Genetic and phenotypic analysis of reassortants of high growth and low growth strains of influenza B virus.

The yield of influenza virus in eggs is critical to influenza vaccine production and availability, but the contribution of specific genes to the growth properties of influenza B viruses is not well understood. Influenza B/Beijing/184/93 and B/Shangdong/7/97 were chosen for study because B/Shangdong/7/97 replicated to several fold higher titers in eggs than B/Beijing/184/93 as demonstrated by hemagglutination titers and EID50. A reassortant with the HA, NP and PB2 genes from B/Beijing/184/93 and all other genes from B/Shangdong/7/97 had the high growth phenotype of B/Shangdong/7/97 in eggs, which suggests that NS, M, NA, PB1 or PA, or a combination of these genes derived from B/Shangdong/7/97 were needed for the high growth phenotype of the reassortants. A high degree of homology was found among the genetic sequences of B/Beijing/184/93, B/Shangdong/7/97, and other influenza B viruses. However, differences potentially related to growth characteristics were suggested by analysis of the deduced amino acid (AA) sequences of four genes: NS (NS1, NS2), M (BM2), NA (NA, NB) and PB1. The studies identify multiple genes that may affect growth of influenza B viruses in eggs.

The influenza vaccine licensing process.

Influenza vaccines are unique because they require a licensing process which includes a procedure for rapid annual updates to vaccine strains. The licensing procedures in the European Union and the USA are described as examples. In the event of an influenza pandemic, vaccines will be required urgently and licensing process should reflect such needs.

Immune-globulin prophylaxis of respiratory syncytial virus infection in patients undergoing stem-cell transplantation.

Thirty-two patients undergoing allogeneic hematopoietic stem-cell transplantation were given respiratory syncytial virus (RSV) immune globulin (RSVIG) at the time of transplantation and again 3 weeks later. Antibody titers to RSV, human parainfluenza virus 3, measles, and influenza H1N1, H3N2, and B were measured prior to administration of RSVIG and 6 more times over the course of the subsequent 6 weeks. Baseline antiviral titers and increases in antibody after administration of RSVIG were extremely variable for all the viruses. In 18 patients in whom the baseline titers of antibody titers to RSV-F protein were 1:640-1:2048, there was a 7.7-fold initial increase in these titers after the first dose of RSVIG, compared with a 2.1-fold increase in 14 patients with baseline titers of 1:4096-1:20,840; increases in titers of antibody against the other viruses after the first dose of RSVIG reflected similar variability. The subset of patients with the lowest titers appear to receive the greatest benefit from administration of RSVIG.

Effect of influenza virus matrix protein and viral RNA on ribonucleoprotein formation and nuclear export.

The formation of influenza virus ribonucleoprotein (RNP) is a necessary step in viral assembly and maturation in infected cells, but the mechanism remains incompletely understood. Influenza virus proteins such as matrix (M1) and cellular proteins have been implicated in assembly and transport of RNP. To study the assembly of RNP and the translocation of RNP complexes in cells, RNPs were reconstituted from nucleoprotein (NP), M1, and viral RNA (vRNA) synthesized in vitro. The syntheses were accomplished using specific plasmids in a system coupling transcription and translation under the control of the T7 promoter. The density of the resulting RNP complexes was analyzed by glycerol gradient centrifugation and the morphology was examined by transmission electron microscopy. Protomers of NP self-assembled into circular oligomers regardless of the presence of vRNA or M1. However, helical structures similar in conformation and density to RNPs purified directly from influenza virus were formed only when M1 and vRNA were also present. In the absence of vRNA, no helical structures were formed from NP and M1. The plasmids also contained the CMV promoter, which permitted expression of M1, NP, and vRNA in Madin-Darby canine kidney (MDCK). M1 and NP were both present in the cytoplasm of MDCK also expressing vRNA, but NP was retained in the nucleus of cells expressing M1 without vRNA. Our data demonstrate for the first time that vRNA and M1 together promote the self-assembly of influenza virus NP into the quaternary helical structure typical of the viral RNP. The results also indicate that the interaction of NP with vRNA and M1 in a system devoid of other viral proteins can lead to translocation of RNP from nucleus to cytoplasm.

A comprehensive systematic approach to identification of influenza A virus genotype using RT-PCR and RFLP.

