoclonal antibody-based TR FIA for the rapid diagnosis of acute respiratory viral infections.

Cold-recombinant strains of influenza virus were derived at 25 C using an attenuated cold-adapted (ca) and temperature-sensitive (ts) A/Ann Arbor/6/60 (H2N2) strain and wild-type (wt) strains of epidemic relevance. The cold recombinants were characterized in ferrets in terms of clinical manifestations, viral titers, and histopathologic lesions in turbinates and lungs. The data in ferrets showed that cold recombinants with six genes derived from the ca "master" strain and the two surface antigens of the wt parent strain were predictably attenuated and genetically stable. Attenuation of recombinants with an additional gene derived from the wt parent was less predictable. Recombinants possessing the nonstructural gene of the wt parent showed some reactogenicity at high doses, whereas cold recombinants with RNA 2 from the wt parent did not. A recombinant strain possessing RNA 7 of the wt parent showed reversion to the ts+ phenotype but remained attenuated. Similar results were obtained from testing the recombinants in humans.

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.

The genetic and biological properties of 13 recombinant influenza A clones derived at 25 degrees C from the A/AA/6/60-cold variant (by crosses with 4 different wild type strains) were compared with a set of 5-FU induced ts-mutants. The 5-FU mutants had previously been placed into 7 complementation-recombination groups; the A/AA/6/60-cold parent (PI-7) and the 12 cold recombinant clones which were ts were shown to share a lesion with only one of these groups. The parental strain and 5 recombinant clones were evaluated for replication in the lungs and nasal turbinates of hamsters. Each virus appeared to be attenuated; genetic stability correlated with the level of viral replication in the hamster lung, i.e., viruses which grew best showed a tendency to revert to the ts+ phenotype. Characterization of the ts+ revertants for the presence of the cold adaptation property revealed that these viruses exhibited a spectrum of cold adaptation properties. Two viruses, PI-7 (the parental cold variant) and the CR6 recombinant (A/Queensland/6/72) did not revert in either the lungs or nasal turbinates of hamsters.

Of 45 influenza virus strains (43 type A and 2 type B) detected in conventional tube cell cultures (average time, 4 days), 25 (56%) were detected by immunofluorescence in the shell vial assay 24 h postinoculation. The specific fluorescence produced should allow this procedure to be readily adapted by laboratories with various degrees of experience with immunofluorescence methodology.

Mouse monoclonal antibodies, directed against antigenic sites on influenza A and B viruses and found to be type-specific in an immunoassay, were assessed for use as diagnostic reagents in an indirect immunofluorescence assay on nasopharyngeal secretions. The influenza A antibodies were directed against nucleoprotein or matrix protein antigens and the influenza B antibodies against nucleoprotein and haemagglutinin antigens. The influenza A anti-matrix monoclonal antibody was found to give a strong intranuclear particulate fluorescence in normal baboon kidney cells and cells from nasopharyngeal secretions negative for influenza A virus, including those from a patient infected with respiratory syncytial virus. Pools of the remaining monoclonal antibodies gave satisfactory results on 25 specimens from patients with influenza A H1N1 and H3N2 subtypes and 12 from patients with influenza B.

The hexagonal surface structure believed to be characteristic for influenza C virus was not initially observed for a newly isolated virus of this type. After a few additional egg passages of the virus, however, the hexagonal structure could be readily seen on many virions.

At a time when outbreaks and sporadic cases of influenza caused a A/Victoria/3/75-like and A/Texas/1/77-like H3N2 strain of influenza were occurring in the Rocky Mountain region of the USA, about 60% of the students of a high school in Cheyenne, Wyoming, were involved in an outbreak of influenza-like illness. Six influenza A(H1N1) virus isolates were obtained from throat swabs collected from 12 of these students. Virus isolated from a seventh student, however, contained a mixture of H1 and H3 (A/Victoria/3/75-like) hemagglutinins and N1 and N2 neuraminidases, as shown by the ability to clone from the mixture viruses with antigenic components H1N1, H3N1, and H3N2. An antigenic hybrid virus with H3N1 composition was re-isolated from the original throat swab. The results show that one student was shedding a mixture of A/Victoria/3/75(H3N2)-like and A/USSR/90/77(H1N1)-like viruses at the time his throat swab was taken.

