AU718045B2 - An attenuated vaccination and gene-transfer virus, a method to make the virus and a pharmaceutical composition comprising the virus - Google Patents

Description

Ir a Our Ref: 740404 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT &sow*: 0 46 i*p.

Applicant(s): Prof. Dr. Dr. Gerd Hobom V. V V 9

V.

Arndtstrasse 14 D-35392, Giessen

GERMANY

Address for Service: Invention Title: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 An attenuated vaccination and gene-transfer virus, a method to make the virus and a pharmaceutical composition comprising the virus The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 AN ATTENUATED VACCINATION AND GENE-TRANSFER VIRUS, A METHOD TO MAKE THE VIRUS AND A PHARMACEUTICAL

COMPO-

SITION COMPRISING THE VIRUS The object of the present invention was to make a vaccination virus. This objective has been fulfilled with the segmented virus constructed as described herein.

The genome of influenza A viruses consists of 8 different single-stranded viral 10 RNA (vRNA) molecules of negative polarity, which have in common 5' and 3' terminal sequences largely complementary to each other. These conserved segments 13 and 12 nucleotides in length are known to form double-stranded RNA panhandle structures (Hsu et al., 1987; Fodor et al., 1993) which have been analysed in more detail recently in vitro using internally deleted model RNAs 15 (Baudin et al., 1994; Tiley et al., 1994). In the virion the panhandle ends of all RNA segments are found in specific binding to viral RNA polymerase complexes, while the remaining internal segments stay single-stranded with viral nucleoprotein (NP) in cooperative binding (Compans et al., 1972; Honda et al., 1988; Martin et al., 1992). Upon infection these viral RNPs initially serve as templates for the synthesis of viral mRNAs by a specific cap-snatching mechanism (Plotch et al., 1979; Braam et al., 1983), and later on will direct synthesis of full-length complementary RNAs (cRNAs), probably dependent on the absence or presence of newly synthesized NP protein (Shapiro and Krug, 1988). The plus-strand cRNAs are then used as templates for progeny vRNA synthesis.

The viral RNA polymerase complex consisting of proteins PBI, PB2, and PA is involved in all three different modes of RNA synthesis during the viral replication cycle, following its specific binding to the terminal panhandle segments of both vRNAs and cRNAs. Sequence comparison reveals that the vRNA and cRNA -1termini have similar, but not identical sequences. For that reason vRNA and cRNA recognition may be distinguished because of these structural alterations allowing for asymmetries in initiation of plus and minus strand RNA synthesis, and possibly in viral RNP packaging, which has also been suggested to be controlled by the panhandle RNA sequence (Hsu et al., 1987).

Recently, we reported on an in viv system for the introduction of specific mutations into the genome of influenza viruses: viral cDNA has been inserted in antisense orientation between mouse rDNA promoter and terminator sequences.

This has been derived from in vito transcription experiments based on nuclear extracts from Ehrlich ascites cells, which resulted in transcripts exactly resembling influenza vRNA. For a series of in vivo studies, the viral coding sequence was replaced by the coding sequence for chloramphenicol-acetyltransferase

(CAT),

however, with both influenza terminal non-coding sequences being retained exactly on the resulting vRNA transcripts. After transfection of this recombinant DNA template into mouse cells followed by influenza virus infection, CAT activity was detectable. Transfer of supematants to different cells demonstrated that CAT-vRNAs transcribed in vivo by cellular RNA polymerase I were not only transcribed by viral RNA polymerase into plus-strand mRNA and translated into CAT protein, but also were replicated and packaged into infectious progeny virus 20 particles (Zobel et al., 1993; Neumann et al., 1994).

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25 We have used this system for a stepwise introduction of single and multiple mutations into the conserved panhandle RNA sequence, thereby effectively converting the HA-vRNA promoter sequence into an HA-cRNA promoter sequence and vice versa. For these series of constructs CAT activities have been measured both in primarily transfected and infected B82 cells and, after passaging of B82 supematants, in secondarily infected MDC-K cells. From- the results obtained we propose a model for the terminal RNA sequence as being recognized RNA polymerase in consecutive steps of different structure when used as a template for initiation of viral mRNA synthesis.

-2- The present invention relates to a segmented RNA virus of the Orthomyxoviridae family, said RNA virus comprising at least one segment comprising a vRNA nucleotide sequence comprising a 3' terminal nucleotide sequence and a 5' terminal nucleotide sequence in association therewith, wherein said 3' terminal nucleotide sequence consists of 15 nucleotides corresponding to a wild-type vRNA 3' tennrminal nucleotide sequence modified by replacement of two or three nucleotides naturally occurring in the wild-type 3' terminal nucleotide sequence at positions 3, 5 and 8 by other nucleotides, and said terminal nucleotide sequence consists of 16 nucleotides corresponding to a wild-type vRNA 5' terminal nucleotide sequence optionally modified by replacement of the nucleotides naturally occurring in the wild-type 5' terminal nucleotide sequence at positions 3 and 8 by other nucleotides and further, wherein said RNA virus exhibits as a result of said replacements rates of transcription, replication and/or expression that are higher than those of a wild-type RNA virus of the same species, with the proviso that the 3' terminal nucleotide sequence does not have the sequence CACCCUGUUUUUACU or 5'-CACCCUGUUUCUGCU-3'.

The present invention also relates to a method of enhancing gene transcription and expression in a host cell containing a genetically-engineered segmented RNA virus of the Orthomyxoviridae family, comprising: providing a segmented RNA virus according to claims 1 to 10; and introducing said RNA virus into a host cell, wherein as a result of said .0•replacements said RNA virus exhibits rates of transcription and expression S .that are higher than those of a wild-type RNA virus of the same species.

Throughout this specification and the claims which follow, unless the text requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The virus can be one where one or more modifications have been introduced in the noncoding region(s) and/or one or more modifications have been introduced in the coding region(s). A possibility is that at least one modified segment is derived from an original one by sequence variation(s). It is also possible that at least one modified segment is an artificial addition to the set of original or modified original segments. The virus can be one wherein the modified segment cbmprises a nucleotide sequence which codes for a protein or peptide which is foreign to the original virus. Preferred is that the foreign protein or peptide constitutes an antigen or antigen-like sequence, a T-cell epitope or related sequence. In such a case it is possible that the segment comprises repetitions of an antigen or epitope or other.

Such an antigen or epitope can be for example derived from HIV, Herpes-Virus Human Papilloma virus, Rhinovirus, CMV or Hog Cholera Virus (HCV). The virus of the present invention may be a single stranded negative-strand RNA virus as for example one of the Orthomyxoviridae family, the Bunyaviridae family or of the Arenaviridae family. The most preferred virus is an influenza virus. The virus of the present invention can also be a double-stranded RNA virus, as for example a reovirus, a rotavirus or an orbivirus. The viruses of the present invention can be used in gen-therapy.

The present invention also relates to the virus and use of the virus for the 20 preparation of pharmaceuticals.

Mutational analysis of vRNA 3' terminal sequence positions.

Influenza A viral RNA 5' and 3' ends have similar, but not identical sequences with nucleotide mismatches at positions 3, 5 and 8, and an additional unpaired nucleotide is located at position 10 in the 5' region. Nevertheless, both vRNA 5 termini hybridize into a double stranded panhandle structure made up of twelve and thirteen nucleotides in common for all eight RNA segments, plus in average three additional basepairs specific for each of the vRNA molecules. Due to the deviations mentioned the cRNA or plus strand panhandle structures have to be different from the vRNA structures; however, both are recognized by viral RNA polymerase and are used for initiation of RNA synthesis, i.e. they are constituting a promoter structure. Even if in initial recognition and binding of RNA polymerase the double stranded RNA panhandle structure is known to be the substrate, and is also observed in virion RNPs (Hsu et al., 1987), for the initiation step of transcription at the 3' ultimate template position this terminal region has to -3be separated into a partially single stranded, i.e. 'forked' structure (Fodor et al., 1994). RNA polymerase may be predicted to continue its binding interaction with both, the remaining double stranded segment: nucleotides 10 to 15 versus 11 to 16', and to the.single stranded 3' template segment: nucleotides I up to as well as the 5' single stranded end (Tiley et al., 1994). Introduction of mutations at specific positions in either strand may hence alterate simultaneously both of these consecutive vRNA promoter structures: panhandle and fork in different ways, and will in addition also result in corresponding variations of the cRNA promoter structure.

To investigate the importance of the three mismatch positions, specific single, double or triple nucleotide exchanges were first introduced into the vRNA 3' end sequence at positions 3 5 and 8 thereby approaching a fully double-stranded vRNA promoter structure, in a step-wise manner. At the same time the vRNA 3' end template sequence will become equivalent to the cRNA 3' end in these 15 positions, but not in regard to the additional nucleotide at position 10. Single nucleotide exchanges according to this scheme (pHL1098, pHL1099, pHL1100) abolished the promoter activity, and no CAT activity was observed, as has been reported before with a different method (Luo et al., 1993). Two of the double mutation constructs (pHL1101, pHLI103) also gave negative results.

20 In contrast, for pHLll02 (G3A,C8U) l a significant CAT activity was detected, distinctly higher than for the corresponding wild-type construct (pHL926;) which in the conditions applied (8 hr after infection) resulted in rather low levels of CAT expression.

This activity increase is further enhanced for the final construct of this series carrying the triple exchange G3, U5C and C8U (pHL 1104), i.e. transfection of pHL1004 DNA S 25 followed by influenza virus infection results in a very high level of CAT expression, also considerably above the pHLl 102 results.

These results have been repeated using various conditions of transfection and infection as well as determining kinetic data during the course of infection. While the pHL1104 variant is always observed far superior over any wild-type construct 'Notations concerning nucleotidesT to 15 refer to positions in the vRNA 3' end, e.g. position 2 designates the penultimate nucleotide; 5' end positions are given in ordinary numbers. The notation G3A describes a mutational change of guanosine to adenosine at position 3.

-4that expression ratio may be variable and difficult to quantitate (between around fold and nearly 100 fold). Rather short infective cycles of eight hours as used prevalently appear to put more slowly replicating, i.e. wild-type molecules at a disadvantage, in particular in passaging of packaged pseudo-vRNA molecules via virus progeny, both is found increased for wildtype and related constructs after DNA transfection plus twelve hours of infection (see Neumann et il., 1994).

Remaining deviations in CAT expression ratios may be attributed to variations in growth conditions in individual experiments.

Mutational analysis of vRNA 5' terminal sequence positions We also addressed the question whether the unexpectedly high viral mRNA expression rate of pHL1104 is the consequence of a stabilized panhandle doublestrand structure or may be directly attributed to the point mutations introduced into the vRNA 3' sequence, and active when being used as a single-stranded template segment, e.g. in the 'forked' structure.

S 15 For this purpose we constructed pHL1124, three complementary point mutations introduced at the 5' end of the vRNA sequence again in positions 3, 5 and 8 (U3C, G5A, A8G). Together with a sequence wild-type vRNA 3' end these variations again result in a panhandle structure free of mismatches and, therefore, pHL 1124 is equivalent in this regard to pHL 104, but different in the sequence of 20 its template and non-template single strands. No significant CAT expression was detected for pHL1124. We conclude that the increased CAT activity of pHL1104 is not a consequence of the stabilized panhandle structure itself, but at least in part is a consequence of the individual nucleotide exchanges at positions T, 5 and 8 at the 3' end of the vRNA sequence, it is also more likely then to originate from 25 other structural intermediates of initiation than a stabilized panhandle.

a.

Mutational analyses of concerted exchanges at both ends of the vRNA sequence In order to determine in detail the influence of single, double and triple exchanges at the vRNA 5' end upon CAT expression rates we also used the improved vRNA 3' end sequences of pHL1104 and pHLll02 as starting points rather than the corresponding wild-type sequence. From the series of experiments related to pHL1104 and from the equivalent series related to pHL 1102 it can be concluded that retaining a G residue in position 5 is the most important single feature in these 5' end variations. A single exchange into an A residue at position 5 as in pHL1185 will render the promoter entirely inactive, while single exchanges in positions 3 or 8, as well as a 3 plus 8 double exchange will retain promoter activity even if reduced from the level observed for pHL1104, but still above wild-type expression rates. While the G5A nucleotide substitution opposite nucleotide C5 in the 3' terminus results in losing one basepair (in the panhandle context) within the pHL1104 series, a basepair is indeed gained by exactly the same G5A exchange within the pHL 102 series, i.e. opposite the U5 residue as present in the pHL1102 vRNA 3' end. Since again the G5A exchange results in loss of promoter function inspite of gaining one basepair we conclude that the guanosine at position 5 may be important for RNA polymerase binding within the non-template single strand rather than being part of the panhandle doublestranded structure in this region. The importance of a G residue at this position has been shown earlier in a single-step mutational analysis (Li and Palese, 1992), 15 while non-template strand binding of RNA polymerase has been studied recently in vilro (Tiley et al., 1994). Different from the deleterious effect of an exchange at position 5 exchanges at positions 3 and in particular 8 are of minor importance.

The series of 5' nucleotide exchanges has also been repeated for the pHL1102 version of the vRNA 3' end yielding exactly the same pattern of results, albeit at the somewhat reduced levels characteristic for pHL1102. The only result in both series not quite in agreement with a uniquely important role for a G-residue in position 5 is the triple exchange of pHL1126 which retains low promoter activity inspite of an A residue in that position. Due to altogether six concerted exchanges in positions 3, 5 and 8 as well as 3 ,5 and 8 from the 5' and 3' end of the 25 vRNA sequence the pHL1126 vRNA panhandle structure is indeed nearly equivalent to a wild-type cRNA panhandle, with the exception of an unpaired adenosine being present in position 10 of pHL1126 while an unpaired uridine at position F0 is part of the wild-type cRNA structure. This correlation may indicate a correct structure in pHL1126 for several other residues of (minor) importance which, therefore, apparently allows to compensate for the missing G residue in position 5, even if at a clearly reduced level of activity. In the parallel pHL1102 series the corresponding triple exchange clone pHL1125 does not show any promoter activity; however, because of its deviation at position 5 it does not completely resemble the cRNA panhandle structure.

