**2. The segmented influenza genome**

The Orthomyxoviridae family is comprised of negative-sense, segmented RNA viruses. There are three influenza genera, influenza A, B and C viruses, that belong to the family. Eight negative-sense RNA segments make up the viral genome for the A and B viruses, one more than the influenza C virus genome which has seven segments (Figure 1). The terminal ends of each gene segment are conserved and this allows control over several aspects of the influenza lifecycle. The terminal sequences are partially complementary and can form structures that serve as regulatory signals for transcription and replication (Figure 1). The structures are dynamic and it is thought that switching between the structures allows different steps of replication to occur [1].

Each negative-sense viral RNA is encapsidated with nucleoprotein to form a ribonucleoprotein (RNP). Attached to the 5' end of each segment is the influenza polymerase complex [2]. This arrangement allows a message RNA (mRNA) from each segment to be transcribed independ‐ ently of other segments in the nucleus. Most of the segments encode a single protein but some of the RNA segments are spliced or have alternative translation mechanisms so that usually more than 10 proteins are made during an infection [3;4]. The mRNAs transcribed from all the segments are exported from the nucleus to the cytoplasm where they are translated into the viral proteins. The nucleoprotein and polymerase proteins each contain nuclear localization sequences and are imported into the nucleus where they participate in the production of new viral RNA. Some of the remaining proteins are processed in the secretory pathway and transported to the cell surface for incorporation into the virions while others remain in the cytoplasm or nucleus and modulate the host cell immune response.

The negative-sense segmented genome bestows some advantages and disadvantages to the virus. Having the genome divided up into segments creates some challenges such as ensuring that one of each segment is packaged into one virion. However, it also helps alleviate a problem faced by many RNA viruses, the high error rate inherent in RNA synthesis. The error rate for the influenza viruses has be calculated to be 2.0 x 10-6 and 0.6 x 10-6 mutations per site per infectious cycle for influenza A and B respectively [5]. Rates ranging from 3.72 to 6.77 x 10-4 substitutions per site per year have been calculated for the influenza C segments [6]. Influen‐ za viruses can exist as a quasispecies, that is, a group of diverse viruses that collectively contribute to the characteristics of the population (reviewed in [7]). This enables mutations to exist that, by themselves, may not increase the fitness of the virus and could even be detrimen‐ tal. A combination of these mutations, that together increase the fitness of the virus, may result in a virus with some selective advantage. Such a combination could occur by gene reassort‐ ment. The separation of different mutations on different virus segments facilitates this proc‐

the influenza A and B viruses are made up of eight negative-strand RNA segments. The haemagglutinin (HA) and neuraminidase (NA) proteins found on the surface of the virus are found on two different segments. Usually these two segments from a seasonal virus are combined with another six gene segments from a high yield strain to make a vaccine seed virus. HA is the major immunogenic protein recognized by the host immune system. Because of influenza's high rate of mutation, and the capacity of the genome to tolerate many mutations, there is a need to update the influenza vaccine seed virus strains each year. The influenza virus is able to avoid control by the host immune system via two major types of mutation. Antigenic drift is the process of gradual genetic mutation, especially in the HA gene, that results in newer viruses not being well recognized by antibodies that recognized the progenitor virus. Anti‐ genic shift is the replacement of one or more segments from one influenza virus with those of another. This unpredictable event can lead to a change in host range, transmission or patho‐ genicity. Likewise, genetic reassortment, the mixing of genomic segments from different strains, can generate undesirable characteristics in the influenza vaccine seed viruses. Here we explore possible reasons for this and describe approaches that might be beneficial to the

214 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

The Orthomyxoviridae family is comprised of negative-sense, segmented RNA viruses. There are three influenza genera, influenza A, B and C viruses, that belong to the family. Eight negative-sense RNA segments make up the viral genome for the A and B viruses, one more than the influenza C virus genome which has seven segments (Figure 1). The terminal ends of each gene segment are conserved and this allows control over several aspects of the influenza lifecycle. The terminal sequences are partially complementary and can form structures that serve as regulatory signals for transcription and replication (Figure 1). The structures are dynamic and it is thought that switching between the structures allows different steps of