Amplification of influenza A virus gene segments by reverse transcription-polymerase chain reaction (RT-PCR) can be combined with enzymatic digestion to reveal unique restriction fragment length polymorphisms specific for H1N1 and H3N2 subtype viruses. We have used the method to provide a rapid, specific and reproducible identification of the genotype of high-growth influenza reassortants derived from A/Puerto Rico/8/34 (PR8). Digestion of the gene segments amplified from wild-type viruses, PR8 and reassortants at sites unique to either the wild-type strain or to PR8 provided positive, unambiguous identification of the origin of each of the internal genes, and distinguished the internal genes of both H1N1 and H3N2 strains from those of PR8. This method has also permitted us to quickly confirm that reassorting has occurred and to optimize the selection of reassortant clones with maximum number of PR8 internal genes. Since the method can detect 1-10% of a second strain in a mixed population, the method can also be used to detect samples containing more than one viral subtype and to assess the purity of influenza viruses used for manufacturing vaccines.

Regulatory perspective in the United States on cell cultures for production of inactivated influenza virus vaccines.

The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy. For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important. Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents.

Association of influenza virus matrix protein with ribonucleoproteins.

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.

The efficacy of influenza vaccine in elderly persons. A meta-analysis and review of the literature.

OBJECTIVE: To quantify the protective efficacy of influenza vaccine in elderly persons. DATA SOURCES: A MEDLINE search was done using the index terms influenza vaccine, vaccine efficacy, elderly, mortality, hospitalized, and pneumonia. Appropriate references in the initially selected articles were also reviewed. STUDY SELECTION: Only cohort observational studies with mortality assessment were included in the meta-analysis. In addition, 3 recent case-control studies, 2 cost-effectiveness studies, and 1 randomized, double-blind, placebo-controlled trial were reviewed. DATA EXTRACTION: Vaccine and epidemic virus strains, age and sex of patients, severity of illness, patient status, and study design were recorded. Upper respiratory illness, hospitalization, pneumonia, and mortality were used as outcome measures. DATA SYNTHESIS: In a meta-analysis of 20 cohort studies, the pooled estimates of vaccine efficacy (1-odds ratio) were 56% (95% Cl, 39% to 68%) for preventing respiratory illness, 53% (Cl, 35% to 66%) for preventing pneumonia, 50% (Cl, 28% to 65%) for preventing hospitalization, and 68% (Cl, 56% to 76%) for preventing death. Vaccine efficacy in the case-control studies ranged from 32% to 45% for preventing hospitalization for pneumonia, from 31% to 65% for preventing hospital deaths from pneumonia and influenza, from 43% to 50% for preventing hospital deaths from all respiratory conditions, and from 27% to 30% for preventing deaths from all causes. The randomized, double-blind, placebo-controlled trial showed a 50% or greater reduction in influenza-related illness. Recent cost-effectiveness studies confirm the efficacy of influenza vaccine in reducing influenza-related morbidity and mortality and show that vaccine provides important cost savings per year per vaccinated person. CONCLUSION: Despite the paucity of randomized trials, many studies confirm that influenza vaccine reduces the risks for pneumonia, hospitalization, and death in elderly persons during an influenza epidemic if the vaccine strain is identical or similar to the epidemic strain. Influenza immunization is an indispensable part of the care of persons 65 years of age and older. Annual vaccine administration requires the attention of all physicians and public health organizations.

Time to peak serum antibody response to influenza vaccine.

The time to the appearance of a peak serum antibody response to influenza virus vaccine is not clearly defined. We compared the most commonly used time intervals described in the literature--4 and 6 weeks after vaccination. We studied 118 elderly patients from three different geographic sites. The 1992 to 1993 trivalent inactivated influenza virus vaccine containing influenza virus A/Beijing/353/89 (H3N2), influenza virus A/Texas/36/91 (H1N1), and influenza virus B/Panama/45/90 was used. No statistically significant differences were found at the 4- and 6-week intervals after vaccination.

Vaccine immune response and side effects with the use of acetaminophen with influenza vaccine.

The purpose of this study was to determine whether acetaminophen impairs the immune response to influenza vaccine. Influenza vaccine is an under-utilized preventive measure, partly because of the unfounded perception that fever and myalgias frequently follow vaccination. While acetaminophen may decrease these infrequent side effects, it may also alter the immune response to vaccination. We compare the effect of acetaminophen with placebo on the humoral immune response to the 1991-1992 commercially available influenza vaccine. We studied 60 healthy, elderly subjects from a geriatric clinic and 20 infirm, elderly subjects from a nursing home. The subjects were randomly assigned to receive placebo or acetaminophen (1,000 mg every 6 h) for 2 days. Acetaminophen did not depress or enhance the immune development of serum hemagglutination inhibition antibody to the three vaccine antigens. The systemic side effects of fever and myalgia were uncommon in both groups. The healthy elderly subjects mounted a significantly better immune response to the influenza virus A/Taiwan/1/86 (H1N1) vaccine strain than did the infirm elderly subjects (geometric mean titer, 115 versus 51; P = 0.003). The functional activity score obtained by using the chronic healthy evaluation component of the Acute Physiology and Chronic Health Evaluation system could be used to distinguish the healthy from the infirm elderly (scores of 1.27 versus 3.75, P < 0.001). Acetaminophen neither depressed nor enhanced the serum antibody response to the vaccine in the healthy and infirm elderly subjects studied.

