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Throughout Eastern Asia, there is currently an epidemic of fowl plague or highly pathogenic avian influenza, on an unprecedented scale. The prospects for rapid containment are poor. The causative virus, influenza A of the H5N1 subtype, is of limited infectivity for humans. If infection occurs, however, then the consequences are serious and even fatal in a majority of cases. In view of the receptor specificity of avian influenza viruses, this may be related to individually increased susceptibility, which does not lead to further spread. However, it is known that influenza A viruses can readily adapt to replication in the human host by the acquisition of specific gene segments or even by mutations of the avian virus. The extreme scale of human contact with influenza virus of the H5N1 subtype at present engenders fear that there is a high risk of such adaptation and a subsequent pandemic spread. Adequate precautions are necessary, not only in terms of an acceleration of vaccine production but primarily in arranging for sufficient availability of the new antiviral drugs.

Mouse-adapted (MA) variants of human and avian influenza A (H2) viruses were generated and characterized with respect to acquisition of virulence in mice. From the nucleotide sequence the amino acid sequence was deduced. The HA1 subunit of the hemagglutinin (HA) contained three amino acid substitutions in the A/black duck/New Jersey/1580/78-MA variant (Glu216--Asp, Lys307--Arg, and Thr318--Ile) and two substitutions in the A/JapanxBellamy/57-MA variant (Lys25--Thr and Ser203--Phe). In the M1 protein, there were two substitutions in the A/black duck/New Jersey/1580/78-MA variant (Asn30--Asp and Gln214--His) and a single substitution in the A/JapanxBellamy/57-MA variant (Met179--Lys). The M2 protein amino acid sequences of the parental virus and the MA variants differed by a single identical mutation (Asn93--Ser). The localization and atomic distances of the observed mutations on the three-dimensional (3D) structure of the HA protein were analyzed for influenza H2 viruses. The obtained results were similar to those published earlier on H1, H3 and H5 subtypes. The amino acid changes in the HA protein could be divided into two groups. In one group the substitutions were situated at the top of the molecule, while in the other group they were clustered in the stem area at the interface region between three HA monomers. The analysis revealed that the substitutions observed in the MA variants probably increase the flexibility of the HA molecule and/or weaken the interactions between monomers or subunits in the HA trimer. The relationships of the observed amino acid changes in the HA and M proteins to the biological properties of the respective viruses and possible mechanisms involved in the acquisition of viral virulence are discussed.

We have used the mouse model to monitor the acquisition of virulence of a non-pathogenic influenza A virus upon adaptation to a new mammalian host. An avian strain, A/Mallard duck/Pennsylvania/10218/84 (H5N2) (Mld/PA/84) was adapted to mice by 23 serial lung-to-lung passages until a highly virulent mouse-adapted (MA) variant (Mld/PA/84-MA) emerged. This MA variant was characterized and compared to the parental strain as well as some of its intermediate passage variants. MA variant caused bronchopneumonia in mice with a high mortality rate (the virulence of Mld/PA/84-MA measured as log (EID50/LD50) was 1.75), while the parental, avirulent strain Mld/PA/84 did not cause illness and mortality in mice (log (EID50/LD50) was 7.25). Hemagglutination-inhibition (HAI) test with a set of hemagglutinin- (HA) specific monoclonal antibodies (MAbs) revealed antigenic differences between the parental strain and MA variant. Mld/PA/84-MA reacted with HA-specific MAbs in higher titers than the parental strain. The HA genes of the parental strain Mld/PA/84, the 1st, 3rd, 8th, and 15th intermediate passage variants, and Mld/PA/84-MA were sequenced. Three amino acid changes at positions 203, 273 and 320 were determined in the HA of MA variant. The first of them, Leu--Pro (320), appeared in the HA stem region at the 8th passage. Two other in the HA1 globular region (Ser--Phe (203) and Glu--Gly (273)) appeared at the 15th passage. All of these substitutions were associated with the increase of viral infectivity for mouse lungs and changes in the HA antigenicity. The potential role of these changes in HA with respect to the process of viral interspecies transmission and acquisition of virulence for new host is discussed.

The membrane-inserted hemagglutinin (HA) is the most variable protein of influenza viruses. Here we describe the characterization of a shared epitope in the HA of influenza A virus H1, H2, and H5 subtypes which were completely neutralized by a monoclonal antibody (MAb), directed against this epitope. This MAb (C179) also efficiently precipitated the HAs of these viruses. In addition, MAb C179 did not neutralize H6 subtype strains despite complete amino acid homology of the epitope regions. Furthermore, only the non-glycosylated form of the HA of one of the H6 subtype strains could be precipitated by the MAb. The conformational epitope may be masked by glycosylation, although it could not be excluded that differences in the primary amino acid sequence may cause the decreased accessibility of the epitope in H6 subtype strains.