Each negative-sense viral RNA is encapsidated with nucleoprotein to form a ribonucleoprotein (RNP). Attached to the 5' end of each segment is the influenza polymerase complex [2]. This arrangement allows a message RNA (mRNA) from each segment to be transcribed independ‐ ently of other segments in the nucleus. Most of the segments encode a single protein but some of the RNA segments are spliced or have alternative translation mechanisms so that usually more than 10 proteins are made during an infection [3;4]. The mRNAs transcribed from all the segments are exported from the nucleus to the cytoplasm where they are translated into the viral proteins. The nucleoprotein and polymerase proteins each contain nuclear localization sequences and are imported into the nucleus where they participate in the production of new viral RNA. Some of the remaining proteins are processed in the secretory pathway and transported to the cell surface for incorporation into the virions while others remain in the

The negative-sense segmented genome bestows some advantages and disadvantages to the virus. Having the genome divided up into segments creates some challenges such as ensuring

cytoplasm or nucleus and modulate the host cell immune response.

development of influenza vaccine seed viruses.

**2. The segmented influenza genome**

replication to occur [1].

**Figure 1.** Genomes of influenza viruses. A) The ends of the negative strand influenza genomic RNA are complexed with the three polymerase proteins and the remaining sequence is encapsidated with nucleoprotein (vRNP (-)). The positive strand cRNP is similarly complexed. The mRNA is transcribed with a 5' cap structure and poly-A tail (see main text for details). Figure used with permission from Resa-Infante et al., 2011. B) Schematic of the influenza A virus ge‐ nome. The bold black lines represent the 3' and 5' untranslated regions. The blue and pink boxes represent the major protein coding regions. C) Schematic of the influenza B virus genome. The green and brown boxes represent the ma‐ jor protein coding regions. D) Schematic of the influenza C virus genome. The red and purple boxes represent the ma‐ jor protein coding regions. The protein coding regions are not to scale. Coding regions in a different reading frame are shown above or below each other, coding regions in the same frame are show as contiguous blocks.

ess and allows reassortant viruses to be made if a cell is infected with two influenza viruses at the same time.

appropriate mutants, if any, become manifest" [16]. This was achieved by infecting an egg with a combination of a non-infective strain (with a high yield characteristic) and infective influenza strain (with the desired antigenic trait) in the presence of antiserum to suppress the antigenic proteins of the non-infective strain. This allowed for the selection of virus with the same antigenicity as the infectious strain and the same growth characteristics of the non-infectious strain [17]. This method for making vaccine seed virus strains has been widely used since. One drawback of this approach is that, because the antiserum only suppresses virus expressing the non-infective strain surface proteins, segments from the infective strain, in addition to the HA and NA segments, are often present in the resulting strains [18;19]. This may result in an undesirable trait being present in the vaccine seed virus strain and, as reassortments involving whole gene segments cannot revert like single point mutations, the traits are not as easily

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More recently, with the development of reverse genetics, it has been possible to make reas‐ sortant viruses from cloned viral segments. This allows genetically defined vaccine seed virus strains to be produced and the methodology has been employed extensively for the production of live attenuated vaccine seed virus strains [20]. The cold adaption and attenuation mutations are spread out on multiple segments. A large number of human influenza vaccine seed virus strains have been made with the cold adapted strains A/Ann Arbor/6/60-H2N2 and B/Ann Arbor/1/66 strains [20]. High yield attenuated backbone strains for vaccination of livestock such as birds or pigs have also been developed and are typically made using reverse genetics [21-23]. In addition, viruses expressing the haemagglutinin from highly pathogenic avian strains (H5N1) need to have the polybasic cleavage site removed by mutagenesis and reverse genetic viruses made to reduce pathogenicity. Viruses made by reverse genetics use the same plasmid derived internal gene segments. In constrast, vaccine seed strains made using *in ovo* reassortment are sometimes made with a recent high growth reassortant as the non-infective strain. This could result in the carrying forth of internal segments that are not from the high yield strain Puerto Rico/8/1934 (PR/8) or mutations that have appeared during the passage of the earlier reassortant strain. Although the reverse genetically engineered viruses are geneti‐ cally defined, there is no avenue for reassortment to occur if there is some incompatibility between the glycoproteins from the season strain and the remaining proteins from the

