**3. The gene constellation effect**

It stands to reason that if a certain gene constellation confers some desirable attribute then viruses containing those segments should occur more often in the population over time than other reassortments. That is, the combination of gene segments should occur independently many times in a large enough population if the same parental viruses continue to co-circulate in the population. However, if the reassortant has a relative fitness that is much greater than other circulating strains then the virus may quickly replace other virus strains in the popula‐ tion. An example of this is the emergence and spread of the 2009 pandemic H1N1 strain. Very quickly, and during a season not typically associated with high influenza rates, the 2009 pandemic strain became the prevalent H1N1 strain and prior seasonal H1N1 strains become less common [60].

Possibly more common, but less well documented, are reassortants that have a small increase in fitness compared to the parental strains. A recent analysis of reassortant H3N2 viruses in swine demonstrated that multiple reassortants generating the same gene constellation occurred [61]. Several H3N2 swine lineage viruses were isolated from humans in 2011 and found to contain the segment 7 from the 2009 pandemic H1N1 virus. This prompted Nelson and colleagues to analyze the reassortants present in swine populations. What they discovered was that, in addition to the reassortants that transmitted to humans, reassortants in swine with a range of genetic backbones contained the 2009 pandemic segment 7 [61]. It is not known if the presence of the 2009 segment 7 in swine viruses plays a role in viral fitness in swine or if it has a role in zoonotic infection of humans, but clearly the presence of this segment gives rise to viruses from different reassortment events that are stably represented in the population.

The presence of segment 7 or 8 from differing viruses can alter the phenotype of another virus. For example, addition of different NS segments from an H3N2 virus or different H5N1 viruses into a PR/8 backbone could result in no attenuation or complete attenuation [53;54]. Similarly, the same gene can have different effects on different viruses. For example, replacement of the A/Korea/82 (H3/N2) M segment with the A/Ann Arbor/6/60 M segment attenuated the virus. However, introducing the same A/Ann Arbor/6/60 M segment into A/Udorn/72 (H3/N2) did not attenuate the virus [55]. This clearly demonstrates the greater impact the gene constellation

The characterization of the laboratory generated reassortants provides useful hypothesisdriven information. Natural reassortment of the smallest influenza segments may give additional information about the role these segments play in the virus lifecycle. Some viruses isolated recently from North American pigs contain the 2009 H1N1 M segment in the context of a previously endemic H1N2 strain [56]. This suggests that this particular gene constellation may increase viral fitness. Supporting this Chou et al., [57] were able to show that inclusion of the M segment in a PR/8 backbone was essential for transmission in guinea pigs. Another group reported that the neuraminidase segment from the 2009 H1N1 strain, in addition to the M segment, was required for efficient replication and transmission in pigs [58]. Finally, Hause et al. [59] reported transmission but lower viral titers for the reassortant viruses containing the M segment from the 2009 pandemic strain in pig lung homogenate when compared to infection with the parental strains. The major difference between the viruses analyzed by these groups is that the backbone strains differed; one group used comtemporary H1N2 swine viruses to generate reassortants while the other groups used laboratory adapted strains. The different

It stands to reason that if a certain gene constellation confers some desirable attribute then viruses containing those segments should occur more often in the population over time than other reassortments. That is, the combination of gene segments should occur independently many times in a large enough population if the same parental viruses continue to co-circulate in the population. However, if the reassortant has a relative fitness that is much greater than other circulating strains then the virus may quickly replace other virus strains in the popula‐ tion. An example of this is the emergence and spread of the 2009 pandemic H1N1 strain. Very quickly, and during a season not typically associated with high influenza rates, the 2009 pandemic strain became the prevalent H1N1 strain and prior seasonal H1N1 strains become

Possibly more common, but less well documented, are reassortants that have a small increase in fitness compared to the parental strains. A recent analysis of reassortant H3N2 viruses in swine demonstrated that multiple reassortants generating the same gene constellation occurred [61]. Several H3N2 swine lineage viruses were isolated from humans in 2011 and found to contain the segment 7 from the 2009 pandemic H1N1 virus. This prompted Nelson

has toward the virus phenotype than an individual segment in this instance.

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

outcomes observed are based on gene constellation effects.

**3. The gene constellation effect**

less common [60].

In addition to the appearance of this particular gene constellation in North America pigs, gene constellations involving all segments from the 2009 pandemic strain are becoming dominant in other parts of the world. It has been reported that the 2009 pandemic strain is present in pigs and reassorting with H1N2 and H3N2 strains [62-65]. It is not clear how the combination of gene segments present in strains like the 2009 pandemic strain results in a greater viral fitness but study of different viral characteristics has given us some insight. Here we highlight research describing the effect different gene constellations have on viral fitness.