Antibody responses to influenza B viruses in immunologically unprimed children.

The cocirculation in several parts of the world of influenza viruses B/Yamagata/16/88 and B/Victoria/2/87, which are genetically and antigenically divergent, has prompted the question of whether immunization with one viral antigen is sufficient for protection against both strains. Twenty-three high-risk infants and young children were immunized with a commercial trivalent influenza vaccine containing the antigens of influenza virus B/Yamagata/16/88. When antibodies against influenza viruses B/Yamagata/16/88 and B/Victoria/2/87 were determined, increases developed uniformly to both in the sera of primed children previously exposed to influenza virus B/Victoria/2/87 by immunization or infection. Antibodies against B/Yamagata/16/88 developed in the sera of unprimed children with titers similar to those of the primed children. However, antibodies to B/Victoria/2/87 were not detected in the sera of the unprimed children. These data suggest that children without appropriate immunologic priming may not be protected against an infection with a B/Victoria/2/87 strain after vaccination with a B/Yamagata/16/88 strain. Immunization with more than one influenza B virus strain may be desirable in some high-risk pediatric patients if divergent influenza B viruses circulate.

Rhinovirus induces natural killer-like cytotoxic cells and interferon alpha in mononuclear leukocytes.

Natural killer-like cellular cytotoxicity was augmented by incubation of human rhinovirus serotype 2 with peripheral blood mononuclear leukocytes collected from healthy donors. The production of alpha interferon but not gamma interferon was identified in the same cell cultures. A specific interaction of conformationally intact rhinovirus with peripheral blood mononuclear leukocytes was required for induction of the response, since the response was extinguished at reduced quantities of infectious rhinovirus, and acid inactivated rhinovirus did not augment cellular cytotoxicity. Productive replication of rhinovirus was not observed in cultures of peripheral blood mononuclear leukocytes. The replicative failure was not related merely to interferon production, since the rate of disappearance of rhinovirus was similar to that observed in cell free medium. The findings suggest that natural killer cells should be considered as a potential component of the local nasopharyngeal pathophysiology of rhinovirus infection.

Cross-reactive antibodies induced by a monovalent influenza B virus vaccine.

Influenza viruses related to the markedly antigenically divergent strains B/Yamagata/16/88 and B/Victoria/2/87 are circulating in human populations. Adults develop cross-reacting antibodies against recent and earlier influenza B virus strains after vaccination with B/Yamagata/16/88, probably because of previous influenza B virus infections or immunizations. Vaccines containing B/Yamagata/16/88 should adequately protect adults against B/Victoria/2/87 infections.

Nasal-secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection.

Leukocyte populations in the secretions of volunteers challenged by intranasal inoculation with rhinovirus serotype 25 were evaluated by means of flow cytometry. With the light-scatter properties of peripheral blood leukocytes as the standard of reference, significant increases (P less than .05) of both lymphocytes and phagocytes (polymorphonuclear leukocytes plus monocytes) were detected in the nasal secretions of persons infected by the viral challenge. There was a direct correlation of nasopharyngeal symptom severity with both the percentage of lymphocytes (P less than .05) and the percentage of phagocytes (P less than .001). Monoclonal antibodies for specific cell-membrane antigens identified the lymphocytes and phagocytes as leukocytes and also demonstrated the presence of a population of monocytic cells during the phase of maximal symptoms. The panel of monoclonals chosen did not unequivocally identify a lymphocyte population except in the presence of nosebleed. However, the results show that flow cytometry can be used to investigate nasal-secretion cell populations during the rhinovirus common cold.

Successful treatment with ganciclovir of disseminated cytomegalovirus infection after liver transplantation.

Disseminated cytomegalovirus (CMV) infection in a liver transplant recipient was treated successfully by administration of ganciclovir (BW B759U) at a dosage of 7.5 mg/kg/day for 2 wk in the face of continuation of chemical immunosuppression. The spectrum of illness included symptomatic esophagitis and hepatic dysfunction associated with the appearance of CMV inclusion bodies, retinal lesions, and bone marrow suppression. Clinical improvement during therapy with ganciclovir was prompt and was paralleled by reversal of histological abnormalities. CMV was recovered from none of the cultured tissues after the start of therapy. Ten months after discontinuation of ganciclovir, the patient had no evidence of further CMV disease. The observation suggests that replicative CMV infection in organ-transplanted patients may be suppressed by relatively low dose ganciclovir, even when the patients are maintained on immunosuppressive regimens designed to prevent graft rejection.