Influenza viruses are frequently isolated and propagated in tissue culture. Madin-Darby canine kidney (MDCK) cells are widely used because they are quite susceptible to influenza virus infection. This is because the antiviral activity of MDCK cells is lacking due to inadequate interferon-induced myxovirus resistance protein 1 (Mx1) activity [24]. As noted above, the introduction of HA or NA segments into circulating strains is dominant among naturally occurring reassortants [15]. In contrast, recombinants generated in MDCK cells with no selection show a positive correlation between other segments: most of the segment pairs that segregate with each other in MDCK cells were polymerase combinations [25-27]. This suggests that for naturally occurring reassortants there is some selective pressure that is not present in laboratory-based experiments. One explanation for this bias may be due to the limited MDCK cell antiviral response; without this response the replication efficiency of the virus might be

reverted as point mutations.

backbone virus.

Reassortment occurs in all three influenza virus genera [6;8;9]. But there is no evidence of reassortment between the genera. This is likely due, in part, to the level divergence between the viruses, both in the non-coding regulatory elements and in the proteins which interact with each other. Also, there is evidence that one virus may suppress another through pathways that are not well understood [10]. Reassortment has long been known to occur naturally in humans, swine and birds [11-13].

Often evidence of reassortment is based on incongruence in the phylogenetic trees of each of the segments. Although naturally occurring reassortants can be compared to previously sequenced strains, the actual strains that a particular segment is derived from, and the steps in reassortment, are only deduced. The different segments from the recent 2009 pandemic H1N1 virus were phylogenetically similar to human, avian, and classical, Eurasian and triple reassortant swine virus segments. Using all the available whole virus genome sequences Bokhari et al. [14] used a bioinformatics approach to determine which viruses were most likely homologous to the ancestors to the 2009 pandemic strain, and what reassortments needed to occur. Interestingly, they found that among 92% of the possible paths there were certain bottleneck viruses [14]. That is, these viruses contained the mutations and segment reassort‐ ments that made them most like the next virus in the reassortment path that eventually gave rise to the 2009 pandemic strain. This suggests that there are certain sequence requirements that need to be present before reassortment occurs.

Sequence analysis indicates that a disproportionate number of the naturally occurring reassortants are the result of novel haemagglutinin and/or neuraminidase genes being introduced into a previously circulating strain [15]. The introduction of the HA and NA segments into another strain is also the goal of influenza vaccine seed virus strain construction.

#### **2.1. Making vaccine seed virus**

There are two predominant ways that an influenza virus can be engineered; one, by simulta‐ neous infection of a cell with two viruses each bearing some desired trait, or two, by reverse genetics. Both methods are commonly used to produce influenza vaccine seed viruses. A disadvantage of co-infection is that it may result in the production of viruses with unwanted combinations of the gene segments. A disadvantage to the reverse genetic approach is that it may be difficult to generate a virus if the introduced HA and NA genes cause a detrimental gene constellation effect.

In the late 1950s and throughout the 1960s Edwin Kilbourne pioneered the development of genetic recombination with influenza [16]. He recognised that an influenza vaccine seed virus should have certain desirable characteristics; good growth, low virulence, thermal stability and the proper antigenicity. At that time, development of high yield vaccine seed virus strains was via empirical methods such as mouse-lung passage. Kilbourne developed and promoted the use of "the deliberate mating of 2 or more viruses, each bearing a desired trait" so that "an appropriate progeny virus can be selected without the need for tedious" adaptation "until appropriate mutants, if any, become manifest" [16]. This was achieved by infecting an egg with a combination of a non-infective strain (with a high yield characteristic) and infective influenza strain (with the desired antigenic trait) in the presence of antiserum to suppress the antigenic proteins of the non-infective strain. This allowed for the selection of virus with the same antigenicity as the infectious strain and the same growth characteristics of the non-infectious strain [17]. This method for making vaccine seed virus strains has been widely used since. One drawback of this approach is that, because the antiserum only suppresses virus expressing the non-infective strain surface proteins, segments from the infective strain, in addition to the HA and NA segments, are often present in the resulting strains [18;19]. This may result in an undesirable trait being present in the vaccine seed virus strain and, as reassortments involving whole gene segments cannot revert like single point mutations, the traits are not as easily reverted as point mutations.

