Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia.

Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the "Trojan horse" of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1?

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized.

Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine.

In 1979, an H1N1 avian influenza virus crossed the species barrier, establishing a new lineage in European swine. Because there is no direct or serologic evidence of previous H1N1 strains in these pigs, these isolates provide a model for studying early evolution of influenza viruses. The evolutionary rates of both the coding and noncoding changes of the H1N1 swine strains are higher than those of human and classic swine influenza A viruses. In addition, early H1N1 swine isolates show a marked plaque heterogeneity that consistently appears after a few passages. The presence of a mutator mutation was postulated (C. Scholtissek, S. Ludwig, and W. M. Fitch, Arch. Virol. 131:237-250, 1993) to account for these observations and the successful establishment of an avian H1N1 strain in swine. To address this question, we calculated the mutation rates of A/Mallard/New York/6750/78 (H2N2) and A/Swine/Germany/2/81 (H1N1) by using the frequency of amantadine-resistant mutants. To account for the inherent variability of estimated mutation rates, we used a probabilistic model for the statistical analysis. The resulting estimated mutation rates of the two strains were not significantly different. Therefore, an increased mutation rate due to the presence of a mutator mutation is unlikely to have led to the successful introduction of avian H1N1 viruses in European swine.

Long-term stability of the anti-influenza A compounds--amantadine and rimantadine.

Amantadine and rimantadine hydrochloride were tested for stability after storage at different temperatures and under different conditions for extended periods of time. Both compounds were quite stable after storage for at least 25 years at ambient temperature; they both retained full antiviral activity after long-term storage or after boiling and holding at 65-85 degrees C for several days. Thus, amantadine and rimantadine could be synthesized in large quantities and stored for at least one generation without loss of activity in preparation for the next influenza A pandemic in humans.

How to overcome resistance of influenza A viruses against adamantane derivatives.

We tested two approaches to overcoming resistance of influenza A viruses against adamantane derivatives. First, adamantane derivatives that interfere with the ion channel function of the variant M2 protein of amantadine-resistant viruses may prevent drug resistance, if they are used in mixture with amantadine. Second, amantadine acts on the M2 protein (at low concentrations) and indirectly on the hemagglutinin (at concentrations at least 100 times higher). Identifying and using a drug that reacted with both targets at the same concentration might reduce development of resistance, since, in this case, two mutations, one in each target protein would be necessary at once. Such a double mutation is assumed to be a rare event. We evaluated forty adamantane derivatives and two related compounds to determine whether they interfered with plaque formation by influenza A strains, including A/Singapore/1/57 (H2N2). Variants resistant to drugs that interfered at low concentrations (approximately 1 microg/ml; e.g. amantadine) were cross-resistant with each other, but were sensitive to those agents effective at high concentrations (8 microg/ml; e.g. memantine). The former group of compounds act on the ion channel; the corresponding escape mutants tested had amino acid replacements at positions 27, 30 or 31 of the M2 protein. Hemagglutinin was the indirect target of the latter group of compounds. Variants resistant to these agents lacked amino acid replacements within the ion channel of the M2 protein and the mutants tested had amino acid replacements in the hemagglutinin. Although we failed to identify compounds that interacted with the ion channel of amantadine-resistant variants and inhibited their replication, we were able to construct at least two compounds that interfered with both the ion channel and the hemagglutinin at about the same concentration. After passage in the presence of these compounds, we either failed to obtain any drug-resistant mutants or those obtained had amino acid replacements in the ion channel of the M2 protein and the hemagglutinin.

Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs.

H1N1 influenza A viruses isolated from pigs in Europe since 1981 were examined both antigenically and genetically and compared with H1N1 viruses from other sources. H1N1 viruses from pigs and birds could be divided into three groups: avian, classical swine and 'avian-like' swine viruses. Low or no reactivity of 'avian-like' swine viruses in HI tests with monoclonal antibodies raised against classical swine viruses was associated with amino acid substitutions within antigenic sites of the haemagglutinin (HA). Phylogenetic analysis of the HA gene revealed that classical swine viruses from European pigs are most similar to each other and are closely related to North American swine strains, whilst the 'avian-like' swine viruses cluster with avian viruses. 'Avian-like' viruses introduced into pigs in the UK in 1992 apparently originated directly from strains in pigs in continental Europe at that time. The HA genes of the swine viruses examined had undergone limited variation in antigenic sites and also contained fewer potential glycosylation sites compared to human H1N1 viruses. The HA exhibited antigenic drift which was more marked in 'avian-like' swine viruses than in classical swine strains. Genetic analyses of two recent 'avian-like' swine viruses indicated that all the RNA segments are related most closely to those of avian influenza A viruses.