-6- Mutational analysis of the panhandle bulge structure around nucleotide An extra, unpaired residue in position 10 at the 5' end is a specific feature of the influenza viral RNA panhandle structure. It is causing or at least enforcing a major bulge of the structure, together with unpaired residues at position 9, and might be part of a specific recognition element of that structure by viral RNA polymerase.

In order to investigate the importance of that particular structural feature, a further series of plasmid constructs has been initiated, again based on pHL1104 and its 3' terminal sequence as a reference. A perfectly matched RNA double-strand without any bulge has been achieved either by inserting an additional U residue in the 3' end sequence opposite A10 (pHL1140) or by deleting the A10 residue from the sequence (pHL1152). Finally, a bulge of opposite direction was created in the panhandle structure of pHLl164 with an extra U residue in position 10 of the 3' end, and position 10 deleted from the 5' end sequence. While the latter two constructs proved inactive in the CAT assay, pHL1140 did show some promoter S 15 activity, albeit at a reduced level. We conclude from this result that a bulge in this region may not be recognized directly by viral RNA polymerase but may serve as a flexible joint between two more rigid structural elements that are involved in immediate contact with viral polymerase. The necessary RNA bending may also, but less efficiently be achieved in an A-U-basepaired structure like pHL1140, while the other two structures would not permit such type of interaction with RNA polymerase. This interpretation has also been substantiated in a further series of variations in this region Serial passaging of influenza virus carrying promoter mutants All previous experiments consisted of a first measurement of viral mRNA synthesis in DNA-transfected and infected B82 cells, followed by a second measurement of viral mRNA synthesis in infected MDCK cells, after passaging of progeny virus containing supernatants. CAT expression in infected cells upon viral passaging requires packaging of pseudo-viral vRNAs, in addition to new rounds of viral mRNA synthesis in those cells leading to CAT expression again. All viral promoter mutants analysed and found active in transfected and helper-infected B82 cells also resulted in CAT expression after transfer, and consistently in equivalent ratios of activity. Packaging, therefore, cannot be correlated with any specific element in the vRNA promoter structure so far, and does not appear to be a limiting factor in constructing influenza virus mutants in this system. While CAT expression after passaging in general appeared to be increased over the levels -7before passaging this might have been simply the result of different cells being used for the first and second step of CAT analysis, with MDCK being superior to B82 cells in influenza mRNA synthesis and also in progeny yields. Therefore, several experiments of serial passage have been performed using pHL1104 derived influenza supernatants and others, in MDCK cells. In these serial passages, always done using aliquots of supernatants harvested eight hours after infection for further transfer, a stepwise increase of CAT expression is observed (Fig. Apparently the superior performance of viral RNA promoters carrying sequence deviations according to pHL1104 is not only true for viral mRNA synthesis, but also for viral RNA replication.

Therefore, mutant viral RNAs of this character become accumulated and effectively selected in further passaging, while packaging may be a neutral event in this regard, at least for the variants analysed here.

Serial passaging extended o* 15 During further passaging of supernatants the CAT containing influenza segment carrying the mutationally altered viral promotor sequences became accumulated in a stepwise manner in the population of progeny viruses. In order to demonstrate this effect on the level of individual viruses being transferred we isolated in three indepentent experiments 50 to 85 plaques each after a third round of passage on MDCK cells. Each cell lysate obtained for the individual plaques was assayed for CAT activity according to the standard protocol. While in two of the experiments the fraction of CAT positive plaques was in the range of 4 to 8% (1 out of 50, 4 out of 40 plaques) in one of these series this fraction amounted to 47% (19 of plaques). Both of these results demonstrate a substantial increase over the initial 25 fraction of CAT-segment containing virus, which may be calculated to be in the range of 10- 5 or at most 10- 4 and slight variations in the conditions of growth during three steps of transfer may precipitate to result in the observed differences of CAT positive plaques. While every CAT positive plaque demonstrates the amplification of nine (not eight) viral RNA segments present in the initially infected cell, this may have resulted from a single virus carrying nine or more RNA segments or from coinfection by two defective viruses able to complement each other.

-8- Necessarily, accumulation of a pseudo-viral segment not contributiong to viral growth will, in further steps, become lethal to viral growth, even if a majority of virions may contain an average of eleven rather than eight RNA segments (Hsu et.

al. 1987). Packaging of viral RNA-segments based on a general packaging signal identical for all eight segments and realized via a specific interaction chain: vRNA panhandle structure viral RNA polymerase viral NP protein viral MI protein will reflect the pools of the various vRNA segments in infected cells, and therefore may be biased towards an RNA segment superior in replication and overrepresented in that pool. Biased replication and packaging will, however, lead to accumulation of lethal viral particles due to an imbalance between the eight (or nine) viral RNA segments. This prediction is borne out in continuing the viral passage of pHL1104 derived influenza supernatants beyond step three as exemplified in Fig. 2. While CAT expression based on transcription of the pHL1104 derived pseudoviral RNA segments is increased further up to the fifth 15 passage the number of viable viruses reaches a maximum already after the second step of viral passaging, thus demonstrating the continuous accumulation of an over-replicated foreign segment, based on a superior panhandle sequence.

At a stage representing the third or fourth passage as displayed in Fig. 2 a virus preparation obtained in this way can be regarded as the equivalent of an attenuated 20 viral strain. While the concentration of attenuated virus particles that can be achieved in this way may appear to be limited a stage equivalent to passage 4 in Fig. 2 may be delayed upon coinfection with wildtype helper virus during first or second steps of transfer, and considerably increased concentrations of attenuated virus preparations might be achievable in this way.

pHL1104-mediated high-rate expression of foreign proteins can also be used (after two or more steps of amplification via serial passaging on MDCK cells) for high rate synthesis of foreign proteins in embryonated chicken eggs, following a general method of preparation of viral stocks as used for influenza and other viruses, i. e. injection of virus suspensions into the yolk sac. Protein preparations isolated from those embryonated and infected cells will be glykosylated and modified in other ways according to their origination from eukaryotic cells.

A second method of influenza virus attenuation has been achieved via cleavage of either one of the influenza viral RNAs, preferrably the M or NP gene (segments 7 -9or via ribozyme hydrolysis in a specialised mode of action. The ribozyme RNAs which may be covalently inserted into the pSV2neo early mRNA, located between the neomycin resistance gene and the small t intron sequence originating from SV40 viral DNA, or expressed from similar expression cassettes, are directed against the 5' end sequence of segment 7 (or another of the influenza vRNA segments).

During initiation of mRNA synthesis the 5' terminal sequence which is involved in formation of the panhandle structure is at first covered by viral RNA polymerase in association with that double stranded promoter region. It will, however, become single-stranded and free of protein, since the polymerase molecule will start transcription at the 3' end and move along the 3' template sequence while synthezising a perfectly hybridizing 5' RNA daughter strand, superior in that regard to the parental panhandle 5' segment. Ribozyme RNAs which may be inhibited in their activity either by RNA substrates involved in double strand I: 15 formation or if RNA substrates are covered by protein, have been directed with a .9 3' complementary sequence towards that protein-free 5' sequence of the substrate vRNA molecule for initiation of hybridization, which then will be extended across the entire complementary region of approximately 100 nucleotides i. e. well into the vRNA sequence initially covered by NP protein.

20 A second feature of the ribozyme RNAs as applied for inactivation of influenza vRNA molecules is their double-unit hammerhead character, directed against not one, but two close GUY cleavage sites, e. g. GUU 1 6 and GUU 36 in segment 7 or

GUC

30 and GUC 48 in segment 5, which are also known to be invariable in sequence comparisons of influenza isolates.

25 Both features of anti-influenza ribozymes as pointed out contribute to a reduction of typically two logs (up to three logs) in production of viral progeny in template ribozyme DNA transfected cells as compared to infection of mock transfected cells, both at moi 1 and 20 h after Lipofectamin-DNA treatment. Ribozyme treatment can be applied after two or three rounds in MDCK cells of pHL1104promoted amplification of a pseudo-viral RNA segment originating from RNA- Polymerase I transcription, in the presence of helper virus as used in initial superinfection.

In a simple version ribozyme treatment as described above is employed as a selection technique. Here, its application is appropriate if the pseudoviral RNA is indeed a (foreign) influenza segment carrying particular mutations but capable in principle to act as a functional substitute for the helper viral segment destroyed by ribozyme cleavage. For that purpose the substitute viral segment to be selected in that procedure has to be mutagenized in advance at the two cleavage sites indicated above in order to become resistant against ribozyme interference. In another application ribozyme cleavage of helpervirus vRNA can be used for attenuation of recombinant influenza virus preparations. Here, the pseudo-viral RNA segment may be designed in a way which renders it incapable to substitute for a helpervirus gene. Therefore, viral passaging into ribozyme template DNA transfected cells would lead to an abortive infection only, because of ribozyme mediated destruction of an important viral gene, if its gene product would not be added for complementation via expression from a cDNA construct which is also 1.15 DNA-transfected into the cell together with ribozyme-expressing DNA 20 h before viral infection. In this way viral progeny is obtained that is attenuated because of ribozyme cleavage of one of the vRNA segments, and effectively that segment is missing in the virions because it can no longer be packaged. Viral preparations obtained in this way are capable of only one round of infection because of their inherent Ml M2 protein complementation, and therefore are suited for vaccination purposes. Animal infection with progeny virus as isolated after the ribozyme attenuation step results in abortive infection, but viral proteins synthesized in infected cells are able to induce B-cell and T-cell responses in such animals.

In influenza viral RNA synthesis parental negative-strand vRNA is copied into Splus-strand cRNA, which again is copied into progeny vRNA, from the first to the last nucleotide. This amplification of viral RNAs, however, proceeds in an inherently asymmetric way, since vRNA molecules are synthesized in excess over cRNA molecules. This result is consistent with the idea that cRNA carries a promoter structure more active in binding viral RNA polymerase and in initiation of RNA synthesis, i.e. 'stronger' than does vRNA. While at first simply the two 3' ends of single-stranded vRNA and cRNA templates have been implicated as promoter sequences, the detection of double-stranded panhandle structures involving both ends of the vRNA sequence in virions (Hsu et al., 1987) suggested more complicated substrates for RNA polymerase binding and initation of 11 daughter-strand synthesis. A slightly different panhandle structure has also been observed with model vRNA molecules in the absence of viral proteins in vitro (Baudin et al., 1994), possibly calling for a structural change upon viral RNA polymerase binding, i.e. a bulge may be shifted from position 4 to position 10 in that reaction (see Fig. While originally several of the RNA polymerase vRNA binding experiments in vitro appeared to show recognition only of 3' end oligonucleotides, this has since been shown to be an artifact after pure, recombinant viral polymerase free of residual RNA became available, instead of enzyme preparations from virions. Under these conditions RNA polymerase binding to viral RNA as well as endonucleolytic cleavage of cellular mRNAs by subunit PB2 was observed to depend on vRNA 5' plus 3' terminal sequence binding, with even higher affinity for the 5' non-template segment (Hagen et al., 1994; Tiley et al., 1994).

Different from the employment of both vRNA and cRNA promoter structures in 15 replication physiologically only vRNA promoters will also serve in initiation of viral mRNA synthesis according to the cap-snatching mechanism (Plotch et al., 1979; Braam et al., 1983). While it has been claimed that cRNA promoters would not have the capacity to act according to this scheme (Tiley et al., 1994), the failure to observe viral antisense mRNA molecules may simply reflect the 20 inavailability of cRNA molecules early in infection, i.e. in the absence of surplus viral NP protein, and small amounts of such molecules might even have gone undetected. In this invention we describe a mutagenizational analysis of the vRNA promoter structure in vivo which in approaching the structure of the cRNA promoter via three nucleotide exchanges shows considerably improved activity in viral mRNA synthesis over vRNA promoter wild-type levels. Continuing increase of viral CAT mRNA expression during consecutive steps of. viral passaging suggests that the same vRNA promoter mutants also show increased activity in cRNA' synthesis, both in accordance with the idea that the cRNA promoter structure might be 'stronger' than the vRNA promoter, also in initiation of viral mRNA synthesis.

Additional variations of the 5' terminal sequence clearly indicate the major importance of a G residue in position 5, irrespective of complementarity or not to position 5 at the 3' end. The unique role of this G residue has been observed before in a serial mutagenizational analysis (Li and Palese, 1992). According to 12both data guanosine residue 5 may be involved in single-strand binding of RNA polymerase as has indeed been observed for the non-template strand terminal segment (Tiley et al., 1994). While panhandle double-strand structures are likely to constitute the initial RNA polymerase binding substrate a partial separation of template and non-template strands is expected to take place consecutively resulting in a 'forked structure' such as proposed by Fodor et al. (1994). Specific'and tight binding of RNA polymerase in this structure may predominantly be oriented towards sequence elements in the non-template strand, since the growing point of RNA synthesis will have to move along the entire template strand following its initiation. It is, therefore, possible that such a binding interaction survives most or all of an individual round of mRNA synthesis as has been proposed (Tiley et al., 1994).