ess and allows reassortant viruses to be made if a cell is infected with two influenza viruses at

216 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Reassortment occurs in all three influenza virus genera [6;8;9]. But there is no evidence of reassortment between the genera. This is likely due, in part, to the level divergence between the viruses, both in the non-coding regulatory elements and in the proteins which interact with each other. Also, there is evidence that one virus may suppress another through pathways that are not well understood [10]. Reassortment has long been known to occur naturally in humans,

Often evidence of reassortment is based on incongruence in the phylogenetic trees of each of the segments. Although naturally occurring reassortants can be compared to previously sequenced strains, the actual strains that a particular segment is derived from, and the steps in reassortment, are only deduced. The different segments from the recent 2009 pandemic H1N1 virus were phylogenetically similar to human, avian, and classical, Eurasian and triple reassortant swine virus segments. Using all the available whole virus genome sequences Bokhari et al. [14] used a bioinformatics approach to determine which viruses were most likely homologous to the ancestors to the 2009 pandemic strain, and what reassortments needed to occur. Interestingly, they found that among 92% of the possible paths there were certain bottleneck viruses [14]. That is, these viruses contained the mutations and segment reassort‐ ments that made them most like the next virus in the reassortment path that eventually gave rise to the 2009 pandemic strain. This suggests that there are certain sequence requirements

Sequence analysis indicates that a disproportionate number of the naturally occurring reassortants are the result of novel haemagglutinin and/or neuraminidase genes being introduced into a previously circulating strain [15]. The introduction of the HA and NA segments into another strain is also the goal of influenza vaccine seed virus strain construction.

There are two predominant ways that an influenza virus can be engineered; one, by simulta‐ neous infection of a cell with two viruses each bearing some desired trait, or two, by reverse genetics. Both methods are commonly used to produce influenza vaccine seed viruses. A disadvantage of co-infection is that it may result in the production of viruses with unwanted combinations of the gene segments. A disadvantage to the reverse genetic approach is that it may be difficult to generate a virus if the introduced HA and NA genes cause a detrimental

In the late 1950s and throughout the 1960s Edwin Kilbourne pioneered the development of genetic recombination with influenza [16]. He recognised that an influenza vaccine seed virus should have certain desirable characteristics; good growth, low virulence, thermal stability and the proper antigenicity. At that time, development of high yield vaccine seed virus strains was via empirical methods such as mouse-lung passage. Kilbourne developed and promoted the use of "the deliberate mating of 2 or more viruses, each bearing a desired trait" so that "an appropriate progeny virus can be selected without the need for tedious" adaptation "until

the same time.

swine and birds [11-13].

that need to be present before reassortment occurs.

**2.1. Making vaccine seed virus**

gene constellation effect.

More recently, with the development of reverse genetics, it has been possible to make reas‐ sortant viruses from cloned viral segments. This allows genetically defined vaccine seed virus strains to be produced and the methodology has been employed extensively for the production of live attenuated vaccine seed virus strains [20]. The cold adaption and attenuation mutations are spread out on multiple segments. A large number of human influenza vaccine seed virus strains have been made with the cold adapted strains A/Ann Arbor/6/60-H2N2 and B/Ann Arbor/1/66 strains [20]. High yield attenuated backbone strains for vaccination of livestock such as birds or pigs have also been developed and are typically made using reverse genetics [21-23]. In addition, viruses expressing the haemagglutinin from highly pathogenic avian strains (H5N1) need to have the polybasic cleavage site removed by mutagenesis and reverse genetic viruses made to reduce pathogenicity. Viruses made by reverse genetics use the same plasmid derived internal gene segments. In constrast, vaccine seed strains made using *in ovo* reassortment are sometimes made with a recent high growth reassortant as the non-infective strain. This could result in the carrying forth of internal segments that are not from the high yield strain Puerto Rico/8/1934 (PR/8) or mutations that have appeared during the passage of the earlier reassortant strain. Although the reverse genetically engineered viruses are geneti‐ cally defined, there is no avenue for reassortment to occur if there is some incompatibility between the glycoproteins from the season strain and the remaining proteins from the backbone virus.