The replacement of embryonated chicken eggs by tissue culture cells for the production of influenza vaccines is likely to take place in the near future. Vaccines have already been produced in Madin Darby Canine Kidney (MDCK) cells (Brands et al, in this issue) and extensively tested in phase III trials in humans (Palache et al, in this issue) and it seems a matter of time before such vaccines will become available. For this reason, the generation of high-growth reassortants of influenza A virus strains in MDCK cells has been examined. Influenza A virus reassortants of the field strains A/Taiwan/1/86, A/Johannesburg/82/96 and A/Shenzhen/227/95 (all H1N1) were generated in serum-free cultured MDCK-SF1 cells by dual infection with A/Hong Kong/2/68 (H3N2), a strain selected for its high-growth phenotype. These reassortant viruses all contained at least the matrix gene of A/Hong Kong/2/68 which apparently correlates with an improvement of the viral yield.

The 1997/'98 influenza season in the Netherlands was marked by influenza A/H3N2 activity which never reached a true epidemic level. There was no real peak activity either but a prolonged period of increased activity of approximately eight weeks with a maximum in week 13, when sentinel physicians reported 16.6 cases of influenza-like illness per 10,000 inhabitants. It was not until week 18 of 1998 that the influenza activity declined to baseline levels. During the season, almost exclusively influenza A/H3N2 viruses were isolated, of which the majority resembled the new strain influenza A/Sydney/5/97 (H3N2). Further analysis of these variant viruses revealed that, although there was some cross-reactivity with the vaccine strain (A/Nanchang/933/95), no optimal protection could be expected to be induced by the vaccine. Antigenic characterisation of the sporadic influenza A/H1N1 and influenza B viruses showed that these were related to the vaccine strains. As a result of these findings, the World Health Organization (WHO) recommended to change the H3N2 strain in the influenza vaccine for the season 1998/'99 to an influenza A/Sydney/5/97(H3N2)-like strain. Based on epidemiological data from other countries, it was also decided to change the influenza A/H1N1 component to an influenza A/Beijing/262/95 (H1N1)-like strain.

In 1997, 18 influenza patients were detected who were infected with influenza A(H5N1) virus. Six patients died. Presumably most of the patients had acquired the infection directly from chickens with the fowl plague prevalent in China in 1997. These are the first reported cases of isolation of influenza viruses belonging to one of the H4-H15 subtypes from human influenza patients. Man-to-man transmission of the virus has not been demonstrated but cannot be excluded in every case. Genetic analyses of seven of these virus isolates showed that no reassortment with a human or porcine influenza virus had occurred. It is unpredictable whether the H5N1-virus in question will start a pandemic in the next few years.

From a three-year old boy in Hong Kong who died in May 1997 with an extensive influenza pneumonia an influenza A virus has been isolated which was, first at the National Influenza Centre of the Netherlands, identified as belonging to subtype H5N1. Presumably the patient had acquired the infection directly from an outbreak of fowl plague among chickens. As far as is known this is the first case of the isolation of an influenza virus belonging to one of the subtypes H4-H15 from a human influenza patient. At the end of 1997 seventeen more cases of human A (H5N1) influenza have been detected in Hong Kong, including five fatal cases. Genetic analyses of seven of these virus isolates did not reveal the occurrence of reassortment with a human or porcine influenza virus, which could have rendered the virus potentially pandemic. Man-to-man transmission of the virus has not been demonstrated but cannot be excluded either. This event has shown that the WHO surveillance of influenza viruses, although perhaps not perfect, has functioned well.

The first indication of flu activity in the Netherlands in the 1996/'97 season was the isolation of an A/H3N2 influenza virus in week 48 of 1996. In subsequent weeks influenza viruses were isolated sporadically. The clinical influenza activity increased from week I of 1997 and reached its peak in week 4 of 1997. Simultaneously with the increase of clinical influenza activity, an increasing number of influenza viruses were isolated. The epidemic had a relatively small extent. Initially, A/H3N2 influenza viruses were predominant, but in the second half of the epidemic an increasing number of influenza B viruses were isolated as well. The A/H3N2 viruses were antigenically fairly strongly distinct from the variants prevalent in the preceding years. This season influenza A/HINI viruses did not play a significant role and only one virus of this subtype was isolated. All influenza A/H3N2, A/HINI and B viruses isolated were antigenically similar to the vaccine strains.