The triple nucleotide exchanges as introduced in vRNA molecules derived from S: pHL1104 templates will create three additional basepairs able to stabilize the '.15 panhandle structure in general, but more specifically they will favor a bulged adenosine 10 over the bulged adenosine 4 conformation as observed for the wildtype sequence in vitro (Baudin et al., 1994). Since the changes introduced here lead to a considerable enhancement of promoter activity we propose that a bulged S 10 conformation may be the structure underlying the vRNA polymerase binding :20 reaction, which otherwise would have to be achieved only as a result of that interaction. A bulged 10 adenosine residue may constitute a kind of flexible joint or angular kink which in turn suggests two major, structurally stable binding sites to the left and right of this element. One of these sites has to be the doublestranded sequence element of (in average) six basepairs extending from positions 11 to 16 and 1M to 15 respectively. While the distal three basepairs are known to be variable for the various RNA segments, basepair 13/12 has been shown to be exchangeable experimentally, and also the number of basepairs has been reduced to four without complete loss of function (Luo et al., 1991). With all of these data it seems clear that the main recognition element in this region is an RNA doublestrand of certain stability, while it remains possible that residue 12 guanosine and potentially others are also recognized individually within that structure. A major second binding element for RNA polymerase on the other side relative to position is less evident, but may be located in a distance of nearly one helical turn in the de-bulged region around position 4, since direct contacts are suggested by that initial conformational interaction, and also by the specific requirement of a 13 guanosine residue in position 5, which is likely to interact not only during, but also before partial strand separation in that region, i.e. in the panhandle as well as the forked structure. While an extra adenosine residue in position 10 may be optimal for creating a correctly shaped bulge in this region of RNA, structural variants are possible in this regard (see pHL 1140) which excludes direct interactions between RNA polymerase and residues constituting that bulge.

In summary we are proposing a model (see Fig. 1) of consecutive steps of interaction between a vRNA or cRNA promoter structure and viral RNA polymerase: bulged 4 panhandle bulged 10 panhandle polymerase forked RNA polymerase (bound to 5G and ds element 11-16) initiation of RNA synthesis (recognition of 3' end of template).

Attenuation of influenza viruses for preparation of a live nasal vaccine relies on two mechanisms: 1) preferential amplification of a recombinant viral segment carrying the pHL1104 promoter mutation, which will increase its rate in packaged viral RNP particles and indirectly decrease that of the eight helper virus RNP particles. This competition results in an increase of defective viruses from which sone or more of the regular viral gene segments are missing. 2) Sequence specific ribozyme cleavage of one or more helper virus RNA segments, if compensated through gene product expression for. functional complementation. This dual interaction will result in virus progeny, which is capable of only one round of infection, abortive because of the missing viral protein(s) that are required for their propagation. Ribozyme cleavage of one out of two sister viral gene segments, sensitive and (artificially) insensitive for its hydrolysis may also be used 25 (repeatedly) for selection purposes, including selection for viral gene constructs expressed via RNA polymerase I transcription.

MATERIALS AND METHODS Plasmid constructions Plasmids with mutated vRNA and/or mutated cRNA promoter sequences are derivatives of pHL926 (Zobel et al., 1993; Neumann et al., 1994). In pHL926 a hybrid CAT cDNA with flanking non-coding sequences derived from influenza vRNA segments has been precisely inserted in antisense orientation between 14mouse rDNA promoter and terminator sequences. The CAT reporter gene in this way has been introduced by exactly replacing the coding sequence for hemagglutinin, retaining the untranslated viral 5' and 3' sequences of segment 4.

vRNA 5' end mutations were created by PCR, using a general primer hybridizing to a position in the flanking rDNA promoter sequence, and a specific primer carrying the desired nucleotide substitution to be introduced in the viral terminal sequence. The polymerase chain reaction products were first digested by the restriction enzymes BgII and SpeI, inserted into the left boundary position by exchanging the segment between these appropriate restricton sites in pHL926, and finally confirmed in their constitution by DNA Sanger sequencing.

Generation of vRNA 3' end mutations followed the same general scheme at the right boundary. PCR products were obtained by using a general primer S complementary to a CAT gene internal sequence position, and a specific primer with appropriate nucleotide exchanges inserted into its sequence. Following digestion with restriction enzymes NcoI and Scal, the PCR products were cloned into NcoI- and ScaI(partially)-digested plasmid pHL926. Any PCR derived sequences were investigated by DNA sequencing.

For constructs with both 5' end and 3' end mutations in combination, 5' variation containing fragments were obtained by BglII and SpeI restriction and inserted into ''20 the appropriate 3' terminal variation plasmids.

Cells and viruses Influenza A/FPV/Bratislava viruses were grown in NIH3T3 cells. For transfection and passaging experiments B82 cells (a mouse L cell line) and MDCK cells were used.

Lipofectamin DNA transfection and influenza virus helper infection For DNA transfection 10 7 B82 cells were used. 5 gg of plasmid DNA were mixed with 60 Ltg of Lipofectamin (LipofectaminM, GIBCO/BRL) in serum-free medium and incubated at room temperature for 10-15 min. This mixture was added to the cells washed twice with serum-free medium, and the incubation with Lipofectamin/DNA was continued for 1 hr. After further incubation with DMEM medium for 1 hr the transfected B82 cells were infected with influenza 15 A/FPV/Bratislava at a multiplicity of infection of 0.01 to 1 for another 30-60 min.

Further incubation was performed with DMEM medium.

Passaging of virus containing supernatants Under standard conditions 8 hr after influenza infection (at moi 0.1 to 1) cells were harvested for CAT assays, and supernatants were collected and spun down at 1200 rpm for 5 min for removal of cell debris.

Aliquots of virus containing cleared supematants were used for plaque tests, and another aliquot was adsorbed to 107 MDCK cells for 30-60 min for further passaging. Again 8 hr after infection the CPE was verified, and cells and supernatants were collected and treated as before.

CAT assay Cell extracts were prepared as desribed by Gorman et al. (1982). CAT assays were done with 4 C]chloramphenicol or fluorescent-labeled chloramphenicol S* (borondipyrromethane difluoride fluorophore; FLASH CAT Kit, Stratagene) as 15 substrates.

For 4 C]chloramphenicol the assay mixture contained: 0.1 pCi 4 C]chloramphenicol, 20 pl 4 mM Acetyl-CoA, 25 1 1 M Tris-HCI (pH 7.5) and pl of cell lysate in a total volume of 150 pl. The assay mixture for the fluorescent-labeled substrate contained (in a final volume of 80 pl): 10 pl 0.25 M Tris-HCI (pH 10 pl 4 M Acetyl-CoA, 10 p1 fluorescent-labeled chloramphenicol, and 50 il of cell lysate. After an incubation time of 16 hr the S. :reaction products were separated by chromatography and either autoradiographed or visualized by UV illumination and photography.

REFERENCES

Baudin,F., Bach,C., Cusack,S. and Ruigrok,R.W.H. (1994) The EMBO 13, 3158-3165.

Braam,J., Uhmanen,l. and Krug,R. (1983) Cell, 34, 609-618.

Compans,R.W., Content,J. and Duesberg,P.H. (1972) J. Virol., 10, 795-800.

Fodor,E., Seong,B.L. and Brownlee,G.G. (1993) J. Gen. Virol., 74, 1327-1333.

Fodor,E., Pritlove,D.C. and Brownlee,G.G. (1994) J. Virol., 68, 4092-4096.

Gorman,M., Moffat,L. and Howard,B. (1982) Mol. Cell Biol., 2, 1044-1057.

16- Hagen~m., Chung,T.D.Y. Butcherj.A. and Krystal,M. (1994) Virol., 68, 1509- 1515.

Honda,A., UedaK., NagataK. and Ishihama,A. (1987) J1 Biochem., 102, 1241- 1249.

HsuM., ParvinJ.D., GuptaS., Krystal,M. and Palese,P. (1987) Proc. Nail. Acad Sci. USA, 84, 8140-8144.

Li,X and PaleseP. (1992) J Virol., 66, 4331-4338.

Luo,G., Luytjes,W., EnamiM. and Palese,P. (1991) J1 ViroL, 65, 2861-2867.

Martinj., AlboC., Ortinj., Meleroj.A. and PortelaA. (1992) J Gen. ViroL, 73, 1855-1859.

Neumann,G. ZobelA. and Hobom,G. (1994) Virology, 202, 477-479.

Plotch,S., BouloyM. and Krug,R-M. (1979) Proc Nail. Acad. Sci. USA, 76, 1618- 1622.

Seong,B.L. and Brownlee,G.G. (1992) J. Virol., 73, 3115-3124.

j ShapiroG. and Krug,R. (1988) J1 ViroL., 62, 2285-2290.

Tiley,L.S., Hagen,M., Matthews,J.T. and Krystal,M. (1994) J1 Virol., 68, 5 108- 5116.

YamanakaK. OgasawaraN., Yoshikawa., IshihamaA. and Nagata,K. (1991) Proc. Nail. Acad Sc. USA, 88: 5369-5373.

ZobelA., Neumann,G. and Hobom,G. (1993) Nucl. Acids Res., 21, 3607-3614.

LEGENDS TO FIGURES 99*9 Fig. 1. Proposed scheme of consecutive conformational steps occurring prior to initiation of viral nRINA synthesis in influenza vRNA, in wildtype and pHL1 104 derived mutant sequences. Positions of triple mutation in pHL1 104 vRNA are indicated in bold and larger size letters.

Free RNA panhandle structure, bulged at position 4 (wildtype vRNA; Baudin et al, 1994) or at position 10 (mutant vR.NA). Bulged 10 panhandle structures after binding of viral RNA polymerase; proposed protein binding positions marked by underlignments. Forked structures of partial strand separation. Initiation of viral mRNA synthesis via hybridization of capped primer oligonucleotide.

Fig. 2. Serial passaging of pHL 1104-derived progeny viruses.

17 B82 cells were transfected with 5u of pHL 1104 DNA (in 60 pg Lipofectamin) and infected two hours later with influenza A/FPV/Bratislava 8 hr post-infection the cells were assayed for plaque forming units (dark column) and for CAT activity (hatched column) After sedimentation an other aliquot of the supernatant was adsorbed to 10 7 MDCK cells. Further rounds of passaging were done equivalently by harvesting the cells 8 hr after infection for assaying CAT activities, whereas an aliquot of the supernatant was always adsorbed to fresh MDCK cells. Numbers of serial passages are indicated at the bottom. Plaque forming units per ml refer to the left ordinate, CAT expression rates (relative to primary imfection levels) to the ordinate on the right.

S

C S

S

o*• **ooa 18 SEQUENCE

LISTING

GENERAL

INFORMATION:

Ci) APPLICANT: NAME: Bayer AG STREET: Bayerwerk CITY: Leverkusen COUNTRY: Deutschland POSTAL CODE (ZIP) 51368 TELEPHONE: (0)214-3061455 TELEFAX: C0)214-303482 (ii) TITLE OF INVENTION. Vaccination virus, method of making itan pharmaceutical composition comprising that virusan (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC cmail OPERATING SYSTEM: PC-DOS/Ms.DOS D) SOFTWARE: Patentln Release Version #1.30B

CEPO)

INFORMATION FOR SEQ ID NO: 1: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 5241 base pairs TYPE: nucleic acid CC) STRANDEDNESS. double CD) TOPOLOGY: circular "goo&Cii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: No ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

CA) ORGANISM: Influenza virus, RNA sequence INDIVIDUAL ISOLATE: pHL926.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGGCGGCAG TAGCGCGGTG GTCCCACCTG ACCCCATGCC GAACTCAGAA GTGAAACGCC GTAGCGCCGA TGGTAGTGTG GGGTCTCCCC ATGCGAGAGT AGGGAACTGC CAGGCATCAA 120 ATAAAACGAA AGGCTCAGTC GAAAGACTGG GCCTTTCGTT TTATCTGTTG TTTGTCGGTG 180 AACGCTCTCC TGAGTAGGAC AAATCCGCCG GGAGCGGATT TGAACGTTGC GAAGCAACGG 240 19 CCCGGAGGGT GGCGGGCAGG ACGCCCGCCA TAAACTGCCA GGCATCAAAT TAAGCAGAAG GCCATCCTGA CGGATGGCCT TTTTGCGTTT CTACAAACTC TTTTGTTTAT ACATTCAAAT ATGTATCCGC AAAAAGGAAG AGTATGAGTA ATTTTGCCTT CCTGTTTTTG TCAGTTGGGT GCACGAGTGG GAGTTTTCGC CCCGAAGAAC CGCGGTATTA TCCCGTGTTG TCAGAATGAC

TTGGTTGAGT

AGTAAGAGAA

TTATGCAGTG

TCTGACAACG ATCGGAGGAC TGTAACTCGC CTTGATCGTT TGACACCACG ATGCCTGCAG ACTTACTCTA GCTTCCCGGC 15 ACCACTTCTG

CGCTCGGCC

TGAGCGTGGG TCTCGCGGTA CGTAGTTATc TACACGACGG TGAGATAGGT GCCTCACTGA ACTTTAGATT GATTTAAAAC 20 TGATAATCTC

ATGACCAAAA

CGTAGAAAAG ATCAAAGGAT GCAAACAAAA AAACCACCGC TCTTTTTCCG AAGGTAACTG GTAGCCGTAG TTAGGCCACC GCTAATCCTG TTACCAGTGG CTCAAGACGA TAGTTACCGC rCATGAGACA

TTCAACATTT

CTCACCCAGA

GTTACATCGA

GTTTTCCAAT

ACGCCGGGCA

ACTCACCAGT

CTGCCATAAC

CGAAGGAGCT

GGGAACCGGA

CAATGGCAAC

AACAATTAAT

TTCCGGCTGG

TCATTGCAGC

GGAGTCAGGC

TTAAGCATTG

TTCATTTTTA

TCCCTTAACG

CTTCTTGAGP

TACCAGCGGI

GCTTCAGCAC

ACTTCAAGA)

