Outbreak of influenza amongst residential school students in Malaysia.

In the months of July and August 2003, an outbreak of acute respiratory illness caused by influenza A virus occurred among students in seven residential schools situated in the northern part (Perak) of Peninsular Malaysia. Out of 4989 students, aged 13 to 18 years (mean = 15.9), 1419 (28%) were effected by influenza-like illness. All patients were treated as outpatients except for 36 students who required admission for high fever, severe coughing and shortness of breath. Abnormal chest X-ray findings were noted for those that required inpatient management. Influenza A virus was isolated from 37 sputum specimens, 20 throat swabs and three nasal swab specimens from a total of 278 clinical samples obtained from 180 patients. Isolates from each of the outbreaks were sent to WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia for antigenic and genetic analysis. One school outbreak was due to influenza A (H1N1), A/New Caledonia/20/99-like virus while the other six school outbreaks were due to influenza A (H3N2) viruses which were A/Fujian/411/2002-like).

Influenza virus: an NMR study of mechanisms involved in infection.

High resolution 1H-NMR spectroscopy has been used to study the infection of chicken embryo fibroblasts by influenza virus. Marked changes in the NMR spectrum occur when infectious influenza virus is introduced into the fibroblasts and these changes appear to depend upon the presence of active neuraminidase (EC 3.2.1.18). A crude preparation of neuraminidase from Vibrio cholerae is able to effect similar changes. Only minor spectral changes are observed in the absence of culture medium or when the viral genome material is inactivated by beta-propiolactone. Similarly, little change is seen in the NMR spectrum when amantadine, which is thought to inhibit uncoating of the virus inside the cell, or actinomycin D, which inhibits cellular nucleic acid metabolism, are incubated with fibroblasts prior to the addition of virus. The results suggest that neuraminidase, in co-operation with a factor in the infectious process, initiates a cellular event which can be monitored by NMR. The nature of this cellular mechanism is unknown, but further studies are under way to determine its importance in viral infection.

Effect of monoclonal anti-neuraminidase antibodies on the kinetic behavior of influenza virus neuraminidase.

Neuraminidase from the recombinant influenza virus A/NWSHA-Tokyo/3/67NA HON2 has been shown to exhibit non-Michaelis-Menten kinetics. The multiphasic behaviour was demonstrated for both the isolated neuraminidase heads and for the intact virus. Interaction of the enzyme with two monoclonal anti-neuraminidase antibodies (WANA 1 and RANA 1), which recognize separate antigenic determinants on the molecule, resulted in hyperbolic kinetic behaviour. While both antibodies abolished the multiphasic kinetics of the enzymic reaction, only WANA 1 altered the Vmax and Km values, indicating that it may in some way inhibit the interaction of enzyme and substrate.

Influenza immunization in adults with diabetes mellitus.

The antibody responses to influenza vaccination of a group of adult diabetic patients were compared with responses in a healthy group of regular volunteer vaccinees. The initial and final geometric mean hemagglutination-inhibiting antibody titers were lower in the patient group, but the relative increase in titers was greater for each of the vaccine components. The percentage of fourfold rises in individual titers was greater in the diabetic group than in the control group. It was concluded that patients with diabetes mellitus responded normally to influenza vaccination. This was confirmed in an additional study. There was no significant difference in the antibody responses of patients treated with insulin or oral antidiabetic agents. There was no impairment of diabetic control as a result of influenza vaccination when this was evaluated by measuring the concentration of glycosylated hemoglobin, or by random blood glucose estimations. There was no significant change in the serum insulin level after immunization in patients on oral diabetic agents. It was concluded that influenza vaccination was safe and effective in adult diabetic patients.

Reassortants in recent human influenza A and B isolates from South East Asia and Oceania.

From 2000 to 2002, human influenza A and B viruses that were genetic reassortants of contemporary circulating human strains, were isolated in South East Asia and Oceania. Similar to reports from other regions, A(H1N2) isolates were found to be reassortants of circulating A(H3N2) viruses that had acquired only the haemagglutinin gene of an A(H1N1) virus. Some of these reassortants from Thailand and Singapore predate those previously recorded during the winter of 2001-2002 in Europe and the Middle East and may be precursors of these viruses. The B reassortants had a haemagglutinin similar to an earlier B strain, B/Shangdong/7/97 (B/Victoria/2/87-lineage) and a neuraminidase similar to the recently circulating B/Sichuan/379/99 virus (B/Yamagata/16/88-lineage). Despite the early occurrences of A(H1N2) reassortants and the extensive circulation of A(H1) viruses in South East Asia and Oceania during 2000-2001, these reassortant influenza A viruses have to date not been prominent unlike Europe and the Middle East where they were common in the 2001-2002 winter. In contrast the reassortant B viruses, which first emerged in this region in early 2002, rapidly became the predominant strains isolated from patients with influenza B in South East Asia and Oceania.

Use of oligonucleotide probes for selecting potential high-yielding influenza reassortants.