Molecular epidemiology of influenza.

The genome of the influenza A viruses comprises eight single-stranded RNA segments, and this property makes genetic reassortment after double infection of a host with two different influenza A strains possible. Nature takes advantage of genetic reassortment during antigenic shift creating new pandemic strains. After concurrent infection of a host with both avian and human strains, the hemagglutinin gene of the human virus may be replaced by the allelic gene of the avian virus. This reassortment leads to a human virus strain that has avian hemagglutinin molecules on its surface, significant because the human population lacks neutralizing antibodies to this new glycoprotein. The Hong Kong pandemic of 1968 resulted from just such an event.

Previous H1N1 influenza A viruses circulating in the Mongolian population.

Four influenza A viruses of the subtype H1N1, isolated from Mongolian patients in Ulaanbaatar between 1985 and 1991, were analysed by sequencing of various RNA segments. The isolate from 1985 was found to be highly related in all genes sequenced to strains isolated from camels in the same region and at about the same time. These camel isolates were presumably derived from a UV-light inactivated reassortant vaccine (PR8 x USSR/77) prepared in Leningrad in 1978 and used in the Mongolian population at that time [19]. The human isolate from 1988 was also found to be a derivative of a reassortant between PR8 and USSR/77; in contrast to the 1985 isolate, however, it contained an HA closely related to PR8. One of the Mongolian isolates from 1991 (111/91) was in all genes sequenced closely related to PR8, while the other isolate from 1991 (162/91) was closely related to H1N1 strains isolated around 1986 in other parts of the world. About 12% of 235 convalescent sera collected in various parts of Mongolia contained antibodies against PR8, while none of German control sera contained such antibodies. The mutational and evolutionary rates of the Mongolian strains seem to be significantly lower when compared to the rates of human influenza A strains isolated in other parts of the world. This might indicate that these rates depend to a certain extent on the population density. Thus, viruses from remote areas might keep the potential to reappear in the human population after several years to cause a pandemic as it had happened in 1977.

European swine virus as a possible source for the next influenza pandemic?

According to phylogenetic data, about 100 years ago an avian influenza virus passed the species barrier (possibly first) to pigs and (possibly from there) to humans. In 1979 an avian influenza A virus (as a whole, without reassortment) again entered the pig population in northern Europe, forming a stable lineage. Here it is shown that the early North European swine viruses exhibit higher than normal evolutionary rates and are highly variable with respect to plaque morphology and neutralizability by monoclonal antibodies. Our results are consistent with the idea that, in order to pass the species barrier, an influenza A virus needs a mutator mutation to provide an additional number of variants, from which the new host might select the best fitting ones. A mutator mutation could be of advantage under such stress conditions and might enable a virus to pass the species barrier as a whole even twice, as it seems to have happened about 100 years ago. This stressful situation should be over for the recent swine lineage, since the viruses seem to be adapted already to the new host in that the most recent isolates--at least in northern Germany--are genetically stable and seem to have lost the putative mutator mutation again.

Abortive infection of Vero cells by an influenza A virus (FPV).

We have discovered a new type of abortive replication in Vero cells infected with fowl plague virus. In these cells there is an enhanced splicing of the colinear mRNAs of segment 7 and presumably also of segment 8, leading to an extreme overproduction of M2 and NS2 proteins. The cleavage of the hemagglutinin (HA) into HA1 and HA2 and the processing of its carbohydrate side chains are markedly retarded and incomplete. Although some of the HA is incorporated into the plasma membrane, leading to a positive hemadsorption, most of it accumulates in a discrete compartment close to the nuclear membrane, representing presumably the reticuloendothel and/or the Golgi network. Neuraminidase activity in Vero cells is extremely low. The nucleoprotein is normally released from nuclei late in infection. Very little infectious virus is released, and its spread is highly impeded.

Effect of methyltransferase inhibitors on the regulation of baculovirus protein synthesis.

In the presence of the methyltransferase inhibitor 3-deazaadenosine (3DA-Ado) the production of infectious Autographa californica nuclear polyhedrosis virus (AcMNPV) in tissue culture was only slightly affected, while the synthesis of very late proteins (polyhedrin and p10) was abolished. The synthesis of the influenza virus proteins NS1 and HA, expressed under the polyhedrin promoter, was also abolished by 3DA-Ado. Furthermore, 3DA-Ado interfered with the shut-off of early and late AcMNPV proteins. Most of these results were also obtained with 5-azadeoxycytidine (5A-dCyt). In cells in which NS1 was produced abundantly, at least one specific AcMNPV protein was not synthesized. However, if the production of NS1 was inhibited by 3DA-Ado, or if HA was synthesized instead, this AcMNPV protein showed up normally.