Acute-phase decrease of T lymphocyte subsets in rhinovirus infection.

Populations of peripheral blood leukocytes were enumerated in 15 volunteers challenged by intranasal inoculation with rhinovirus serotype 25. The results demonstrated a significant decrease in total lymphocyte count among infected persons on the third day after challenge with the virus (P less than .01). The change in lymphocyte count was associated with a significant decrease in total T cells, as determined by monoclonal antibodies (both T11+ and T3+, P less than .02), but not in B cells (B7+). Among the subsets of T cells, T4+ (T helper/inducer) and T8+ (T suppressor/cytotoxic) lymphocytes both declined in number, but only the change in the T4+ subset was significant. For each of the lymphocyte populations that decreased significantly (T3+, T11+, and T4+) there was a strong correlation with increased severity of symptoms. Persons who had the greatest decrease in total lymphocyte count also shed virus most frequently. The number of nonlymphocyte leukocytes increased with the severity of the symptoms. These data show that T lymphocytes (particularly the T4+ population) are related to both the progression of infection and the symptoms of the rhinovirus common cold.

The effects of acute respiratory virus infection upon tracheal mucous transport.

Tracheal mucous velocity was measured in 13 healthy non-smokers using a radioisotope-labeled aerosol and a multidetector probe during respiratory virus infections. The movement of boluses of tracheal mucous were either absent or reduced in number in five subjects with myxovirus infection (four influenza and one respiratory syncytial virus) within 48 hr of the onset of symptoms and in four subjects 1 wk later. One subject with influenza still had reduced bolus formation 12-16 wk after infection. Frequent coughing was a feature of those subjects with absent tracheal boluses. In contrast, four subjects with rhinovirus infection had normal tracheal mucous velocity at 48 hr after the onset of symptoms (4.1 +/- 1.3 mm/min). Tracheal mucous velocity was also normal (4.6 +/- 1.1 mm/min) in four subjects in whom no specific viral agent could be defined but of respiratory viral infection. During health tracheal mucous velocity was (4.8 +/- 1.6 mm/min) in the eleven subjects who had measurements made. Disturbances in tracheal mucous transport during virus infection appear to depend upon the type of virus and are most severe in influenza A and respiratory syncytial virus infection.

Modifications of lung clearance mechanisms by acute influenza A infection.

Four volunteers with naturally acquired, culture-proved influenza A infection inhaled a radiolabeled aerosol to permit investigation of lung mucociliary clearance mechanisms during and after symptomatic illness. Mucus transport in the trachea was undetectable when monitored with an external multidetector probe within 48 hours of the onset of the illness, but was found at a normal velocity (4.9 +/- 1.9 mm/min) by 1 week in three of the four subjects. In two volunteers who coughed 23 to 48 times during the 4.5-hour observation period, whole lung clearance was as fast within the first 48 hours of illness as during health 3 months later in spite of the absence of measurable tracheal mucus transport. Conversely, in spite of the return 1 week later of mucus transport at velocities expected in the trachea, whole lung clearance for the 4.5-hour period was slowed in two volunteers who coughed less than once an hour. The data offer evidence that cough is important in maintaining lung clearance for at least several days after symptomatic influenza A infection when other mechanisms that depend on ciliary function are severely deficient.

Streptococcus milleri endocarditis complicated by myocardial abscess.

A patient with Streptococcus milleri endocarditis and myocardial abscess was bacteriologically cured after surgical intervention and six weeks of parenteral penicillin therapy plus two weeks of gentamicin therapy. Studies of the bacterial isolate suggest that gentamicin added no therapeutic advantage to the antimicrobial regimen. This case demonstrates that local cardiac suppurative complications can occur during endocarditis caused by S milleri, and suggests that speciation of alpha-hemolytic streptococci may in some instances complement antimicrobial susceptibility testing.

Prognostic implications of therapy for necrotizing external otitis.

Necrotizing external otitis is a slowly progressive infection of the ear canal and basal skull caused by Pseudomonas aeruginosa. Treatment with aminoglycoside and antipseudomonal penicillin antibiotics significantly reduces extension of infection, decreases the severity of the associated cranial nerve injury, and limits disease-related mortality. Combined antimicrobial and surgical treatment appears to be more efficacious than antibiotics alone when evaluated for comparable stages of the disease. However, invasive surgical procedures may promote the spread of infection, particularly in the absence of appropriate antibiotic therapy. A high index of suspicion for the syndrome should be aroused when external otitis is present for longer than two weeks, especially after local debridement and topical antibiotic treatment. Aggressive use of systemic antibiotic therapy in diabetic patients, who are at greatest risk, should reduce disease extension and lessen the need for multiple surgical procedures.