Influenza viruses are frequently isolated and propagated in tissue culture. Madin-Darby canine kidney (MDCK) cells are widely used because they are quite susceptible to influenza virus infection. This is because the antiviral activity of MDCK cells is lacking due to inadequate interferon-induced myxovirus resistance protein 1 (Mx1) activity [24]. As noted above, the introduction of HA or NA segments into circulating strains is dominant among naturally occurring reassortants [15]. In contrast, recombinants generated in MDCK cells with no selection show a positive correlation between other segments: most of the segment pairs that segregate with each other in MDCK cells were polymerase combinations [25-27]. This suggests that for naturally occurring reassortants there is some selective pressure that is not present in laboratory-based experiments. One explanation for this bias may be due to the limited MDCK cell antiviral response; without this response the replication efficiency of the virus might be the main limiting factor. Thus, viruses with combinations of polymerase factors that are most efficient will become dominant. One report of recombinants generated in eggs without selection described a preference for cosegregation of the HA and M segments [28]. Although the number of reports regarding reassortment without selection is limited, the current data suggests that egg-based experiments may more closely reflect naturally occurring events.

additional subtype was recently identified in bats [29]. The HA from the bat virus is more similar to the Group 1 HAs (subtypes H1, 2, 6, 8, 9, 11, 12, 13 and 16) than the Group 2 subtypes but the NA shows no similarity to any previously identified NA subtypes [29]. It remains to

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Infection of humans with viruses containing swine origin HA and NA is known to occur. Like other zoonoses, most of these swine viruses do not spread efficiently in humans. However, the swine origin 2009 pandemic H1N1 virus spread around the world supplanting the prior seasonal human H1N1 strains. This virus was a reassortant derived from a North American

Four proteins are required for influenza virus replication; the nucleocapsid protein (NP) and the three polymerase proteins PB1, PB2 and PA. The polymerase proteins are the larger influenza proteins and are encoded in the largest segments 1-3. NP is encoded in segment 5. The RNA from each viral segment form ribbon-like closed superhelical structures (reviewed in [30]). The 5' and 3' ends of the RNA are in close proximity to one another and are associated with the three polymerase proteins. Nucleoprotein is associated with the remaining genomic RNA and there is one NP monomer present for each 24 nucleotides. Nuclear localization sequences on NP facilitate import of each ribonucleoprotein complex (RNP) into the nucleus (reviewed in [31]). Inside the nucleus mRNA transcription and viral replication take place.

Two of the polymerase proteins, PB1 and PB2, have biochemical interactions with NP proteins. In addition, the three polymerase proteins interact with each other. The carboxyl-terminus of PA interacts with the amino-terminal end of PB1 and the carboxyl-terminal end of PB1 interacts with the amino-terminal end of PB2. The same arrangement is described for both the negative strand viral RNA (vRNP) and the positive strand copy RNA (cRNP) with the polymerase being associated with the 5' end of the RNAs. New negative-strand viral genomes are derived from the cRNP and also have a newly synthesized polymerase complex associated with the 5' end. The NP and three polymerase proteins all have nuclear localization signals which enable them to be imported into the nucleus after they are synthesized in the cytoplasm. There is evidence that PA and PB1 associate with each other before localizing to the nucleus (reviewed in [31]).