Inhibition of the intracellular transport of influenza viral RNA by actinomycin D.

In primary chicken embryo cells infected with fowl plague virus addition of actinomycin D at defined times during the infection cycle has different consequences on viral replication. If actinomycin D is added immediately after infection with a concentration, which inhibits viral RNA synthesis only partially, it interferes with the nucleo-cytoplasmic transport of all viral RNA species (mRNA and vRNA) so far tested. If actinomycin D is present during infection (adsorption, penetration and uncoating) no viral RNA is synthesized, and the nucleocapsid of the infecting virus does not reach the nucleus, as shown by fluorescent antibodies. Therefore the primary effect of actinomycin D on influenza virus replication is on the transport of the incoming vRNPs from the cytoplasm to the cell nucleus, which is the cell compartment where transcription takes place.

Amino acid replacements leading to temperature-sensitive defects of the NS1 protein of influenza A virus.

The nonstructural (NS) genes of two influenza virus temperature-sensitive (ts) reassortants have been sequenced and compared with the corresponding wild type sequences. Ts 412 has a single base substitution (G100-->A) leading to an amino acid replacement (Arg 25-->Lys) in the NS1 protein. Ts 451 also has a single base substitution (U273-->C) leading to an amino acid replacement (Ser 83-->Pro) in the NS1 protein. In ts 412 infected cells at the nonpermissive temperature very little M1 and HA mRNA and proteins are synthesized, suggesting that NS1 is involved in a transcriptional regulation process. The ts mutation in ts 451 could be extragenically suppressed by replacement of the PB1 and/or PA protein genes of the mutant by the allelic genes of PR8. Both observations suggest that NS1 cooperates with the polymerase complex.

Potential hazards associated with influenza virus vaccines.

There is general agreement that human reassortant vaccine strains should be used only for the preparation of the viral glycoproteins (split vaccine), and not in toto after UV-inactivation. Live vaccines with lowered pathogenicity obtained by reassortment between human and avian strains may carry a risk of causing epizootics in other species, even though they are useful for the host for which they were designed. It has been argued that this dangerous situation is avoided when both parent strains are of human origin. For this reason, cold-adapted human master strains are used for reassortment with the most recent isolates from cases of human influenza. There is, however, convincing evidence that a reassortant between two human strains has caused severe epizootics among camels, which were not regarded as natural hosts for influenza A viruses. This sudden appearance of a reassortant camel virus had a precedent in experiments in which, starting from parent strains that are non-pathogenic for mice, highly pathogenic reassortants for this species were created. Safety requirements for cold-adapted reassortants must therefore take account of the fact that these new strains may have a high pathenogenicity for other species.

The methyltransferase inhibitor Neplanocin A interferes with influenza virus replication by a mechanism different from that of 3-deazaadenosine.

Neplanocin A (NeplA) and 3-deazaadenosine (3DA-Ado) are both inhibitors of methyltransferases, and both interfere with influenza virus replication. Their modes of action, however, are different. In chicken embryo cells NeplA inhibits only in media depleted of or low in methionine, while 3DA-Ado acts independently of the concentration of methionine. While homocysteine partially reverses the effect of NeplA, it strongly potentiates the effect of 3DA-Ado. While NeplA inhibits the synthesis of all viral proteins to nearly the same extent, 3DA-Ado interferes only with the production of late proteins (Fischer et al. (1990) Virology 177, 523-531). In NeplA-pretreated cells there is an extreme accumulation of S-adenosylhomocysteine, independent of the concentration of methionine in the medium, although NeplA inhibits influenza virus replication only in methionine-depleted medium. Therefore an accumulation of this intermediate by NeplA cannot account for the inhibitory effect, as has been implicated in the inhibition of the replication of other viruses. Our results indicate that at least two different methyltransferases are involved in influenza virus replication.

Molecular evolution of influenza viruses.