A new method for rapid screening of high-yielding reassortants of influenza virus, as candidates for vaccine production, is described. Oligonucleotide probes specific for all the parent genes of A/PR/8/34 (PR8), except the HA and the NA were designed based on database information available for different influenza strains. Digoxigenin labelled probes were tested by slot-blot hybridizations to purified RNA from a panel of A/PR/8/34 wild type and A/PR/8/34 reassortant viruses. The results show that the vast majority of reassortants selected for their high growth yield had acquired the non-structural (NS), matrix (M) and RNA polymerase 2 (PB2) genes from the PR8 parent. It is proposed that probes for these genes provide the potential for a simple and rapid procedure for selection of candidate high-yield reassortants for vaccine production.

Enzyme immunoassay for antibodies to membrane associated antigen of varicella zoster virus.

An in situ enzyme immunoassay to viral membrane antigen was developed to enable the specific estimation of antibodies to varicella zoster (VZ) virus. The technique was compared with a modified fluorescent antibody to membrane antigen (FAMA) procedure and with the complement fixation (CF) test by parallel assay of 352 plasma samples. The enzyme immunoassay (EIA) procedure showed very good correlation with the modified FAMA procedure, and both were far more specific than the CF test. This specificity was achieved by the use, in the EIA, of VZ virus-infected cells grown and fixed in situ with glutaraldehyde. Thus the only virus antigens accessible to antibody were the VZ-specific antigens expressed at the cell membrane, cross-reactions with herpes simplex virus antibodies thereby being avoided.

Identification of genetic diversity by cultivating influenza A(H3N2) virus in vitro in the presence of post-infection sera from small children.

Antigenic variants probably arise in the field by escaping herd immunity. We have earlier found that sera from small children are more strain-specific than sera from adults and could therefore, provide favourable conditions for selecting antigenic escape mutants. We had access to small volumes of anonymous sera collected in Norway after the epidemic season 1999/00, which was dominated by the A/Panama/2007/99 (H3N2) variant. The HA gene of the representative strain of that season was genetically identical to A/South Australia/147/99 (H3N2) and was selected for this study. Two sera from children aged 4 and 3 years, respectively, and one adult (64 years old) were used to attempt selecting antigenic escape mutants. Virus was grown in MDCK cells in the presence of human serum and escaped variants were tested by haemagglutination-inhibition tests. Although variant strains were occasionally identified, their HA1 genetic sequence did not identify obvious changes at known antigenic sites. However, by cloning and subsequent sequencing, the genetic diversity of the parent virus was found to be significantly reduced when grown in the presence of human sera. Data also showed that the two children's sera selected additional mutants from those already present in the parent pool and that the two sera selected different mutants. On a community level, it is possible that antigenic changes could be accumulated in a step-wise manner when epidemic virus is transmitted from one small child to the next, each with a restricted and possibly variant antibody repertoire.

Isolation of avian influenza viruses from two different transhemispheric migratory shorebird species in Australia.

Shorebirds on their southerly migration from Siberia to Australia, may pass through Asian regions currently experiencing outbreaks of highly pathogenic H5N1 influenza. To test for the presence of avian influenza viruses in migratory shorebirds arriving in Australia during spring 2004, 173 cloacal swabs were collected from six species. Ten swabs were positive for influenza A, with H4N8 viruses detected in five red-necked stints and H11N9 viruses detected in five sharp-tailed sandpipers. No H5N1 viruses were detected. All isolated viruses were non-pathogenic in domestic chickens. These results further demonstrate the potential for migratory shorebirds to carry and potentially spread influenza viruses.

An influenza A(H3) reassortant was epidemic in Australia and New Zealand in 2003.

During 2003, Australia and New Zealand experienced substantial outbreaks of influenza. The strain responsible was an A(H3N2) influenza virus described as A/Fujian/411/2002-like, which had circulated as a minor variant in the previous Northern Hemisphere (NH) winter, mainly in Korea and Japan. Early in the year the isolates were very similar to those that had been previously isolated in the NH, however, a reassortant strain emerged early in the New Zealand winter, followed by the appearance of similar viruses in Australia and other regional areas. While the hemagglutinin HA1 sequence of these viruses demonstrated only minor differences from the A/Fujian/411/2002 reference strain, the neuraminidase gene was clearly different from that of other recently circulating H3 viruses and most closely matched an earlier reference strain A/Chile/6416/2001. Three internal genes (NS, NP, M) in the reassortant viruses were also more closely related to the A/Chile/6416/2001 lineage. This reassortant A(H3) virus predominated in Australia and New Zealand in 2003 was also seen in Brazil and Malaysia during 2003 and was widespread in the United States and Europe during their 2003-04 winter. Interestingly most of the strains of A(H3) that were isolated at the beginning of the 2004 winter in Australia, did not have this earlier A/Chile/6416/2001-like neuraminidase but had a neuraminidase that was similar to that of the reference strain A/Fujian/411/2002. This was suggestive of the re-introduction of influenza A(H3) from other countries, however, there was still low level circulation of the reassortant virus in 2004 with isolates detected in Australia and Singapore.

Surveillance for pandemic influenza.