**Figure 2.** Coding strategies of influenza viruses (adapted from Lamb & Takeda, 2001). A) Multiple splice variants are transcribed from one segment to enable the production of multiple proteins. As many as four mRNAs (and proteins) are produced from influenza A segment 7. The mRNA from the smallest segment of all influenza species is spliced for the translation of the nuclear export protein xlink. B) A larger protein is translated and processed to produce the influ‐ enza C protein CM2. The M1 protein is produced from a spliced mRNA. C) Different AUG start codons are used to pro‐ duce up to three proteins from influenza A segment 1. A second membrane protein, NB, is produced from an alternative start codon in segment 6 of influenza B. D) The termination and start codons of the respective M1 and BM2 open reading frames in segment 7 of influenza B overlap. A stop/start translation strategy is used to produce the second protein.

## **3.1. Altered pathogenicity**

There have been many efforts to understand which gene segments contribute to pathogenicity. If a particular segment were known to contribute to pathogenicity in a vaccine strain then safeguards could be put in place to prevent the generation of the gene constellations containing the offending segment(s). The current recommendation for Institutional Biosafety Committees in the United States is that gene constellation be included in the evaluation for determining the biocontainment level for influenza work [66]. One difficulty in applying this recommen‐ dation is that the pathogenicity of a virus in one host species may differ greatly from the pathogenicity in another host species. Another difficulty is that new strains emerge and evolve faster than the pathogenicity of the gene combinations can be assessed. Here we reflect on what is known about gene constellation and pathogenicity.

addition of an H5N1-derived NS segment to a PR/8 backbone attenuated the virus when tested in mice [53]. Because PR/8 is a high growth strain these results may not be truly representative of the effect the NS gene can have. When the NS segment from a highly pathogenic H5N1 strain was added to a highly pathogenic H7N1 strain the virus was more pathogenic in mice [76]. Here the observed increase in virulence was also associated with enhanced cell tropism. This demonstrates the potential for reassortants created in a host specific manner to gain the

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The 2009 H1N1 pandemic virus arose by reassortment of swine influenza viruses of different lineages. The NA and M gene segments were from the Eurasian avian-like swine virus lineage and the remaining segments were from the North American triple reassortant lineage. The triple reassortant lineage emerged in the 1990s with the PB2 and PA segments derived from an avian virus, the PB1 from a human virus and the remaining segments from the classical swine lineage. With all three swine lineages circulating, and reassorting, there is a concerted effort to characterize current and possible reassortants for their potential to infect humans [62;65]. The ferret is a widely used model for assessing pathogenicity and transmission of viruses. Unlike mice, but like humans, ferrets infected with a seasonal influenza strain present with an increase in temperature, nasal secretions, sneezing and sometimes with a cough, making them suitable for study of human viruses. Triple reassortant swine viruses isolated before 2009 did not produce clinical signs of respiratory symptoms like sneezing and nasal secretions [77]. The pathogenicity of these viruses was similar to the 2009 pandemic virus with more lung pathology than seasonal viruses [77]. All the triple reassortant viruses transmitted between ferrets by direct contact but only those with human-like HA and NA were transmitted efficiently by respiratory droplets [65;78]. Addition of a seasonal H3N2 NA to a 2009 pandemic virus gave rise to more severe pulmonary lesions in ferrets demonstrating the importance of gene constellation [79]. Further, it has been shown that the tropism of such a virus is linked to the balance between HA and the NA even when the replication competence is lower [62]. For influenza vaccine seed viruses it has been hypothesized that a lower NA content in the virion

A high growth phenotype in a virus seed strain is beneficial for vaccine production. There are many proteins that might have an effect on the virus growth rate. The envelope proteins determine the infectivity through effects on attachment, entry and budding. The replicase proteins affect the speed of transcription and replication. And finally, NS1 protein modulates the host response. Thus, differing combinations can result in changes in growth rates. Many vaccine seed strains have been made using PR/8 as the backbone, that is, replacement of the HA and NA segments whilst retaining the six remaining PR/8 segments. Usually the intro‐ duced HA and NA segments come from the same virus so one would assume that the encoded proteins are compatible with each other. Thus, any reduction in growth rate is due to the change in interactions between the HA and NA combination and other proteins. Increased growth rates are often achieved by passaging the virus but the enhanced growth sometimes

results in antigenic changes to HA rather than adaptation of the other proteins.

ability to jump the species barrier.

can increase HA content [80].