CTGCTGCCA(

ATAACCCTGA

CCGTGTCGCC

AACGCTGGTG

ACTGGATCTC

GATGAGCACT

AGAGCAACTC

CACAGAAAAG

CATGAGTGAT

AACCGCTTTT

GCTGAATGAA

AACGTTGCGC

AGACTGGATG

CTG.GTTTA TT

ACTGGGGCCA

AMCTATGGAT

GTAACTGTCA

*ATTTAAAAGCG

TGAGTTTTCC

LTCCTTTTTTI

*GGTTTGTTTC

AGCGCAGATI

k CTCTGTAGCI

TAAATGCTTC

CTTATTCCCT I AAAGTAAAAG1

AACAGCGGTA

TTTAAAGTTC

GGTCGCCGCA

CATCTTACGG

AACACTGCGG

TTGCACAACA

GCCATACCAA

AAACTATTAA

GAGGCGGATA

GCTGATAAAT

GATGGTAAGC

GAACGATA

GACCAAGTTT

ATCTAGGTGA

*TTCCACTGAG

*CTGCGCGTAA

CCGGATCAAG

k. CCAAATACTrG k. CCGCCTACAT rTTTCTAT

LATAATATTG

TTTTGCGGC

i'TGCTGAAGA kGATCCTTGA

P'GCTATGTGG

rACACTATTC

A.TGGCATGAC

CCAACTTACT

rGGGGGATCA

ACGACGAGCG

CTGGCGAACT

AAGTTGCAGG

CTGGAGCCGG

CCTCCCGTAT

GACAGATCG C

ACTCATATAT

AGATCCTTTT

CGTCAGACCC

TCTGCTGCTT

AGCTACCAAC

TCCTTCTAGT

ACCTCGCTCT

CCGGGTTGGA

360 420 480 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 TGGCGATAAG TCGTGTCTTA ATAAGGCGCA GCGGTCGGGc TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCATTG 20 AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA

GCGGCAGGGT

CGGAACAGGA

TGTCGGGTTT

GAGCCTATGG

TGGCGGACGC

AGAATTGATT

CATTCAGGTC

GTATAGGGCG

AATCGCCGTG

TCCTTGAAGC

GGGCATCCCG

CGTCGCGAAC

TTACATTAAT

TGCATTAATG

1:5 GTTTTTCTTT

GAGAGTTGCA

GTGGTTGACG

ATATCCGCAC

TGATCGTTGG

TGTTGAAAAC

TTGCGAGTGA

GGGCCCCCGG

GATCCAAJAGC

CTTACACATC

TTACGTTGAC

AGAGAGTCA

TGCCGGTGTC

GAGCGCACGA

CGCCACCTCT

AAA.AACGCCA

GATGGATATG

GGCTCCAATT

GAGGTGGccc

GCGCCTACAA

ACGATCAGCG

TGTCCCTGAT

ATGCCGCCGG

GCCAGCAAGA

TGCGT-TGCGC

AATCGGCCAA

TCACCAGTGA

GCAAGCGGTC

GCGGGATATA

CAACGCGCAG

CAACCAGCAT

CGGACATGGC

GATATTTATG

TGACAGGGAC

TCCAGGGCGA

CCAGCCCTGA

ACCATCGAAT

TTCAGGGTGG

TCTTATCAGA

GGGAGCTTCC

GACTTGAGCG

GCAACGCGGC

TTCTGCCAAG

CTTGGAGTGG

GGCTCCATGC

TCCATGCCAA

GTCCAGTGAT

GGTCGTCATc

AAGCGAGAAG

CGTAGCCCAG

TCACTGCCCG

CGCGCGGGGA

GACGGGCAAc

CACGCTGGTT

ACATGAGCTG

CCCGGACTCG

CGCAGTGGGA

ACTCCAGTCG

CCAGCCAGCC

AGAGAGGGCT

GCTCGAATTC

AAAAGGG CAT

GGTGCAAAAC

TGAATGTGAA

CCGTTTCcCG

AGGGGGAAAC

TCGATTTTTG

CCGAGATGCG

GGTTGGTTTG

TGAATCCGTT

ACCGCGACGC

CCCGTTCCAT

CGAAGTTAGG

TACCTGCCTG

AATCATAATG

CGCGTCGGCC

CTTTCCAGTC

GAGGCGGTTT

AGCTGATTGC

TGCCCCAGCA

TCTTCGGTAT

GTAATGGCGC

ACGATGCCCT

CCTTCCCGTT

AGACGCAGAC

TCTGGAGGAA

CCCGGTAAAG

CAAAATAAAC

CTTTCGCGGT

ACCAGTAACG

CGTGGTGAAC

GCCTGGTATC

TGATGCTCGT

CCGCGTGCGG

CGCATTCACA

AGCGAGGTGC

AACGCGGGGA

GTGCTCGCCG

CTGGTAAGAG

GACAGCATGG

GGGAAGGCCA

AGCTTGCAAT

GGGAAACCTG

GCGTATTGGG

CCTTCACCGC

GGCGAAAATC

CGTCGTATCC

GCATTGCGCC

CATTCAGCAT

CCGCTATCGG

GCGCCGAGAC

AAAAGAAAAA

CCGCTTAAGA

CACACCTATG

ATGGCATGAT

TTATACGATG

CAGGCCAGCC

TTTATAGTCC

CAGGGGGGCG

CTGCTGGAGA

GTTCTCCGCA

CGCCGGCTTC

GGCAGACAAG

AGGCGGCATA

CCGCGAGCGA

CCTGCAACGC

TCCAGCCTCG

TCGCGCTAAC

TCGTGCCAGC

CGCCAGGGTG

CTGGCCCTGA

CTGTTTGATG

CACTACCGAG

CAGCGCCATC

TTGCATOGTT

CTGAATTTGA

AGAACTTAAT

AAAAAAAAAA

CATTCCCGCT

GTGTATGCAT

AGCGCCCGGA

TCGCAGAGTA

ACGTTTCTGC

1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 21

GAAAACGCGG

GGCACAACAA

CCTGCACGCG

CAGCGTGGTG

CAATCTTCTC

CGCTGGATGA

TTCTTGATGT

CGCGACTGGG

GCCCTGTACG

GGTCTTATCA

TTCGTTATGG

GCCTGTCAC-T

GACCTGGAGA

CCTGCCACTC

15

CACAGACGGC

GAA.AAAGTGG

CTGGCGGGCA

CCGTCGCAIA

GTGTCGATGG

GCGCAACGCG

CCAGGATGC

CTCTGACCAG

CGTGGAG CAT

TCTGAGGCCG

GTTCTCCGGG

GGTCATTTTT

TTCCTCCCTG

TAGGTAGTAG

ATCGCAGTAC

ATGATGAACC

a a a. a a

PLAGCGGCGAT

!ACAGTCGTT

rTGTCGCGGC rAGAACGAAG rTCAGTGGGC 1TTGCTGTGG

ACACCCATCA

CTGGTCGCAT

AGGGAAAGCT

TTGTCAGGTC

GGGCCACCTC

TCTCTTTTAT

AAACAAGGGT

TGTTGTAATT

TGAATCGCCA

ACGGGGGCGA

CAGGGATTGG

TTTTCACCGT

TGGTATTCAC

GGGTGAACAC

TGAGCATTCA

TTCTTTACGG

TGAGCAACTG

GTGGTATATC

CCCCCAACTT

GATGCTGGAG

GGCGGAGCTG

GCTGATTGGC

GATTAAATCT

CGGCGTCGAA~

TAGATCTAC

AAGCTGCCTG

ACAGTATTAT

TGGGTCACCA

ATGGGCGCGG

GACCAGTTGT

CCCAGGTATG

GCTTGTGATC

GTTTTTAAAT

CATTAhGCAT

GCGGCATCAG

AGAAGTTGTC

CTGAGACGAA

AACACGCCAC

TCCAGAGCGA

TATCCCATAT

TCAGGCGGGC

TCTTTAAAAA

ACTGAAATGC

CAGTGATTTT

CGGAGGTCGA

GTCGACCAGA

AATTACATTC

GTTGCCACCT

CGCGCCGATC

GCCTGTAAAG

GCGGTAGATC

CACTAATGTT

TTTCTCCCAT

GCAA.ATCGCG

TTTTCTTTCA

TCCTTTGAGG

ACTTCCAGGT

TTTTAGATCT

ACTAGTACAT

TCTGCCGACA

CACCTTGTCG

CATATTGGCC

AAACATATTC

ATCTTGCGAA

TGAAAACGTT

CACCAGCTCA

AAGAATGTGA

GGCCGTAATA

CTCAAAATGT

TTTCTCCATG

CCAGTACTCC

CCAACCGCGT

CCAGTCTGGC

AACTGGGTGC

CGGCGGTGCA

ATiTAACTATC

CCGGCGTTAT

GAAGACGGTA

CTGTTAGCGG

TTGACCTGTC

TCCGGTTCTT

ATTCTCTGTG

GGTCCTATTG

TACGCCCCGC

TGGAAGCCAT

CCTTGCGTAT

ACGTTTAAAT

TCAATAAACC

TATA4TGTGTA

TCAGTTTGCT

CCGTCTTTCA

ATAAAGGCCG

TCCAGCTGAA

TCTTTACGAT

ATTAATAGAA

GGGCGAAACT

3540 36Co~ 3660 3720 378o 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 AATATTTGCC

CATGGTGA

CAAAACTGGT

CTTTAGGGA3A

GAAACTGCCG

20 CATGGAAAAC

TTGCCATACG

GATAAAACTT

CGGTCTGGTT

GCCATTGGGA

TTATCCCCTG

TTGTTTTTTT

GAAACTCACC

ATAGGCCAGG

GAAATCGTCG

GGTGTAACAA

GAATTCCGGA

GTGCTTATTT

ATAGGTACAT

TATATCAACG

TTTCTACTCC

TTTTTCCCCC

CCCCCCCCCC 'CCCGGCGCGG AACGGCGGGG CCACTCTGGA CTCTTTTTTT

TTTTTTTTTT

22 .I15 TTTTTTTTTG GGGATCCTCT AGAGTCGACC TGCAGCCCAAJ GCTAGCGGCC

GCTACGCTTCT

GTTTTGGCGG ATGAGAGAAG ATTTTCAGCC TGATACAGAT TAAATCAGA

CGCAGAAGCG

GTCTGATAAA ACAGAATTTG

C

INFORMATION FOR SEQ ID NO: 2: SEQUENCE

CHARACTERISTICS:

LENGTH: 5241 base pairs TYPE: nucleic acid STRANDEDNESS. double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Influenza virus, RNA sequence INDIVIDUAL ISOLATE: pHL11O4 5160 5220 5241 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTGGCGGCAG T .AGCGCGGTG GTCCCACCTG ACCCCATGCC GAACTCAGAA

GTGAAACGCC

C.

C

GTAGCGCCGA

ATAAAACGAA

AACGCTCTCC

CCCGGAGGGT

GCCATCCTGA

ACATTCAAAT

AAAAAGGAAG

ATTTTGCCTT

TCAGTTGGGT

GAGTTTTCGC

CGCGGTATTA

TCAGAATGAC

TGGTAGTGTG

AGGCTCAGTC

TGAGTAGGAC

GGCGGGCAGG

CGGATGGCCT

ATGTATCCGC

AGTATGAGTA

CCTGTTTTTG

GCACGAGTGG

CCCGAAGAAC

TCCCGTGTTG

TTGGTTGAGT

GGGTCTCCCC

GAAAGACTGG

AAATCCGCCG

ACGCCCGCCA

TTTTGCGTTT

TCATGAGACA

TTCAACATTT

CTCACCCAGA

GTTACATCGA

GTTTTCCAAT

ACGCCGGGCA

ACTCACCAGT

ATGCGAGAGT

GCCTT1ZCGTT

GGAGCGGATT

TAAACTGCCA

CTACAAACTC

ATAACCCTGA

CCGTGTCGCC

AACGCTGGTG

ACTGGATCTC

GATGAGCACT

AGAGCAACTC

CACAGAIAG

AGGGAACTGC

TTATCTGTTG

TGAACGTTGC

GGCATCAAAT

TTTTGTTTAT

TAAATGCTTC

CTTATTCCCT

AAAGTAAAAG

AACAGCGGTA

TTTAAAGTTC

GGTCGCCGCA

CATCTTACGG

CAGGCATCAA

TTTGTCGGTG

GAAGCAACGG

TAAGCAGAAG

TTTTCTAAAT

AATAATATTG

TTTTTGCGGC

ATGCTGAAGA

AGATCCTTGA

TGCTATGTGG

TACACTATTC

ATGGCATGAC

120 180 240 300 360 420 480 540 600 660 720 780 23

S

S. S AGTAAGAGA TTATGCAGTG

C

TCTGACAACG ATCGGAGGAC

C

TGTAACTCGC CTTGATCGTT

G

TGACACCACG ATGCCTTCAG

C

ACTTACTCTA

GCTTCCCGGC

ACCACTTCTG CGCTCGGCCC

I

TGAGCGTGGG TCTCGCGGTA

I

CGTAGTTATC

TACACGACGG

TGAGATAGGT

GCCTCACTGA

ACTTTAGATT

GATTTAAAAC

TGATAATCTC ATGACCAAAJA CGTAGAAAAG

ATCAAAGGAT

GCAAACAAAA

AAACCACCGC

TCTTTTTCCG

AAGGTAACTG

15 GTAGCCGTAG

TTAGGCCAC

GCTAATCCTG

TTACCAGTGG

CTCAAGACGA

TAGTTACCGG

ACAGCCCAGC

TTGGAGCGAA

AGAAAGCGCC

ACGCTTCCCG

CGGAACAGGA

GAGCGCACGA

TGTCGGGTTT

CGCCACCTCT

GAGCCTATGG

AAAAACGCCA

TGGCGGACGC

GATGGATATG

AGAATTGATT

GGCTCCAATT

CATTCAGGTC

GAGGTGGCCC

GTATAGGGCG

GCGCCTACAA

AATCGCCGTG ACGATCAGCG TGCCATAAC C

GAAGGAGCT

~GGAACCGGA

G

AATGGCAAC

LACAATTAAT A ~TCCGGCTGG C

~CATTGCAGC

;GAGTCAGGC

L'TAAGCATTG C rTCATTTTTA rCCCTTAACG

CTTCTTGAGA

rACCAGCGGT

GCTTCAGCAG

ACTTCAAGAA

CTGCTGCCAG

ATAAGGCGCA

CGACCTACAC

AAGGGAGAAA

GGGAGCTTCC

GACTTGAGCG

GCAACGCGGC

TTCTGCCAAG

CTTGGAGTGG

GGCTCCATGC

TCCATGCCAA

GTCCAGTGAT

ATGAGTGAT A .ACCGCTTTT T1 CTGAATGAA G

ACGTTGCGC

~GACTGGATG C TGGTTTATT C

LCTGGGGCCA

LACTATGGAT

;TAACTGTCA

kTTTAAAAGG rGAGTTTTCG rCCTTTTTTT

;GTTTGTTTG

AGCGCAGATA

CTCTGTAGCA

TGGCGATAAG

GCGGTCGGGC

CGAACTGAGA

GGCGGACAGG

AGGGGGAAAC

TCGATTTTTG

CCGAGATGCG

GGTTGGTTTG

TGAATCCGTT

ACCGCGACGC

CCCGTTCCAT

CGAAGTTAGG

~ACACTGCGG C TGCACAACA I ICCATACCAA I hAACTATTAA

;AGGCGGATA

CTGATAAAT

*ATGGTAAGC

;AACGAAATA

"ACCAAGTTT

ATCTAGGTGA

rTCCACTGAG

CTGCGCGTAA

CCGGATCAAG

CCAAATACTG

CCGCCTACAT

TCGTGTCTTA

TGAACGGGGG

TACCTACAG C

TATCCGGTAA

GCCTGGTATC

TGATGCTCGT

CCGCGTGCGG

CGCATTCACA

AGCGAGGTGC

AACGCGGGGA

GTGCTCGCCG

CTGGTAAGAG

CAACTTACT

~GGGGGATCA

CGACGAGCG

TGGCGAACT

LkGTTGCAGG

:TGGAGCCGG

:CTCCCGTAT

;ACAGATCGC

ACTCATATAT

A.GATCCTTTT

CGTCAGACCC

rCTGCTGCTT

AGCTACCAAC

TCCTTCTAGT

ACCTCGCTCT

CCGGGTTGGA

GTTCGTGCAC

GTGAGCATTG

GCGGCAGGGT

TTTATAGTcC

CAGGGGGGCG

CTGCTGGAGA

GTTCTCCGCA

CGCCGGCTTC

GGCAGACAAG

AGGCGGCATA

CCGCGAGCGA

840 900) 96() 102() 1080) 1140.