Concerns that a new influenza strain may arise that would exhibit similar properties to the 1918-1919 pandemic virus prompted the decision in 1947 to establish a World Health Organization global program for influenza surveillance. This program has contributed greatly to understanding of the epidemiology of influenza and provides the basis for the timely updating of influenza vaccine formulations during interpandemic periods. The spread of pandemic influenza, however, is extremely rapid and, in 1957 and 1969, occurred before sufficient supplies of vaccine could be prepared and administered. Recent evidence regarding the origin of new influenza strains provides some opportunities for improving surveillance for pandemic influenza, but there is a danger that the benefits may be offset by even more rapid spread of a future pandemic due to changes in worldwide transportation and commerce.

A simple endpoint dilution method for evaluating serum used for cell culture.

A simple endpoint dilution method for evaluating foetal calf serum quality is described. The test uses a series of doubling dilutions of cells on microtitre trays with the test sera added to replicate dilution series. After five to six days of incubation the cells are stained with crystal violet and the end points read macroscopically. The cell growth-promoting property of serum may be expressed as a reciprocal of the cell dilution resulting in an approximately 50% coverage of cells.

Influenza vaccination: cost-effective health care for the older adult?

Influenza is a serious cause of morbidity and mortality, particularly in the older adult, and it represents a significant economic burden for health-care services. We have reviewed the published literature relating to influenza vaccine effectiveness and the cost-effectiveness of influenza vaccination in comparison with other health-care interventions and conclude that influenza vaccination is one of the most cost-effective interventions possible in the older adult population.

Planning for the next pandemic of influenza.

Worldwide influenza pandemics have occurred at irregular and unpredictable intervals throughout history and it is confidently expected that they will continue to occur in the future. It is now recognised that these pandemics result when avian influenza A viruses succeed in adaptation to and transmission between humans. The impact of pandemic influenza is substantial in terms of morbidity, mortality and economic cost and there is the potential for serious social disruption. Influenza vaccines remain the most effective defence against influenza but will be in short supply during a pandemic, as will the new specific anti-influenza drugs, due to the lead-time required for production and rapid spread of the virus. To minimise the impact of pandemics it is imperative to maximise the availability of both vaccines and antivirals and to ensure that they are used optimally. This requires planning at both the international and national levels. The World Health Organization has, therefore, developed a staged plan for responding to a pandemic threat which is based principally on its surveillance program. It has also prepared guidelines to assist national agencies in their planning. However, there may be further options for increasing our preparedness which should also be considered.

The single radial immunodiffusion assay highlights small antigenic differences among influenza virus hemagglutinins.

The results of single radial immunodiffusion assays of influenza virus hemagglutinin were found to be greatly altered by small antigenic differences between test and reference strains. When such differences were present, the precise specificity of the antiserum used had a critical effect on the measured hemagglutinin antigen content obtained.

An immunofluorescence study of influenza virus filament formation.

A study is described in which filamentous forms of influenza virus were observed budding from host cell surfaces. Cell cultures infected with influenza virus were stained by indirect immunofluorescence using an antiserum to purified haemagglutinin. Filaments greater than 100 micrometers in length, with several branch points along their length were observed; the number and length of filaments varied according to the virus strain and the time after infection. Examination of infected cells by electron microscopy confirmed the presence of branched structures with an ultrastructure typical of filamentous forms of influenza virus. The immunofluorescence technique was quicker than thin section electron microscopy and was a more sensitive procedure for the detection of filamentous forms of influenza virus than electron microscopy using negative stain. It also enabled the antigenic composition of the filaments to be observed.

Antibody responses to influenza vaccines containing A/USSR/90/77.

Studies were undertaken in adult groups aged 17-24 years, 25-64 years and 66-100 years to determine the haemagglutination-inhibiting antibody responses to sub-unit influenza containing A/USSR/90/77 (H1N1). Antibody responses to A/USSR/90/77 were low in all groups. The young adult group (17-24 years) produced a primary response to A/USSR/90/77 and showed a significant response to a second dose of vaccine, whereas their responses to the A/Texas/1/77 (H3N2) and B/Hong Kong/8/73 components were of the anamnestic type and showed no significant increase to a second dose. The adult (25-64 years) and aged (66-100 years) groups responded anamnestically to all three vaccine components. There was no impairment of the antibody response in the aged group in comparison with the response in the adult group. A comparative assay in microtitre trays and WHO plates showed two- to four-fold differences in antibody titre to A/USSR/90/77 in these systems.

Effect of dose on antibody response to subunit influenza vaccine.

In a group of third-year medical students who were given subunit influenza virus vaccine, the haemagglutination-inhibition (HI) antibody response to A/Victoria/3/75, and the boosting of HI antibody to previously circulating A strains of the Hong Kong subtype were independent of the dosage in the tested range. There was minimal boosting of antibody levels to the Asian strains which circulated during the childhood of the volunteers. The antibody responses to the B/Hong Kong/8/73 component were lower and were dose-related. There was no appreciable increase in the HI geometric mean titre to any of the strains after a second dose.