When increases in influenzalike illnesses were first detected during the 1984 to 1985 and 1985 to 1986 influenza seasons, throat swab specimens were mailed by physicians across the United States to one hospital laboratory, and each specimen was inoculated into a single tube of cell culture. Of 165 specimens, 52 (32%) were positive for influenza A or B virus within 3 days of receipt when tested by fluorescence microscopy, permitting positive results to be received by the physicians within 1 week on the average. Approximate direct cost was $16 per specimen.

Antigenic analysis of influenza A(H1N1) virus isolates by hemagglutination inhibition reactions with postinfection ferret sera and mouse monoclonal antibodies showed that, although the predominant virus in 1978 to 1979 had an A/Brazil/11/78-like hemagglutinin, several new antigenic variants could be detected which differed from those identified in 1977 to 1978. Most of the variants showed very minor changes and did not achieve epidemiological significance. Genome analysis by ribonucleic acid-ribonucleic acid hybridization, and supported by oligonucleotide mapping, indicated that nonrecombinant H1N1 viruses cocirculated in the world with recombinant H1N1 viruses, identified in 1978 to 1979. The results of our antigenic analysis and genomic analysis, in conjunction with genomic analysis by other authors, showed that recombinants with A/USSR/90/77-like and A/Brazil/11/78-like hemagglutinin had different genome compositions, indicating that H3N2 virus recombined separately with these different antigenic variants. Prevalence of the nonrecombinant and recombinant H1N1 viruses varied between regions of the world, and influenza A(H1N1) virus with a genotype almost identical to that of nonrecombinant A/Brazil/11/78 virus reappeared as a cause of local outbreaks in the United States in 1980 despite the predominance of recombinant H1N1 strains in the preceding winter.

Direct biochemical evidence has been obtained for the existence of mutations in all eight RNA segments of the A/Ann Arbor/6/60 cold-adapted (ca) mutant influenza virus strain as compared with its wild-type (wt) progenitor. Polyacrylamide gel electrophoresis (PAGE) of viral RNA revealed a change in the electrophoretic migration of RNA 2 (PB1). T1 oligonucleotide mapping revealed changes in two polymerase genes (the PB2 and PA genes), the hemagglutinin (HA) gene and the nucleoprotein (NP) gene. Analysis of S1 nuclease-treated RNA hybrids on polyacrylamide gels detected changes in the HA and neuraminidase (NA) genes. Partial DNA sequence analysis demonstrated a base sequence change in the matrix (M) protein gene that predicts an amino acid change in the M2 protein and a silent mutation in the non-structural (NS) protein gene. In addition, analysis of viral polypeptides by PAGE has so far revealed changes in the viral protein, PA. These findings directly demonstrate the existence of multiple mutations in the ca vaccine strain, a property that may provide reliably and stably attenuated vaccines that derive their six internal genes from the ca A/Ann Arbor/6/60 donor strain.

A solid-phase immunofluorescence assay was evaluated for the identification of viruses isolated in tissue culture, with influenza virus as a model. Purified immunoglobulin G (IgG) from hyperimmune rabbit sera specific for contemporary strains of influenza A or B was covalently attached to microscopic plastic beads to capture virus. Fluorescein isothiocyanate (FITC)-conjugated antibodies of different specificities were then reacted with bound antigen, and the resulting complexes were quantified in a suitable filter fluorometer. The assay, with appropriately absorbed FITC-conjugated second antibody, reliably identified virus present in harvests from cell cultures infected with clinical specimens. For influenza A (H1N1) virus, sensitivity of detecting antigen was about 8- to 32-fold less when an FITC-conjugated monoclonal Igg antibody pool specific for epitopes in three different antigenic sites on influenza hemagglutinin was used as the second antibody as compared to when IgG from hyperimmune sera specific for virus or its components was used as the second antibody. The immunofluorometric assay provides a method for quantitative detection of viral antigen in tissue culture fluids and objective identification of virus type and subtype with FITC-conjugated reagents.