120o 1260 1320, 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 .5 S S

S

SS

24

TCCTTGAAGC

GGGCATCCCG

TGTCCCTGAT

ATGCCGCCGG

GGTCGTCATC

AAGCGAGAAG

CGTCGCGAAC GCCAGCAAGA CGTAGCCCAG

TTACATTAAT

TGCATTAATG

GTTTTTCTTT

a

GAGAGTTGCA

GTGGTTGACG

ATATCCGCAC

TGATCGTTGG

TGTTGAAAAC

TTGCGAGTGA

GGGCCCCCGG

GATCCAAAGC

CTTACACATC

TTACGTTGAC

AGAGAGTCA"

TGCCGGTGTC

GAAAACG CG(

GGCACAACAI

CCTGCACGC(

TGCGTTGCGC

AATCGGCCAA

TCACCAGTGA

GCAAGCGGTC

GCGGGATATA

CAACGCGCAG

CAACCAGCAT

CGGACATGGC

GATATTTATG

TGACAGGGAC

TCCAGGGCGA

CCAGCCCTGA

*ACCATCGAAT

TTCAGGGTGC-

*TCTTATCAGII

GAAAAAGTGC

k CTGGCGGGCJ

;CCGTCGCAAJ

CACTGCCCG

GCGCGGGGA

ACGGGCAAC

:ACGCTGGTT

kCATGAGCTG

CCCGGACTCG

:GCAGTGGGA

A.CTCCAGTCG

CCAGCCAGCC

PIGAGAGGGCT

GCTCGAATTC

AAAAGGGCA I

GGTGCAAAAC

TGAATGTGA)

CCGTTTCCC(

AAGCGGCGA'.

AACAGTCGT',

TTGTCGCGGi

TAGAACGAAI

TTCAGTGGG

ATTGCTGTG

ACACCCATC

CTGGTCGCA

AGGGAAAGC

TACCTGCCTG

AATCATAATG

CGCGTCGGCC

CTTTCCAGTC

GAGGCGGTTT

AGCTGATTGC

TGCCCCAGCA

TCTTCGGTAT

GTA.ATGGCGC

ACGATGCCCT

CCTTCCCGTT

AGACGCAGAC

TCTGGAGGAA

CCCGGTAAAG

CAAAATAAAC

CTTTCGCGGT

ACCAGTAACG

;CGTGGTGAAC

r~ GGCGGAGCTG r GCTGATTGGC

-GATTAAATCI

G CGGCGTCGAI C TAGATCTAC( G AAGCTGCCT( A ACAGTATTA' .T TGGGTCACC T ATGGGCGCG,

GACAGCATGGC

GGGAAGGCCA I AGCTTGCAAT I GGGAAACCTG I

GCGTATTGGG

CCTTCACCGC

GGCGAAAATC

CGTCGTATCC

GCATTGCGCC

CATTCAGCAT

CCGCTATCGG

GCGCCGAGAC

AAAAGAAAAA

CCGCTTAAGA

CACACCTATG

ATGGCATGAT

TTATACGATG

CAGGCCAGCC

AATTACATTC

GTTGCCACCT

CGCGCCGATC

GCCTGTAAAG

GCGGTAGATC

CTGCAACGC

~CCAGCCTCG

CGCGCTAAC

'CGTGCCAGC

:GCCAGGcfrG

:TGGCCCTGA

CTGTTTGATG

CACTACCGAG

CAGCGCCATC

TTGCATGGTT

CTGAATTTGA

AGAACTTAAT

CATTCCCGCT

GTGTATG CAT

AGCGCCCGGA

TCGCAGAGTA

ACGTTTCTGC

CCAACCGCGT

CCAGTCTGGC

AACTGGGTGC

CGGCGGTGCA

ATTAACTATC

2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 34S0 3540 3600 3660 3720 3780 3S40 3900 3960 4020 a.

a q a. a,.

a.

CAGCGTGGTG GTGTCGATGG

CAATCTTCTC

CGCTGGATGA

TTCTTGATGT

CGCGACTGGG

GCCCTGTACG

GCGCAACGCG

CCAGGATGCC

CTCTGACCAG

CGTGGAGCAT

TCTGAGGCCG

CACTAATGTT CCGGCGTTAT TTTCTCCCAT GAAGACGGTA

GCAAATCGCG

TTTTCTTTCA

CTGTTAGCGG

TTGACCTGTC

25 be..

be C. C eq..

p 9. b.c.

C

ebb.

C

be..

be..

be..

b.c.

GGTCTTATCA

TTCGTTATGG

GCCTGTCACT

GACCTGGAGA

CCTGCCACTC

CACAGACGGC

AATATTTGCC

CAAAACTGGT

CTTTAGGGA

10 GAAACTGCCG

CATGGAAAAC

TTGCCATACG

GATAAAACTT

CGGTCTGGTT

15

GCCATTGGGA

TTATCCCCTG

TTGTTTTTTT

CCCCCCCCCC

TTTTTTTTTG

20 GTTTTGGCGG

GTCTGATAAA

GTTCTCCGGG

GGTCATTTTT

TTCCTCCCTG

TAGGTAGTAG

ATcGCAGTAC

ATGATGAACC

CATGGTGAAA

GAAACTCACC

ATAGGCCAGG

GAAATCGTCG

GGTGTAACAA

GAATTCCGGA

GTGCTTATTT

ATAGGTACAT

TATATCAACG

TTTCTACTCC

TTTTTCCCCC

CCCGGCGCGG

GGGATCCTCT

ATGAGAGAAG

TTGTCAGGTC

GGGCCACCTC

TCTCTTTTAT

AAACAAGGGT

TGTTGTAATT

TGAATCGCCA

ACGGGGGCGA

CAGGGATTGG

TTTTCACCGT

TGGTATTCAC

GGGTGAACAC

TGAGCATTCA

TTCTTTACGG

TGAGCAACTG

GTGGTATATC

CCCCCAACTT

GATGCTGGAG

AACGGCGGGG

AGAGTCGACC

ATTTTCAGCC

GACCAGTTGT

CCCAGGTATG

GCTTGTGATC

GTTTTTAAAT

CATTAAGCAT

GCGGCATCAG

AGAAGTTGTC

CTGAGACGAA

AACACGCCAC

TCCAGAGCGA

TATCCCATAT

TCAGGCGGGC

TCTTTAAAAA

ACTGAAATGC

CAGTGATTTT

CGGAGGTCGA

GTCGACCAGA

CCACTCTGGA

TGCAGCCCAA

TGATACAGAT

TCCTTTGAGG

ACTTCCAGGT

TTTTAGATCT

ACTAGTACAT

TCTGCCGACA

CACCTTGTCG

CATATTGGCC

AAACATATTC

ATCTTGCGAA

TGAAAACGTT

CACCAGCTCA

AAGAATGTGA

GGCCGTAATA

CTCAAAATGT

TTTCTCCATG

CCAGTACTCC

TGTCCGAAAG

CTCTTTTTTT

GCTAGCGGCC

TAAATCAGAA

TCCGGTTCTT

ATTCTCTGTG

GGTCCTATTG

TACGCCCCGC

TGGAAGCCAT

CCTTGCGTAT

ACGTTTAAAT

TCAATAAACC

TATATGTGTA

TCAGTTTGCT

CCGTCTTTCA

ATAAAGGCCG

TCCAGCTGAA

TCTTTACGAT

ATTAATAGAA

GGGCGAAACT

TGTCCCCCCC

TTTTTTTTTT

GCTAGCTTCT

CGCAGAAGCG

4080 4140 4200 426o 4320 4380 4440 4S00 4S60 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 S220 5241 a. be b C

C

Sb Cb ACAGAATTTG C INFORMATION FOR SEQ ID NO: 3: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: RNA (genoruic) (i)HYPOTHETICAL:

NO

26 (iv) ANTI-.SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Influenza virus, 31 RNA sequence INDIVIDUAL ISOLATE: Wild Type vRNA Promotor Element (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCC UGCUUUUGCU 13 INFORMATION FOR SEQ ID NO: 4: Ci) SEQUENCE CHARA =,EAISTICS.

LENGTH: 13' base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genoniic) (iii) HYPOTHETICAL:

NO

1 xo:Civ) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Influenza virus, vRNA 3' sequence INDIVIDUAL ISOLATE: pHI 104 vRNA Promoter Element (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ~~CCC UGUUUCUACU 13 0000.0 INFORMATION FOR SEQ ID NO: .to SEQUENCE

CHARACTERISTICS:

LENGTH: 14 base pairs AL~YiE~iiucJ~icac-id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-.SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Influenza virus, VRNA INDIVIDUAL ISOLATE: vRNA Promoter Element 27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGUAGAAACA AGG INFORMATION FOR SEQ ID NO: 6: Mi SEQUENCE CHARACTERISTICS: LENGTH: 6802 base pairs TYPE; nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Influenza virus, RNA sequence INDIVIDUAL ISOLATE: pHL1191 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTAGACTCTT CAAGCAAAAG CAGGTAGATC TTGAAAGATG AGTCTTCTAA CCGAGGTCGA ec..

q 0@ p.

SO 0 0 4I~* a a S *0 AACGTACGTT

CTCTCTATCA

a. op a

U

*0 0 0~

TGAAGATGTC

AAGACCAATC

20 GCCCAGTGAG

GGATCCAAAT

ATTCCATGGG

GGGCCTCATA

TGCAACCTGT

AACCAACCCA

TATGGAGCAA

GGCTAGGCAA

TCTGAAAAAT

TTTGCAGGGA

CTGTCACCTC

CGAGGACTGC

AACATGGACA

GCCAAAGAAA

TACAACAGGA

GAACAGATTG

CTAATCAGAC

ATGGCTGGAT

ATGGTGCAAG

TCCCGTCAGG

AGAACACCGA

TGACTAAGGG

AGCGTAGACG

AAGCAGTTAA

TCTCACTCAG

TGGGGGCTGT

CTGACTCCCA

ATGAGAACAG

CGAGTGAGCA

CGATGAGAAC

GATTTTAGGA

CTTTGTCCAA

ACTGTATAGG

TTATTCTGCT

GACCACTGAA

GCATCGGTCT

AATGGTTTTA

AGCAGCAGAG

CATTGGGACT

GGCCTATCAG

TTTGTGTTCA

AATGCCCTTA

AAGCTCAAGA

GGTGCACTTG

GTGGCATTTG

CATAGGCARA

GCCAGCACTA

GCCATGGAGG

CATCCTAGCT

CCCCCTCAAA GCCGAGATCG TCTTGAGGTT CTCATGGAAT

CACAGAGACT

GGCTAAAGAC

CGCTCACCGT

ATGGGAACGG

GGGAGATAAC

CCAGTTGTAT

GCCTGGTATG

TGGTGACXAC

CAGCTAAGGC

TTGCTAGTCA

CCAGTGCTGG

.120 180 240 300 360 420 480 540 600 660 720 780 840 GCTCTTCTTG

AAAATTTGCA

AAACGAATGG GGGTGCAGAT GCAACGGTTC AAGTGATCCT CTCGCTATTG CCGCAAATAT CATTGGGATC TTGCACTTGA 28 TATTGTGGAT TCTTGATCGT CTTTTTTTCA AATGCATTTA CCGTCGCTTT AAATACGGAC TGAAAGGAGG G AACAGCAGAG T AAACTACCAT3 GGGATGCGGA3

ATAGAAAATG

GAGGGCACAG

GGGAAACTGA

TTCTCAGAAG

13. ATAGTGTCAC

GCCATCTGTT

TGTCCTTTCC

TCTGGGGGGT

TGCTGGGGAT

GGGGTATCCC

CAGCGTGACC

CTTTCTCGCC

GTTCCGATTT

ACGTAGTGGG

I CTTTAATAGT

CTTTTGATTT

ACAAAAATTT

CCAGGCTCCc

GTGTGGAAAG

GTCAGCAACC

CGCCCATTCT

CTCTGCCTCT

;CCTTCTACG

~GCTGTGGAT

LTGGGGCATG

kTGTACCAGA

TTGGGAGGG

;ACAAGCAGC

kTAGGGTAAT rAGAAGGGAG

:TAAATGCTA

GTTTGCCCCT

rAATAAAATG

GGGGTGGGGC

GCGGTGGGCT

CACGCGCCCT

GCTACACTTG

ACGTTCGCCG

AGTGCTTTAC

CCATCGCCCI

GGACTCTTGI

ATAAGGGATI

AACGCGAATI

CAGGCAGGC)