The decline of maternal respiratory syncytial virus (RSV) specific serum antibodies was studied in 45 children during the first 6 months of life, using a virus neutralization assay and competition ELISAs measuring fusion protein and glycoprotein specific antibodies. In all children RSV neutralizing antibodies were demonstrated at birth, with titers ranging from 33 to 1382. The calculated mean half life of these antibodies was 26 days. Furthermore, in a group of 38 children with suspected RSV infection, all younger than 6 months of age on admission, the diagnostic value of serological assays was evaluated. In 32 children RSV infection was confirmed by virus isolation, direct immune fluorescence and RT-PCR. In 7 patients of this group a significant titer rise in virus neutralization assay was demonstrated. Six additional RSV infected children could be identified by showing the presence of RSV-specific IgM or IgA serum antibodies or by showing an increase in fusion protein or glycoprotein specific antibodies. All serological tests together identified 13 (41%) of the 32 RSV infected patients. It is concluded that in children of this age group, which represent the majority of patients hospitalized with RSV infections, serological assays not only have a limited diagnostic value but are of limited value for sero-epidemiological studies.

The 1995/'96 season in the Netherlands was marked by an influenza A/H3N2 epidemic that peaked in week 5I. In this week, 39 patients with influenza-like illness per 10,000 inhabitants contacted the sentinel physicians. With two exceptions, influenza A/H3N2 viruses exclusively were isolated during this epidemic period. In the first few months of 1996, a substantial number of influenza A/H1N1 and influenza B viruses were isolated as well. Serological characterization of the circulating viruses revealed that they all resembled the virus strains of the influenza vaccine of 1995/'96, which therefore probably will have provided good protection. Based on the epidemiological data from other countries and the fact that similar H3N2 viruses have been circulating since 1993, the World Health Organization has recommended to exchange the H3N2 component of the 1996/'97 vaccine for a Wuhan/353/95 (H3N2)-like strain.

The 1994/'95 season in the Netherlands was marked by a limited influenza activity which only emerged in late February. The influenza activity remained elevated until the end of April, which is unusually late, and epidemic activity was only reported in the south of the country. Both influenza A/H3N2 and B viruses were isolated in this period. In addition, influenza A/HINI viruses were isolated for the first time since March 1993, from two patients. The majority of the influenza A strains that circulated in the Netherlands in 1994/'95 reacted well with ferret antiserum raised against the strains of the 1994/'95 influenza vaccine, which therefore probably offered good protection. The reactivity of the B strains to antiserum raised against the vaccine strain, B/Panama/45/90, was only moderate, which implies that the protection against the Dutch influenza B strains was not optimal. Based on the results of the worldwide influenza surveillance, the World Health Organization (WHO) has recommended an alteration in both the A/H3N2 and the B component for the vaccine of 1995/1996.

The influenza season 1993/'94 in the Netherlands and the rest of Northwestern Europe was marked by an influenza A/H3N2 epidemic. The morbidity of this epidemic was moderate, but a high mortality rate was observed. The epidemic viruses, represented by A/Netherlands/241/93 (H3N2), were characterised by haemagglutination inhibition assays and nucleotide sequence analysis. The viruses were related to A/Beijing/32/92 (H3N2), the vaccine strain for 1993/'94, but clear antigenic differences were detected. Therefore, the WHO has recommended a new A/H3N2 component, A/Shangdong/9/93, for the vaccine of 1994/'95. The onset of the epidemic was unusually early in the influenza season. An increase in the influenza activity was already noticed in the second week of November and it reached its peak in week 49. As a result of the early epidemic, the influenza vaccination programme had not been completed yet. Therefore, the point of time for vaccinating people at risk may have to be reconsidered and moved up in order to complete the vaccination programme earlier.

The polymerase chain reaction (PCR) is used for human papillomavirus (HPV) detection in paraffin-embedded tissue. The specificity of the reaction is unaffected by the method of fixation used before embedding into paraffin. Five HPV 16, 18, 31, and 33 DNA in situ hybridization (DISH)-negative cervical carcinomas were subjected to the PCR. In two patients, HPV 16 DNA could be detected in the cervical squamous cell carcinomas and also in their lymph node metastases. One patient with an adeno-carcinoma of the cervix was found positive for HPV-18. A lymph node of this patient was HPV 18 positive as well. In the tumors of the remaining two patients, no HPV 16, 18, or 33 DNA was detected by the PCR. Both negative patients had cervical squamous cell carcinomas. One had a bladder metastasis, whereas the other had a lymph node metastasis and an additional distant metastasis in the lung. HPV DNA positivity in cervical carcinomas correlated with HPV prevalence in the metastases. This relationship can be of use for diagnostic purposes in the pathologic analysis of metastases.