The sequence of the HA1 subunit region of the haemagglutinin gene of influenza A/USSR/90/77, and A/Brazil/11/78, A/Lackland/3/78, A/England/333/80 and A/India/6263/80 was determined by dideoxy-sequencing methods using total virion RNA and specific oligonucleotide primers for reverse transcriptase. These 1977-1980 strains share a minimum of 85% amino acid sequence homology with influenza A/PR/8/34. Most of the surface amino acid substitutions which occurred during the evolution of A/PR/8/34 to A/USSR/90/77 and subsequently in the 1978-1980 strains are located in the 4 antigenic sites previously defined by an analysis of laboratory-selected mutants of A/PR/8/34. We deduce an evolutionary pathway for the 1977-80 strains and suggest their different epidemic properties may be a consequence of only a few amino acid changes.

To determine risk factors for outbreaks of influenza virus infections in chronic-care facilities for the elderly, the authors compared the characteristics of two groups of nursing homes in Genesee County, Michigan, in 1982-1983, following a community-wide epidemic caused by A/Bangkok/1/79-like (H3N2) viruses: seven homes in which an outbreak occurred (case homes) and six homes with sporadic illnesses only (control homes). The two groups were similar in many respects, including the physical characteristics of the facilities, visitation and staffing patterns, infection control practices, and demographic and clinical profiles of residents. Case homes, however, had larger resident populations than control homes (median 160 vs. 92, respectively; p = 0.028) and a lower proportion of residents who had been vaccinated with A/Bangkok antigen the previous autumn (median 51% vs. 81%, respectively; p = 0.047). Based on a retrospectively determined estimate of the efficacy of influenza vaccine in preventing illness (43%), the number of residents presumed to be susceptible to the epidemic strain was higher in case homes than in control homes (median 102 vs. 61, respectively; p = 0.0047) and was the most important factor selected by a stepwise, multivariate logistic regression model (p less than 0.0005). These findings are consistent with mathematical models of vaccine-induced herd immunity in closed populations and suggest that high rates of vaccination can reduce the risk of influenza outbreaks in this setting.

The influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) virus was evaluated as a donor of attenuating genes to new variants of influenza A virus. This ca donor virus was mated with the A/Alaska/6/77 (H3N2) wild-type virus, and three A/Alaska/6/77 (H3N2) ca recombinant viruses were produced. The parental origin of the genes in the three ca recombinants had been determined previously (2), and their virulence for adult seronegative volunteers was assessed in the present study to identify the genes present in the ca donor virus that confer attenuation. Each of the recombinants received the hemagglutinin and neuraminidase genes from the A/Alaska/6/77 (H3N2) wild-type parent. One ca recombinant (CR-29) received all six transferable genes from the ca parent and was found to be satisfactorily attenuated in the volunteers. The two other ca recombinants received five of the six transferable genes with a wild-type gene at the M or NS locus. The pattern of infection in humans with these latter two ca recombinants was similar to the CR-29 ca recombinant. These findings demonstrate that inheritance of a gene in ca recombinants at the M or NS locus segregates independently of attenuation and suggest that the M and NS genes present in the ca donor virus are not the major determinants of attenuation conferred by this virus.

A recombinant virus AA-CR19 was produced that contains RNA segments coding for one polymerase protein, the nucleoprotein and the matrix protein of cold-adapted A/Ann Arbor/6/60, but HA, NA and two other genes coding for polymerase proteins from A/Victoria/3/75. The nonstructural protein gene is probably derived from A/Ann Arbor/6/60, but may contain a spontaneous mutation. Biological characterization of AA-CR19 showed that it possesses the gene(s) responsible for cold-adaptation and temperature-sensitivity of the A/Ann Arbor/6/60 parent. At a dose of 10(7.2) TCID/50. AA-CR19 was found to be immunogenic and attenuated for seronegative volunteers.