TCCCCAGGC'

ATAGTCCCGI

CCGCCCCATi

GAGCTATTC

GAAGGAGTGC

GCTGACGATG

TCCCAAGTAT

GAAACAAACT

AATGATAGAC

AGATCTTAAA

CGAGAAGACG

AATTCAGGAC

GAGCTCGCTG

CCCCCGTGCC

AGGAAATTGC

AGGACAGCAA

CTATGGCTTC

GTAGCGGCGC

CCAGCGCCCT

GCTTTCCCCG

GGCACCTCGA

GATAGACGG7

TCCAAACTGG

TGGGGATTTC

AATTCTGTGC

k GAAGTATGCJ r' CCCCAGCAG(

:CCCTAACTCI

3 GCTGACTAIV C AGAAGTAGTi

CAAAGTCTAT

GTCATTTTGT

GTTAAGCAAA

AGAGGCATAT

GGTTGGTACG

AGCACTCAAG

AACGAGAAAT

CTCGAGCATG

ATCAGCCTCG

TTCCTTGACC

ATCGCATTGT

GGGGGAGGAT

TGAGGCGGAA

ATTAAGCGCG

AGCGCCCGCT

TCAAGCTCTA

CCC CAAAAAA TTTTCGCCC7

AACAACACTC

GGCCTATTGC

AATGTGTGTc k~ AAGCATGCAI

CAGAAGTAT(

GAGGGAAGAA

CAGCATAGAG

ACACTCTGAA

TCGGCGCAAT

GTTTCAGGCA

CAGCCATCGA

TCCATCAAAT

CATCTAGAGG

ACTGTGCCTT

CTGGAAGGTG

CTGAGTAGGT

TGGGAAGACA

AGAACCAGCT

GCGGGTGTGG

CCTTTCGCTT

AATCGGGGCA

*CTTGATTAGG

TTGACGTTGG

*AACCCTATCI

*TTAAAAAATG-

AGTTAGGGTC

rCTCAATTAGI

CAAAGCATG(

ATCGAAAGG

:TGGAGTAAA

3TTGGCAACA kGCAGGTTTC rCAAAATTCC

CCAAATCAAT

CGAAAAGGAA

GCCCTATTCT

CTAGTTGCCA

CCACTCCCAC

GTCATTCTAT

ATAGCAGGCA

GGGGCTCTAG

TGGTTACGCG

TCTTCCCTTC

TCCCTTTAGG

GTGATGGTTC

AGTCCACGTT

CGGTCTTATT

AGCTGATTTA

TGGAAAGTCC

CAGCAACCAG

ATCTCAATTA

CGCCCAGTTC

CCGAGGCCGC

TAGGCTTTTG

960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 GCCCATCCCG CCCCTAACTC

TTTTTTTATT

AGGAGGCTTT

TATGCAGAGG

TTTGGAGGCC

29 CAAAAAGCTC CCGGGAGCTT GTATATCCAT TTTCGGATCT GATCAAGAGA CAGGATGAGG *ae.

9

ATCGTTTCGC

GAGGCTATTC G CCGGCTGTCA G GAATGAACTG

C

CGCAGCTGTG C GCCGGGGCAG C

TGATGCAATG

GAAACATCGC2 10 TCTGGACGAA CATGCCCGAC4 GGTGGAAAAT4

CTATCAGGAC

TGACCGCTTC

15 TCGCCTTCTT

ACGCCCAACC

TTCGGAATCG

GAGTTCTTCG

AGCATCACAA

20 AAACTCATCA

TAATCATGGT

ATACGiGCCG

TTAATTGCGT

TAATGAATCG

TCGCTCACTG

AAGGCGGTAA

AAAGGCCAGC

,TGATTGAAC

GCTATGACT

CGCAGGGGC

AGGACGAGG

:TCGACGTTG

;ATCTCCTGT

:GGCGGCTGC

kTCGAGCGAG

;AGCATCAGG

GGCGAGGATC

GGCCGCTTTT

ATAGCGTTGG

CTCGTGCTTT

GACGAGTTCT

TGCCATCACG

TTTTCCGGGA

,CCCACCCCA)b

ATTTCACAA)

ATGTATCTT;

CATAGCTGTI

GAAGCATAA)

TGCGCTCAC',

GCCAACGCGI

AAGATGGATT

GGGCACAACA

GCCCGGTTCT

CAGCGCGGCT

TCACTGAAGC

CATCTCACCT

ATACGCTTGA

CACGTACTCG

GGCTCGCGCC

TCGTCGTGAC

CTGGATTCAT

CTACCCGTGA

ACGGTATCGC

TCTGAGCGGG

AGATTTCGAT

CGCCGGCTGG

*CTTGTTTATT

TAAAGCATTT

TCATGTCTGT

TCCTGTGTGA

kGTGTAAAGCC r' GCCCGCTT'C Z GGGGAGAGGC CACGCAGGT TCTCCGGCCG CTTGGGTGGA IACAATCGGC TGCTCTGATG CCGCCGTGTT rTTTGTCAAG ACCGACCTGT CCGGTGCCCT kTCGTGGCTG GCCACGACGG GCGTTCCTTG ;GGAAGGGAC TGGCTGCTAT TGGGCGAAGT rGCTCCTGCC GAGAAAGTAT CCATCATGGC L'CCGGCTACC TGCCCATTCG ACCACCAAGC GATGGAAGCC GGTCTTGTCG ATCAGGATGA '.GCCGAACTG TTCGCCAGGC

TCAAGGCGCG

CCATGGCGAT GCCTGCTTGC CGAATATCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG rATTGCTGAA GAGCTTGGCG

GCGAATGGGC

CGCTCCCGAT TCGCAGCGCA

TCGCCTTCTA

ACTCTGGGGT TCGAAATGAC CGACCAAGCG TCCACCGCCG CCTTCTATGA

AAGGTTGGGC

ATGATCCTCC AGCGCGGGGA

TCTCATGCTG

GCAGCT'TATA ATGGTTACAA

ATAAAGCAAT

TTTTCACTGC ATTCTAGTTG

TGGTTTGTCC

ATACCGTCGA CCTCTAGCTA

GAGCTTGGCG

AATTGTTATC CGCTCACAAT

TCCACACAAC

TGGGGTGCCT AATGAGTGAG

CTAACTCACA

CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT GGTTTGCGTA TTGGGCGCTC TTCCGCTTCC CGGCTGCGGC GAGCGGTATC AGCTCACTCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 2520 2580 2640 2700) 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 ACTCGCTGCG CTCGGTCGTT

TACGGTTATC

AAAAGGCCAG

CACAGAATCA

GA.ACCGTAAA

30 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT 4140 CCGACCCTGC CGCTTACCGG TCTCAATGCT CACGCTGTAG TGTGTGCACG AACCCCCCGT GAGTCCAACC CGGTAAGACA AGCAGAGCGA GGTATGTAGG TACACTAGAA GGACAGTATT AGAGTTGGTA GCTCTTGATC

TGCAAGCAGC

ACGGGGTCTG

TCAAAAAGGA

AGTATATATG

TCAGCGATCT

ACGATACGGG

TCACCGGCTC

AGATTACGCG

ACGCTCAGTG

TCTTCACCTA

AGTAAACTTG

GTCTATTTCG

AGGGCTTACC

CAGATTTATC

RTACCTGTCC

GTATCTCAGT

TCAGCCCGAC

CGACTTATCG

CGGTGCTACA

TGGTATCTGC

CGGCAAACAA

CAGAAAAAAA

GAACGAAAAC:

GATCCTTTTA

GTCTGACAGT

TTCATCCATA

ATCTGGCCCC

AGCAATAAAC

CTCCATCCAC

TTTGCGCAA(

GCCTTTCTCC

TCGGTGTAGG

CGCTGCGCCT

CCACTGGCAG

GAGTTCTTGA

GCTCTGCTGA

ACCACCGCTG

GGATCTCAAG

TCACGTTAAG

AATTAAAAAT

TACCAATGC7

GTTGCCTGAC

AGTGCTGCA)

CAGCCAGCCC

;TCTATTAAT,

GTTGTTGCC;

CTTCGGGAAG

TCGTTCGCTC

TATCCGGTAA

CAGCCACTGG

AGTGGTGGCC

AGCCAGTTAC

GTAGCGGTGG

AAGATCCTTT

GGATTTTGGT

GAAGTTTTAA

*TAATCAGTGA

*TCCCCGTCG7

TGATACCGCG,

GAAGGGCCG;

r' GTTGCCGGGW k TTGCTACAG(

CGTGGCGCTT

CAAGCTGGGC

CTATCG.TCT

TAACAGGATT

TAACTACGGC

CTTCGGAAAA

TTTTTTTGTT

GATCTTTTCT

CATGAGATTA

ATCAATCTAA

GGCACCTATC

GTAGATAACT

AGACCCACGC

kGCGCAGAAGT k AGCTAGAGTA

;CATCGTGGTG

4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400- 5460 S520 5580 GGTCCTGCAA CTTTATCCGC

AGTAGTTCGC

TCACGCTCGT

ACATGATCCC

AGAAGTAAGT

ACTGTCATGC

TGAGAATAGT

GCGCCACATA

CAGTTAATAG

CGTTTGGTAT

CCATGTTGTG

TGGCCGCAGI

CATCCGTAAC

GTATGCGGC(

GCAGAACTT~r GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT

CAAAAAAGCG

GTTATCACTC

ATGCTTTTCT

ACCGAGTTGC

AAAAGTGCTC

GTTAGCTCCT

ATGGTTATGG

GTGACTGGTG

TCTTGCCCGG

ATCATTGGAA

TCGGTCCTCC

CAGCACTGCA

AGTACTCAAC

CGTCAATACG

AACGTTCTTC

GATCGTTGTC

TAATTCTCTT

CAAGTCATTC

GGATAATACC

GGGGCGAAAA

CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGA .TGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC AATGCCGCAA AAAAGGGAAT

TTTCACCAGC

AAGGGCGACA

GTTTCTGGGT

CGGAA-ATGTT

GAGCAAAAAC

GAATACT CAT

AGGAAGGCAA

ACTCTtCCTT 5640 5700 31

TTTCAATATT

TGTATTTAGA

ATTGAAGCAT

AAAATAAACA

q

S

GACGTCGACG

GATCGGGAGA

CTGATGCCGC

ATAGTTAAGC

AGTGCGCGAG;

CAAAATTTAA

ATCTGCTTAG

GGTTAGGCGT

GACATTGATT

ATTGACTAGT

CATATATGGA

GTTCCGCGTT

ACGACCCCCG

CCCATTGACG

10 CTTTCCATTG

ACGTCAATGG

AAGTGTATcA

TATGCCAAGT

GGCATTATGC

CCAGTACATG

TAGTCATCGC

TATTACCATG

GGTTTGACTC

ACGGGGATTT

GGCACCAAAA

TCAACGGGAC

TGGGCGGTAG

GCGTGTACGG

AACCCACTGC TTACTGGCTT GGTACCGAGC

TCGGATCCAC

rTATCAGGGT

PATAGGGGTT

rCTCCCGATC

CAGTATCTGC

GCTACAACAA

TTTGCGCTGC

TATTAATAGT

ACATAACTTA

TCAATAATGA

GTGGACTATT

ACGCCCCCTA

ACCTTATGGG

GTGATGCGGT

CCAAGTCTCC

TTTCCAAAAT

TGGGAGGTCT

ATCGAAATTA

TAGTAACGGC

TATTGTCTCA

CCGCGCACAT

CCCTATGGTC

TCCCTGCTTG

GGCAAGGCTT

TTCGCGATGT

AATCAATTAC

C!GGTAAATGG

CGTATGTTCC

TACGGTAAC

TTGACGTCAA

ACTTTCCTAC

TTTGGCAGTA

ACCCCATTGA

GTCGTAACAA

ATATAAGCAG

ATACGACTCA

CGCCAGTGTG

TGAGCGGATA

TTCCCCGAAA

GACTCTCAGT

TGTGTTGGAG

GACCGACAAT

ACGGGCCAGA

GGGGTCATTA

CCCGCCTGGC

CATAGTAACG

TGCCCACTTG

TGACGGTAAA

TTGGCAGTAC

CATCAATGGG

CGTCAATGGG

CTCCCCCCCA

AGCTCTCTGG

CTATAGGGAG

CTGGAATTCT

CATATTTGAA

AGTGCCACCT

ACAATCTGCT

GTCGCTGAGT

TGCATGAAGA

TATACGCGTT

GTTCATAGCC

TGACCGCCCA

CCAATAGGGA

GCAGTACATC

TGGCCCGCCT

ATCTACGTAT

CGTGGATAGC

AGTTTGTTTT

TTGACGCAAA

CTAACTAGAG

ACCCAAGCTT

GCAGATTCTT

5760 S82 0 5880 S940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 678-0 6802 CTCTCATCCG CCAAACAGA

AG

INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 5825 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Influenza virus, RNA sequence 32 INDIVIDUAL ISOLATE: pHL1489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGATGGTCAT TTTGTCCTGA TGAGTCCGTG AGGACGAAAC ATAGAGCTGG AGTAAAACTG ATGAGTCCGT GAGGACGAAA CAACCTGCCA TCACGAGATT o

AATCGTTTTC

CTTCGCCCAC

CGACCTCGCG

CTATCCGCGC

CCCGGATCTT

CAGAGATTTA

TGATTCTAAT

GGTGGAATGC

ATGAGGCTAC

ACCCCAAGGA

GAACTCTTGC

AAATTATGGP

TACTGTTTTI

AATTGTGTAC

GTGCCTTGA(

AAAAACCTCC

AACTTGTTTJ

AATAAAGCA'