Both types of positive-strand RNA, cRNP and mRNA, are generated from the vRNP. In constrast to the vRNP and cRNP, the mRNA is not associated with NP. The viral mRNAs also have a capped 5' leader sequence snatched from a cellular mRNA and a polyadenylated 3' end. It is not yet known exactly what regulates the polymerase complex so that it makes two distinct products from one template. The cap-binding domain is in PB2 and the endonuclease domain is in PA. Together these parts of the polymerase complex capture and remove the 5' capped region of cellular mRNA and this is used as the priming sequence for viral mRNA production. The RNA-dependent RNA polymerase domain required for all RNA production

Combining the polymerase proteins from different strains to produce chimeric polymerase complexes has been studied with regard to polymerase activity and pathogenicity. It is

be seen if these viruses can reassort and cause disease in humans.

**2.3. Replicase proteins**

is in the PB1 protein.

triple reassortant H1N2 swine strain and a Euroasiatic H1N1 swine strain.

The replicase proteins may play a role in reassortment via their independent interaction with each RNA segment. Each genomic segment has it's own replicase proteins associated with it when it enters the nucleus. A doubly infected cell is capable of producing each of the segments from both viruses independently of each other. The timing of the overall replication will depend on many factors such as which segments are imported into the nucleus first, translation promoter sequences and replication signals on each segment, and the induced host response. If a cell is simultaneously infected with two viruses, it is possible that early in the infection the polymerase that transcribes RNA more efficiently will control the dynamics of the infection. The resulting dynamics between the host cell and the viruses may favor the production of either virus or a reassortant. It is unknown if the dynamics of transcription and replication play a role in reassortment but, given that the transciption and replication signals on each segment can differ, it is easy to imagine the dynamics of an infection being altered when a cell is infected with two viruses. One could also imagine polymerase proteins from one strain transcribing or replicating another strain's RNA, or a polymerase protein from one virus interacting with, and altering the activity of, a polymerase protein from the other virus.

### **2.2. Glycoproteins**

There are two major surface glycoproteins for the influenza A and B viruses. The haemagglu‐ tinin (HA) protein is a sugar-binding protein that facilitates virus entry into epithelial cells that have sialic acid sugars on the cell surface. After the HA is cleaved by a protease, the virion is imported into the cell by endocytosis. Virus replication culminates with the accumulation of new virions at the cell surface. The neuraminidase (NA) cleaves the glycosidic linkages of the sialic acids to mediate virion release. The HA protein is encoded by segment 4 and the NA protein by segment 6 in influenza A and B viruses. Influenza C has only seven segments. The haemagglutinin-esterase-fusion (HEF) glycoprotein is encoded on segment 4 and this protein performs the functions analogous to HA and NA of influenza A and B viruses.

In contrast to the influenza B and C viruses which are only sorted by type and strain, the influenza A viruses are sorted into subtypes. The HA and NA proteins are used for virus classification. There are at least 16 different HA subtypes and 9 different NA subtypes among the influenza A viruses. The HA subtypes are divided into two groups. Certain subtypes from both groups are able to infect and transmit among humans. Most human influenza A infections are from H1N1, H2N2, and H3N2 subtypes. Occasionally a strain will jump the species barrier. A limited number of avian subtypes (H5, H7 and H9) have infected humans. Sometimes the disease is much more severe than that from a human influenza strain but these strains seem to lack the ability to transmit from human to human efficiently. There is a fear that these highly virulent viruses may reassort with human viruses creating virulent viruses that spread easily among humans. New pandemic strains arising by reassortment is clearly a concern. An additional subtype was recently identified in bats [29]. The HA from the bat virus is more similar to the Group 1 HAs (subtypes H1, 2, 6, 8, 9, 11, 12, 13 and 16) than the Group 2 subtypes but the NA shows no similarity to any previously identified NA subtypes [29]. It remains to be seen if these viruses can reassort and cause disease in humans.

Infection of humans with viruses containing swine origin HA and NA is known to occur. Like other zoonoses, most of these swine viruses do not spread efficiently in humans. However, the swine origin 2009 pandemic H1N1 virus spread around the world supplanting the prior seasonal human H1N1 strains. This virus was a reassortant derived from a North American triple reassortant H1N2 swine strain and a Euroasiatic H1N1 swine strain.