Influenza type A nucleoprotein (NP) derived from the full length cloned gene expressed in E. coli was evaluated in a solid phase enzyme immunoassay (EIA) for detection of human antibody to influenza. Monoclonal antibody to human IgG was used for detection. Direct and indirect assays were developed and sera were tested in serial and single dilution formats. Preliminary results indicated that recombinant-and virion-derived NP antigens were comparable in binding ability to plastic and binding human antibody. Eighty-seven paired sera from influenza patients were tested. The most sensitive assay (indirect-serial dilution) detected 56 (64%) rises and the simplest assay (direct-single dilution) detected 43 (49%) rises, compared to 36 (41%) for complement fixation. Paired sera from 18 control patients showed no evidence of antibody rises by any of the assays. Forty-nine paired sera from influenza B infected patients were negative for antibody rises except for one borderline rise by the indirect-serial dilution assay. These results indicate that the use of recombinant DNA derived nucleoprotein for immunoassay is feasible. The sensitivity of immunoassays using NP adsorbed to the solid phase and monoclonal antibody specific for human IgG to detect bound antibody exceeded that of conventional complement fixation testing for establishing serologic evidence of influenza type A infection.

A series of trials was conducted in which wild-type A/USSR/90/77 (H1N1) influenza A virus and a few of its antigenic variants were inoculated into volunteers. Infections readily occurred in people of all ages who had initial low antibody titers, but clinical effects were generally mild in comparison with those of the previously tested subtypes, H0N1, H1N1, H2N2, H3N2. There was, however, an inverse relationship between severity of symptoms and age of volunteers, although the incidence of virus excretion and of increase in anti-hemagglutinin was apparently not age related. Naturally occurring recombinant viruses with H3 hemagglutinin and one or more genes of A/USSR/098/77-like strains were likewise studied in volunteers. These clones also produced mild symptoms, providing evidence of an attenuating effect on H3N2 viruses by the substitution of some of its genes with the genes of an H1N1 virus.

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.

The evolutionary relationships of epidemic influenza A (H1N1) viruses isolated between 1982 and 1986 have been examined by oligonucleotide mapping and partial DNA sequencing. The T1 mapping studies confirmed our previous report that the evolution of the influenza virus genome generally results in an average of four to six oligonucleotide changes per year. Between 1982 and 1986, however, two apparent exceptions to this finding occurred. H1N1 antigenic variants (including the A/Chile/83 and A/Victoria/83 reference strains) that caused influenza outbreaks and epidemics from 1983 to 1984 differed by 20 to 30 oligonucleotides from viruses isolated during the previous influenza season. T1 mapping of individual RNA segments and sequencing revealed that all six internal genes of a representative 1983 A/Chile-like virus were more closely related to genes of non-reassortant H1N1 viruses that circulated from 1977 to 1982 than to genes of H3N2 viruses. Therefore, the 1983 variant viruses were not H1N1-H3N2 reassortants. The A/Taiwan/86-like H1N1 antigenic variants that emerged in south-east Asia in the spring of 1986 and caused epidemic activity the following winter also exhibited changes of 20 to 30 oligonucleotides from the A/Chile/83-like or A/Victoria/83-like H1N1 viruses that circulated during the previous influenza season. Fewer oligonucleotide changes were observed between the 1986 A/Taiwan/86-like and H1N1 viruses isolated before 1983, however, suggesting that the former evolved from viruses that circulated before the 1983 antigenic variants became the predominant H1N1 epidemic virus strains. This was confirmed by sequencing the HA1 domain of the haemagglutinin genes of three A/Taiwan/86-like viruses. These studies provide evidence that other genes of influenza A viruses, in addition to the haemagglutinin gene, may evolve concurrently along two or more separate pathways.