CGGGACGCCG

CCCGGGCTCG

GAGTTCTACC

ATCCATG CCC

TGTGAAGGAA

AAGCTCTAAG

TGTTTGTGTA

CTTTAATGAG

TGCTGACTCT

CTTTCCTTCA

TTGCTTTGCT

AAAATATTCT

CTACCTTGTT I rCGATTCCAC

GCTGGATGAT

ATCCCCTCGC

GGCAGTGCAA

CCGAACTGCA

CCTTACTTCT

GTAAATATAA

TTTTAGATTC

GAAAACCTGT

CAACATTCTA

GAATTGCTAA

ATTTACACCA

GTAACCTTTA

'CTATTCGAA

'GCCGCCTTC T :CTCCAGCGC G

;AGTTGGTTC

kTCCGTCGGC 2GAGTGGGGA C

;TGGTGTGAC

kATTTTTAAG

CAACCTATGG

TTTGCTCAGA

CTCCTCCAAA

GTTTTTTGAG

CAAAGGAAAA

TAAGTAGGCA

GAGTGTCTGC

AAGGGGTTAA

ACCACATTTG

AAACATAAAA

AAATAAAGCA

,TGACCGACC AAGCGACGCC ATGAAAGGT I]

;GGGATCTCA

LGCTGCTGCC

kTCCAGGAAA

;GCACGATGG

kTAATTGGAC rGTATAATGT kACTGATGAA

FGAAATGCCA

A~AAGAAGAGA

TCATGCTGTG

AGCTGCACTG

TAACAGTTAT

TATTAATAAC

TAAGGAATAT

TAGAGGTTTT

TGAATGCAAT

[GGGCTTCGG

cGCTGGAGTT rGAGGCTGGA

CCAGCAGCGG

CCGCTTTGGT

AAACTACCTA

GTTAAACTAC

TGGGAGCAGT

TCTAGTGATG

AAGGTAGAAG

TTTAGTAATA

CTATACAAGA

AATCATAACA

TATGCTCAAA

TTGATGTATA

ACTTGCTTTA

TGTTGTTGTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 TCTTACTCCA CACAGGCATA

CTTTAGCTTT

TAGAGATCAT

CACACCTCCC

TTGCAGCTTA

TTTTTTCACT

TTAATTTGT&A

AATCAGCCAT

CCTGAACCTG

TAATGGTTAC

ATAGCATCAC AAATTTCACA CCAAACTCAT CAATGTATCT GCATTCTAGT TGTGGTTTGT TATCATGT.CT GGATCCCCAG GAAGCTCCTC TGTGTCCTCA TAAACCCTAA CCTCCTCTAC

TTGAGAGGAC

GTCACTTA~c

ATTCCAATCA

AAAAAGGAAA

TAGGCTGCCC

TTGGGTAGGG

ATCCAC.CCTC

GTTTTTCACA

TGTGTCCTCC

GACCGCTTTC

TGTTAATTAG

TAAGGGTAAT

33

TTTAAAATAT

ACAA.ATGTCA

CTCATCAAGA

CCCACCTGTG

GCACTCCACT

CTGACTGTCA

GTTTGCTAAC

TGACCCTTGA

GTTTAACATA

10 AATATTTCCA

GGCCTCGTGA

TCAGGTGGCA

CATTCAAATA

AAAAGGAAGA

TTTTGCCTTC

CAGTTGGGTG

CTGGGILAGTC

ACAGCAGAAA

AGCACTGTGG

TAGGTTCCAA

GGATAAGCAT

ACTGTAGCAT

ACACCCTGCA

ATGGGTTTTC

GCAGTTAcCCC

CAGGTTAAGT

TACGCCTATT

CTTTTCGGGG

TGTATCCGCT

GTATGAGTAT

CTGTTTTTGC

CACGAGTGGG

a 9.

9 .CTTCCACTG C ,ATACAAGCT G rTGCTGTGTT 7 kATATCTAGT C

LATCCTTATC

rTTTTGGGGT

GCTCCAAAGG

CAGCACCATT

CAATAACCTC

CCTCATTTAA

TTTATAGGTT

AAATGTGCGC

CATGAGACAA

TCAACATTTC

TCACCCAGAA

TTACATCGAA

TTTTCCAATG

CGCCGGGCAA

CTCACCAGTC

TGCCATAACC

GAAGGAGCTA

GGAACCGGAG

AATGGCAACA

ACAATTAATA

TCCGGCTGGC

TGTGTTCCA G ;TCAGCTTTG C

LGTAATGTGC

;TTTTCATTT I :AAAJCAGCC I LACAGTTTGA C rTCCCCACCA2 rTCATGAGTT

PLGTTTTAACA

ATTAGGCAAA

A4ATGTCATGA

GGAACCCCTA

TAAccCTGAT

CGTGTCGCCC

AcGCTGGTGA

CTGGATCTCA

ATGAGCACTT

GAGCAACTCG

ACAGAAAAGC

ATGAGTGATA

ACCGCTTTTT

CTGAATGAAG

ACGTTGCGCA

GACTGGATGG

TGGTTTATTG

CTGGGGCCAG

AAGTGTTGG I ACAAGGGCC C LAAACAGGAG C

'TACTTGGAT

TGTGGTCAG

;CAGGATATT

kCAGCAAAAA rTTTGTGTCC 3TAACAGCTT

GGAATTCTTG

TAATAATGGT

TTTGTTTATT

AAATGCTTCA

TTATTCCCTT

AAGTAAAAGA

ACAGCGGTAA

TTAAAGTTCT

GTCGCCGCAT

ATCTTACGGA

ACACTGCGGC

TGCACAACAT

CCATACCAAA

AACTATTAAC

AGGCGGATAA

CTGATAAATC

ATGGTAAGCC

~AAACAGCC

AACACCCTG

;CACATTTTC

:AGGAACCCA

rGTTCATCTG

L'GGTCCTGTA

!ATGAAAATT

CTGAATGCAA

CCCACATCAA

A~AGACGAAAG

TTCTTAGACG

TTTCTAAATA

ATAATATTGA

TTTTGCGGCA

TGCTGAAGAT

GATCCTTGAG

GCTATGTGGC

ACACTATTCT

TGGCATGACA

CAACTTACTT

GGGGGATCAT

CGACGAGCGT

TGGCGAACTA

AGTTGCAGGA

TGGAGCCGGT

CTCCCGTATC

1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400C 2460 252C 2 58 0.

2640 2700 2760 2820 288C 2940 300C 9 5 5 9 a -a q AG7TTTCGCC CCGAAGAACG

GCGGTATTAT

CAGAATGACT

GTAAGAGAAT

CTGACAACGA

GTAACTCGCC

GACACCACGA

CTTACTCTAG

CCACTTCTGC

GAG CGTGGGT

CCCGTGTTGA

TGGTTGAGTA

TATGCAGTGC

TCGGAGGACC

TTGATCGTTG

TGCCTGCAGC

CTTCCCGGCA

GCTCGGCCCT

CTCGCGGTAT CATTGCAGCA GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT 3060 34 q i..

GAGATAGGTG

CTTTAGATTG

GATAATCTCA

GTAGAAAAGA

CAAACAAAAA

CTTTTTCCGA

TAGCCGTAGT

CTAATCCTGT

TCAAGACGAT

CAGCCCAGCT

GAAAGCGCCA

GGAACAGGAG

GTCGGGTTTC

AGCCTATGGA

TTTGCTCACA

TTTGAGTGAG

GAGGAAGCGG

CACCGCATAT

ACACTCCGCT

CTGACGCGCC

TCTCCGGGAG

TGTGGAATGT

TGCAAAGCAT

CAGGCAGAAG

CTCCGCCCAT

TAATTTTTTT

CCTCACTGAT

ATTTAAAACT

TGACCA.AAAT

TCAAAGGATC

A.ACCACCGCT

AGGTAACTGG

TAGGCCACCA

TACCAGTGGC

AGTTACCGGA

TGGAGCGAAC

CGCTTCCCGA

AGCGCACGAG

GCCACCTCTG

AAAACGCCAG

TGTTCTTTCC

CTGATACCGC

AAGAGCGCCT

GGTGCACTCT

ATCGCTACGT

CTGACGGGCT

CTGCATGTGT

GTGTCAGTTA

GCATCTCAAT

TATGCAAAGC

CCCGCCCCTA

TATTTATGCA

TAAGCATTGG

TCATTTTTAA

CCCTTAACGT

TTCTTGAGAT

ACCAGCGGTG

CTTCAGCAGA

CTTCAAGAAC

TGCTGCCAGT

TA.AGGCGCAG

GACCTACACC

AGGGAGAAAG

GGAGCTTCCA

ACTTGAGCGT

CAACGCGGCC

TGCGTTATcC

TCGCCGCAGC

GATGCGGTAT

CAGTACAATC

GACTGGGTCA

TGTCTGCTCC

CAGAGGTTTT

GGGTGTGGAA

TAGTCAGCAP

ATGCATCTC)

ACTCCGCCC;

GAGGCCGAGc

TAACTGTCAG

TTTAAAAGGA

GAGTTTTCGT

CCTTTTTTTC

GTTTGTTTGC

GCGCAGATAC

TCTGTAGCAC

GGCGATAAGT

CGGTCGGGCT

GAACTGAGAT

GCGGACAGGT

GGGGGAAACG

CGATTTTTGT

TTTTTACGGT

CCTGATTCTG

CGAACGACCG

TTTCTCCTTA

TGCTCTGATG

TGGCTGCGCC

CGGCATCCGC

CACCGTCATC

AGTCCCCAGG

CCAGGTGTGG

ATTAGTCAGC

GTTCCGCCC2A

CCGCCTCGGC

ACCAAGTTTA

TCTAGGTGAA

TCCACTGAGC

TGCGCGTAAT

CGGATCAAGA

CAA.ATACTGT

CGCCTACATA

CGTGTCTTAC

GAACGGGGGG

ACCTACAGCG

ATCCGGTAAG

CCTGGTATCT

GATGCTCGTC

TCCTGGCCTT

TGGATAACCG

AGCGCAGCGA

CGCATCTGTG

CCGCATAGTT

CCGACACCCG

TTACAGACAA

ACCGAAACGC

CTCCCCAGCA

AAAGTCCCCA

*AACCATAGTC

*TTCTCCGCCC

CTCTGAGCTA

CTCATATATA

GATCCTTTTT

GTCAGACCCC

CTGCTGCTTG

GCTACCA3ICT

CCTTCTAGTG

CCTCGCTCTG

CGGGTTGGAC

TTCGTGCACA

TGAGCTATGA

CGGCAGGGTC

TTATAGTCCT

AGGGGGGCGG

TTGCTGGCCT

TATTACCGCC

GTCAGTGAGC

CGGTATTTCA

AAGCCAGTAT

CCAACACCCG

GCTGTGACCG

GCGAGGCAGC

GGCAGAAGTA

GGCTCCCCAG

CCGCCCCTAA

CATGGCTGAC

TTCCAGAAGT

3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 9.

9** 9 9..

9. 9 '9 AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA GCTTCACGCT GCCGCAAGCA 35 CTCAGGGCGC AAGGGCTGCT AAAGGAAGCG GAACACGTAG AAAGCCAGTC

CGCAGAAACG

4q #9 4 *4i.

4ib* 4 4.

4* .4 4e** 9 *4*9 4 9. .4 9 4 .9 4 a 9* GTGCTGACCC

CGGATGAATG

AAAGAGAAAG CAGGTAGCTT ATGGACAGCA AGCGAACCGG CTGCAAAGTA

AACTGGATGG

ATCTGATCAA GAGACAGGAT AGGTTCTCCG

GCCGCTTGGG

CGGCTGCTCT GATGCCGCCG CAAGACCGAC

CTGTCCGGTG

10 GCTGGCCACG

ACGGGCGTTC

GGACTGGCTG

CTATTGGGCG

TGCCGAGAAA

GTATCCATCA

TACCTGCCCA TTCGACCACc AGCCGGTCTT

GTCGATCAGG

15 ACTGTTCGCC

AGGCTCAAGG

CGATGCCTGC

TTGCCGAATA

TGGCCGGCTG

GGTGTGGCGG

TGAAGAGCTT GGCGGCGAA1 CGATTCGCAG

CGCATCGCCI

20 GGGTT

TCAGCTACTG

GCAGTGGGCT

AATTGCCAGC

CTTTCTTGCC

GAGGATCGTT

TGGAGAGGCT

TGTTCCGGCT

CCCTGAATGA

CTTGCGCAGC

AAGTGCCGGG

TGGCTGATGC

AAGCGAAACA

ATGATCTGGA

CGCGCATGCC

TCATGGTGGA

ACCGCTATCA

GGGCTGACCG

TCTATCGCC7

GGCTATCTGG

TACATGGCGA

TGGGGCGCCC

GCCAAGGATC

TCGCATGATT

ATTCGGCTAT

GTCAGCGCAG

ACTGCAGGAC

TGTGCTCGAC

GCAGGATCTC

AATGCGGCGG

TCGCATCGAG

CGAAGAGCAT

CGACGGCGAG

AAATGGCCGC

GGACATAGCG

CTTCCTCGTG

TCTTGACGAG

ACAAGGGAAA

TAGCTAGACT

TCTGGTAAGG

TGATGGCGCA

GAACAAGATG

GACTGGGCAC

GGGCGCCCGG

GAGGCAGCGC

GTTGTCACTG

CTGTCATCTC

CTGCATACGC

CGAGCACGTA

CAGGGGCTCG

GATCTCGTCG

TTTTCTGGAT

TTGGCTACCC

CTTTACGGTA

TTCTTCTGAG

ACGCAAGCGC

GGGCGGTTTT

TTGGGAAGCc

GGGGATCMAG

GATTGCACGC

AACAGACAAT

TTCTTTTTGT

GGCTATCGTG

AAGCGGGAAG

ACCTTGCTCC

TTGATCCGGC

CTCGGATGGA

CGCCAGCCGA

TGACCCATGG

TCATCGACTG

GTGATATTGC

TCGCCGCTCC

CGGGACTCTG

4740 480C) 4860 4920 4980 5040 5100 5160 S220 5280 S340 5400 S460 5520 5580 5640 5700 5760 5820 5825 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 4023 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

36 (vi) ORIGINAL

SOURCE.