There are two different mechanisms by which influenza viruses might evolve: (1) Because the RNA genome of influenza viruses is segmented, new strains can suddenly be produced by reassortment, as happens, for example, during antigenic shift, creating new pandemic strains. (2) New viruses evolve relatively slowly by stepwise mutation and selection, for example, during antigenic or genetic drift. Influenza A viruses were found in various vertebrate species, where they form reservoirs that do not easily mix. While human influenza A viruses do not spread in birds and vice versa, the species barrier to pigs is relatively low, so that pigs might function as "mixing vessels" for the creation of new pandemic reassortants in Southeast Asia, where the probability is greatest for double infection of pigs by human and avian influenza viruses. Phylogenetic studies revealed that about 100 years ago, an avian influenza A virus had crossed the species barrier, presumably first to pigs, and from there to humans, forming the new stable human and classical swine lineages. In 1979, again, an avian virus showed up in the North European swine population, forming another stable swine lineage. The North European swine isolates from 1979 until about 1985 were genetically extremely unstable. A hypothesis is put forward stating that a mutator mutation is necessary to enable influenza virus to cross the species barrier by providing the new host with sufficient variants from which it can select the best fitting ones. As long as the mutator mutation is still present, such a virus should be able to cross the species barrier a second time, as happened about 100 years ago. Although the most recent swine isolates from northern Germany are again genetically stable, we nevertheless should be on the lookout to see if a North European swine virus shows up in the human population in the near future.

Source for influenza pandemics.

There are three ways how influenza A viruses can escape the immune response in the human population: (1) By antigenic drift. This means by mutation and selection of variants under the selection pressure of the immune system. These variants have amino acid replacements mainly in the epitopes of the hemagglutinin. (2) By antigenic shift. This means replacement of at least the hemagglutinin gene of the prevailing human strain by the allelic gene of an avian influenza virus by reassortment. (3) As a rare event, direct or indirect introduction of an avian influenza virus in toto into the human population. A prior introduction of an avian virus into pigs and an adaptation to the new host might be a presupposition for its final passage to humans. In this sense the nowadays situation is reminiscent to that of about 100 years ago, when an avian virus was presumably first introduced into pigs, and from there into humans. Immediately or some time thereafter the disastrous Spanish Flu in 1918/19 had killed at least 20,000,000 people in one winter. Pandemic strains can be created by all three means, however the most common way is by reassortment. In order to recognize a pandemic strain as soon as possible a worldwide surveillance system and collaborating laboratories equipped with corresponding modern technologies are required.

Recent influenza A (H1N1) infections of pigs and turkeys in northern Europe.

The most recent introduction of an avian influenza A virus without reassortment into mammals occurred in 1979 when H1N1 strains could be isolated from diseased pigs in northern Europe. This newly introduced avian virus formed a stable lineage in pigs and, in the meantime, spread all over Europe. In 1991 highly pathogenic H1N1 strains closely related to a contemporary swine virus were isolated from turkeys of a breeding farm near Bremen, Germany. Outbreaks in several farms in Germany, France, and the Netherlands indicate that the "avian-like" swine viruses can easily be reintroduced into an avian population causing severe economical losses.

Influenza A virus late mRNAs are specifically retained in the nucleus in the presence of a methyltransferase or a protein kinase inhibitor.

The synthesis of influenza A virus RNA and proteins represents a highly regulated process whereby variable amounts of early and late viral RNAs and proteins may be produced. This regulation is upset by the presence of the methyltransferase inhibitor 3-deazaadenosine (3DA-Ado) or the protein kinase inhibitor H7, resulting in complete or partial inhibition of synthesis of late proteins but normal production of early proteins. Although the total yield of viral mRNAs is somewhat reduced by treatment with 3DA-Ado, the mRNAs that are produced can still be translated in vitro. Both 3DA-Ado and H7 interfere specifically with the transport of the late viral mRNAs from the nucleus to the cytoplasm, but do not affect transport of early mRNA. From these results we conclude that during influenza virus replication, posttranscriptional regulation takes place on the level of mRNA transport. Since hemagglutinin mRNA migrates to the cytoplasm in the presence of 3DA-Ado plus cycloheximide, we assume that a viral protein is involved in the regulation mechanism.

A reassortant H1N1 influenza A virus caused fatal epizootics among camels in Mongolia.

In the autumn of 1979 a severe influenza epizootic started among camels in Mongolia (Lvov et al., 1982; Viprosi Virusol. 27, 401-405.) Between 1980 and 1983 13 independent isolates of H1N1 viruses were obtained from diseased camels, which were virtually indistinguishable from the human A/USSR/90/77 strain by serological means. Two hundred and seventy-one samples of camel sera collected between 1978 and 1983 contained antibodies against the human A/USSR/90/77 isolate. After experimental infection of camels with some of these isolates, the animals developed similar symptoms as those found during natural infection: coughing, bronchitis, fever, discharge from nose and eyes. A genetic sequence analysis revealed that among the eight segments (genes) the PB1, HA, and NA genes were almost identical with allelic genes of the USSR/77 strain, and the PB2, PA, NP, M, and NS genes were almost identical with those of the A/PR/8/34 strain.