ORGANISM: Influenza virus, RNA sequence INDIVIDUAL ISOLATE: pHL149O (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CAGGTACATA TTGAAAGATG AGTCTTCTAA CCGAGGTCGA AACGTACGTT CTIr'rzf

TCCCGTCAGG

AGAACACCGA

TGACTAAGGG

AGCGTAGACG

AAGCAGTTAA

TCTCACTCAG

TGGGGGCTGT

CTGACTCCCA

ATGAGAACAG

CGAGTGAGCA

CGATGAGAC

AAAATTTGCA

CTCGCTATTG

CTTTTTTTCA

GAAGGAGTGC

GCTGACGATG

CCTATCTCCA

GGAAAGTGAC

GAcCCCATAA

GAACTGATA

CAGACGTACA

CACGCCCAGT

CCCCCTCAAA

TCTTGAGGTT

GATTTTAGGA

CTTTGTCCAA

ACTGTATAGG

TTATTCTGCT

GACCACTGAA

GCATCGGTCT

AATGGTTTTA

AGCAGCAGAG

CATTGGGACT

GGCCTATCAG

CCGCAAATAT

AATGCATTTA

CAAAGTCTAT

GTCATTTTGT

GGTCCAATAG

AGGCCACAGA

CGAAAAGAAC

GACCGACAGG

GGGCCCGCTA

CGCGTACCGT

GCCGAGATCG

CTCATGGAAT

TTTGTGTTCA

AATGCCCTTA

AAGCTCAAGA

GGTGCACTTG

GTGGCATTTG

CATAGGCAAA

GCCAGCACTA

GCCATGGAGG

CATCCTAGCT

AAACGAATGG

CATTGGGATC

CCGTCGCTTT

GAGGGAAGAA

CAGTATAGAG

GACCAGATCT

GAATACCTGG

CGGACCTCAA

TCAATGAAAG

ACAGCGCGAT

CTTCATGGGA

CACAGAGACT

GGCTAAAGAC

CGCTCACCGT

ATGGGAACGG

GGGAGATAAC

CCAGTTGTAT

GCCTGGTATG

TGGTGACA~C

CAGCTAAGGC

TTGCTAGTCA

CCAGTGCTGG

GGGTGCAGAT

TTGCACTTGA

AAATACGGAC

TATCGAAAGG

CTGGAGTAAA

AAAAGATCAC

AAGTCATACC

AGGAACAACT

AAAACCGCGC

TTGCTGGTGA

GAAAATAATA

TGAAGATGTC

AAGACCAATC

GCCCAGTGAG

GGATCCAAAT

ATTCCATGGG

GGGCCTCATA.

TGCAACCTGT

AACCAACCCA

TATGGAGCAA

GGCTAGGCAA

TCTGAAAAAT

GCAACGGTTC

TATTGTGGAT

TGAAAGGAGG

AACAGCAGAG

AAAGTACCTT

AAGCATAAAA

TGGGGAGGTG

GGTCGACCTG

CCATAGCTTT

CCCAATGCGA

CTGTTGATGG

TTTGCAGGGA

CTGTCACCTC

CGAGGACTGC

AACATGGACA

GCCAAAGAAA

TACAACAGGA

GAACAGATTG

CTAATCAGAC

ATGGCTGGAT

ATGGTGCAAG

GCTCTTCTTG

AAGTGATCCT

TCTTGATCGT

GCCTTCTACG

TGCTGTGGAT

GTTTCTACTA

GAGACAGGGA

GCCCAAAAAT

ACAACCCGGA

CCCTCGGCCT

CCAGATGCTC

GTGTCTGGTC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 37 AGAGACATCA AGAAATAACG CCGGAACATT AGTGCAGGCA GCTTCCACAG'

CAATGGCATC

9 9.

9 -eCi.

a 99 C.

*9 C 99 9 9

C.

CTGGTCATCC

AGCGGATAGT

CGGCACCTCG

CTAACGGATT

ACTGTGAATG

CGCAAACCAA~

AGCCGCACGC

GGCGCATCTC

TGACGAGCAT

CACAAAJATC

AAGATACCAG

GCGTTTCCCC

GCTTACCGGA

TACCTGTCCG

ACGCTGTAGG

TATCTCAGTT

10 ACCCCCCGTT

CAGCCCGACC

GGTAAGACAC

GACTTATCGC

GTATGTAGGC

GGTGCTACAG

GACAGTATTT

GGTATCTGCG

CTCTTGATCC

GGCAAACA

15 GATTACGCGC

AGAAAAAAAG

CGCTCAGTGG

AACGAAAACT

CTTCACCTAG

ATCCTTTTAA

GTAAACTTGG

TCTGACAGTT

TCTATTTCGT

TCATCCATAG

20 GGGCTTACCA

TCTGGCCCCA

AGATTTATCA GCAAT AAACC TTTATCCGCC

TCCATCCAGT

AGTTAATAGT

TTGCGCAJACG

GTTTGGTATG

GCTTCATTCA

CATGTTGTGC

AAAAAAGCGG

GGCCGCAGTG

TTATCACTCA

ATCCGTAAGA

TGCTTTTCTG

TAATGATCTA

CACCACTCCA

CCCTTGGCAG

GGGCCGCGTT

GACGCTCAAG

CTGGAAGCTC

CCTTTCTCCC

CGGTGTAGGT

GCTGCGCCTT

CACTGGCAGC

AGTTCTTGAA

CTCTGCTGAA~

CCACCGCTGG

GATCTCAAGA

CACGTTAAGG

ATTAAAAATG

ACCAATGCTT

TTGCCTGACT

GTGCTGCAAT

AGCCAGCCGG

CTATTAATTG

TTGTTGCCAT

GCTCCGGTTC

TTAGCTCCTT

TGGTTATGGC

TGACTGGTGA

CCGCGGTAGA

AGAATTGGAG

AACATATCCA

GCTGGCGTTT

TCAGAGGTGG

CCTCGTGCGC

TTCGGGAAGC

CGTTCGCTCC

ATCCGGTAAc

AGCCACTGGT

GTGGTGGCCT

GCCAGTTACC

TAGCGGTGGT

AGATCCTTTG

GATTTTGGTC

AAGTTTTAAA

AATCAGTGAG

CCCCGTCGTG

GATACCGCGA

AAGGGCCGAG

TTGCCGGGAA

TGCTGCAGGC

CCAACGATrCA

CGGTCCTCCG

AGCACTGCAT

GTACTCAACC

TCTAGCCCAC

CCAATCMATT

TCGCGTCCGC

TTCCATAGGC

CGAAACCCGA

TCTCCTGTTC

GTGGCGCTTT

AAGCTGGGCT

TATCGTCTTG

AACAGGATTA

AACTACGGCT

TTCGGAAA

TTTTTTGTTT

ATCTTTTCTA

ATGAGATTAT

TCAATCTAAA

GCACCTATCT

TAGATAACTA

GACCCACGCT

CGCAGAAGTG

GCTAGAGTAA

ATCGTGGTGT

AGGCGAGTTA

ATCGTTGTCA

AATTCTCTTA

AAGTCATTCT

TGAACGCGGG

CTTGCGGAGA

CATCTCCAG;c

TCCGCCCCCC

CAGGACTATA

CGACCCTGCC

CTCAATGCTC

GTGTGCACGA

AGTCCAACc

GCAGAGCGAG

ACA6TAGAAG

GAGTTGGTAG

GCAAGCAGCA

CGGGGTCTGA

CAAAAAGGAT

GTATATATGA

CAGCGATCTG

CGATACGGGA

CACCGGCTCC

GTCCTGCAAC

GTAGTTCGCC

CACGCTCGTc CATGATCCCc

GAAGTAAGTT

CTGTCATGCC

GAGAATAGTG

1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 38

TATGCGGCGA

CAGAACTTTA

CCGAGTTGCT

AAAGTGCTCA

CTTGCCCGGC

TCATTGGAAA

GTCAACACGG

ACGTTCTTCG

GATAATACCG CGCCACATAG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA ATCTTTTACT TTCACCAGCG AAAGGGAATA AGGGCGACAC TTGAAGCATT TATCAGGGTT AAATAAACAA AAGAGTTTGT.

TAATTTGATG CCTGGCAGTT TCGCAACGTT CAAATCCGCT AACAACAGAT AAAACGAAAG

GTTCGATGTA

TTTCTGGGTG

GGAAATGTTG

ATTGTCTCAT

ACCCACTCGT

AGCAAAAACA

AATACTCATA

GAGCGGATAC

GCACCCAACT

GGAAGGCAAA

CTCTTCCTTT

ATATTTGAAT

GATCTTCAC

ATGCCGCAAA

TTCAATATA

GTATTTAGAA

GCCTTCTGCT

GGCCGTTGCT

TTCACCGACA

ATTTGATGCC

AGAAACGCAA AAAGGCCATC CGTCAGGATG TATGGCGGGC GTCCTGCCCG

CCCGGCGGAT

GCCCAGTCTT

TGGCAGTTCC CTACTCTCGC ATGGGGAGAC

TTGTCCTACT

TCGACTGAGC

CCCACACTAC

ACCACCGCGC

TTAATCTGTA

AGCTTGGGCT

CCACCCTCCG

CAGGAGAGCG

CTTTCGTTTT

CATCGGCGCT ACGGCGTTTC 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4023

ACTTCTGAGT

TGTTTTATCA

CATCCGCCAA

CCC CAAAAAA

TCGGCATGGG

GACCGCTTCT

AACAGAAGCT

GTCAGGTGGG

GCGTTCTGAT

AGCGGCCGCT

TACTGCCGCC

TCAGGCTGAA

GCAGGTCGAC

AGGCAAATTC

AATCTTCTCT

TCTAGAGGAT

a. a GAGTCCAGAG TGGCCCCGCC GTTCCGCGCC GGGGGGGGGG GGGGGGGGGG ACACTTTCGG ACATCTGGTC GACCTCCAGC ATCGGGGGAA AAAAAAAAAA CAAAGTTTCG CCCGGAGTAC TGGTCGACCT CCGAAGTTGG GGGGGAGTAG

AAA

4 a 9* a a 4 a.

39

Claims (12)

1. A segmented RNA virus of the Orthomyxoviridae family, said RNA virus comprising at least one segment comprising a vRNA nucleotide sequence comprising a 3' terminal nucleotide sequence and a 5' terminal nucleotide sequence in association therewith, wherein said 3' terminal nucleotide sequence consists of 15 nucleotides corresponding to a wild-type vRNA 3' terminal nucleotide sequence modified by replacement of two or three nucleotides naturally occurring in the wild-type 3' terminal nucleotide sequence at positions 3, 5 and 8 by other nucleotides, and said terminal nucleotide sequence consists of 16 nucleotides corresponding to a wild-type vRNA 5' terminal nucleotide sequence optionally modified by replacement of the nucleotides naturally occurring in the wild-type 5' terminal nucleotide sequence at positions 3 and 8 by other nucleotides and further, wherein said RNA virus exhibits as a result of said replacements rates of transcription, replication and/or expression that are higher than those of a wild-type RNA virus of the same species, with the proviso that the 3' terminal nucleotide sequence does not have the sequence 5'-CACCCUGUUUUUACU or 5'-CACCCUGUUUCUGCU-3'.

2. The segmented RNA virus according to claim 1, wherein the replacements in the 3' terminal nucleotide sequence comprises G 3 A and C 8 U. o*

3. The segmented RNA virus according to claim 2, wherein the replacements in the 3' terminal nucleotide sequence consists of G 3 A, C 8 U and U 5 C.

4. The segmented RNA virus according to claim 3, which comprises a 3' terminal nucleotide sequence of 5'-CCUGUUUCUACU-3'. The segmented RNA virus according to claim 1, which comprises a 5' terminal nucleotide sequence consisting of a corresponding wild-type vRNA 5' terminal nucleotide sequence modified by replacement of the nucleotides naturally occurring in the wild-type 5' terminal nucleotide sequence at positions 3 and 8 by other nucleotides.

6. The virus of claim 1, wherein, at least one modified segment is an artificial addition to the set of original segments.

7. The virus of claims 1 to 6, wherein the modified segment comprises a nucleotide sequence which codes for a protein or peptide which is foreign to the original virus.

8. The virus of claim 7, wherein the foreign protein or peptide constitutes an antigen or antigen-like sequence, a T-cell epitope or related sequence.

9. The virus of claims 7 or 8, wherein the segment comprises repetitions of an antigen or epitope or other peptide or protein. The virus of any of claims 7 to 9, wherein the antigen or epitope is derived from HIV, Herpes-Virus, Rhinovirus, CMV, papilloma viruses, Hepatitis viruses and other human viruses or Hog Cholera Virus.

11. A pharmaceutical preparation of a vaccine comprising the virus of any of claims 1 to 7 -10 in association with a pharmaceutically acceptable carrier and/or diluent.

12. Use of the virus of any of claims 1 to 10 for the preparation of pharmaceuticals.

13. A method of enhancing gene transcription and expression in a host cell containing a genetically-engineered segmented RNA virus of the Orthomyxoviridae family, comprising: a) providing a segmented RNA virus according to claims 1 to 10; and b) introducing said RNA virus into a host cell, wherein as a result of said replacements said RNA virus exhibits rates of transcription and expression that are higher than those of a wild-type RNA virus of the same species.

14. The method of claim 17, wherein said RNA virus exhibits a replication rate that is higher than that of a wild type RNA virus of the same species. DATED this 27 th day of July 1999 PROF. DR. DR. GERD HOBOM By his Patent Attorneys DAVIES COLLISON CAVE