**3.2. Altered growth rates**

In the 1970s it became clear that both the glycoproteins and the internal proteins play a role in pathogenicity. In many experiments segments from a human or animal origin virus were introduced into a pathogenic avian influenza virus and pathogenicity tested in chickens. Often the pathogenicity was reduced [67;68]. However, increased monitoring of avian influenza viruses in Hong Kong indicated that most naturally occurring reassortant H5N1 viruses were lethal to birds [69]. Serial passage of pathogenic avian influenza in MDCK cells and selection of large plaques resulted in reduced pathogenicity in mice suggesting that differences in the host cell type that the virus is propagated in play a role in pathogenicity [70]. Interestingly, the attenuated variants all had common mutations in the polymerase genes and grew to higher titers on MDCK cells than virus purified from small plaques. This suggests that an equilibrium between replication efficiency and pathogenicity is being altered when a virus is adapting to a new host. This is in contrast to the increased pathogenicity seen when faster growing viruses are compared to slower growing viruses in the same host; for example, avian viruses grown in eggs [71;72].

There are other examples of changes in the replication machinery of the influenza virus affecting pathogenicity. In one study it was found that reassortant avian H5N1 viruses containing the PB2 gene from a human H3N2 virus had increased pathogenicity in mice [73]. It was also shown that the introduction of a human PB1 segment alone did not enhance pathogenicity but had a cooperative effect when the human PB2 was present. As noted above, the segments containing the polymerase often cosegregate, which could be troubling if this enhances pathogenicity.

Pathogenicity is not only dependent on the virus, it also depends on the host species and even where in the host the virus replicates [74]. The pathogenicity caused by a virus is often due to the host response to the virus and is perhaps best exemplified by the cytokine storm. In such instances an excessive amount of proinflammatory cytokines are released and inflammation spreads from the site of infection. Acute lung injury, or the more severe acute respiratory distress syndrome, is associated with influenza infections (reviewed in [75]). As the NS-1 protein encoded on segment 8 has a role in modulating the host immune response, one would expect that segregation of segment 8 during reassortment might alter virus pathogenicity. However, while addition of an H3N2 NS segment to a 2009 H1N1 virus increased replication efficiency, the virus was not as pathogenic in mice as the parental H1N1 strain [54]. Also, the addition of an H5N1-derived NS segment to a PR/8 backbone attenuated the virus when tested in mice [53]. Because PR/8 is a high growth strain these results may not be truly representative of the effect the NS gene can have. When the NS segment from a highly pathogenic H5N1 strain was added to a highly pathogenic H7N1 strain the virus was more pathogenic in mice [76]. Here the observed increase in virulence was also associated with enhanced cell tropism. This demonstrates the potential for reassortants created in a host specific manner to gain the ability to jump the species barrier.

The 2009 H1N1 pandemic virus arose by reassortment of swine influenza viruses of different lineages. The NA and M gene segments were from the Eurasian avian-like swine virus lineage and the remaining segments were from the North American triple reassortant lineage. The triple reassortant lineage emerged in the 1990s with the PB2 and PA segments derived from an avian virus, the PB1 from a human virus and the remaining segments from the classical swine lineage. With all three swine lineages circulating, and reassorting, there is a concerted effort to characterize current and possible reassortants for their potential to infect humans [62;65]. The ferret is a widely used model for assessing pathogenicity and transmission of viruses. Unlike mice, but like humans, ferrets infected with a seasonal influenza strain present with an increase in temperature, nasal secretions, sneezing and sometimes with a cough, making them suitable for study of human viruses. Triple reassortant swine viruses isolated before 2009 did not produce clinical signs of respiratory symptoms like sneezing and nasal secretions [77]. The pathogenicity of these viruses was similar to the 2009 pandemic virus with more lung pathology than seasonal viruses [77]. All the triple reassortant viruses transmitted between ferrets by direct contact but only those with human-like HA and NA were transmitted efficiently by respiratory droplets [65;78]. Addition of a seasonal H3N2 NA to a 2009 pandemic virus gave rise to more severe pulmonary lesions in ferrets demonstrating the importance of gene constellation [79]. Further, it has been shown that the tropism of such a virus is linked to the balance between HA and the NA even when the replication competence is lower [62]. For influenza vaccine seed viruses it has been hypothesized that a lower NA content in the virion can increase HA content [80].

#### **3.2. Altered growth rates**

**3.1. Altered pathogenicity**

in eggs [71;72].

enhances pathogenicity.

is known about gene constellation and pathogenicity.

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

There have been many efforts to understand which gene segments contribute to pathogenicity. If a particular segment were known to contribute to pathogenicity in a vaccine strain then safeguards could be put in place to prevent the generation of the gene constellations containing the offending segment(s). The current recommendation for Institutional Biosafety Committees in the United States is that gene constellation be included in the evaluation for determining the biocontainment level for influenza work [66]. One difficulty in applying this recommen‐ dation is that the pathogenicity of a virus in one host species may differ greatly from the pathogenicity in another host species. Another difficulty is that new strains emerge and evolve faster than the pathogenicity of the gene combinations can be assessed. Here we reflect on what

In the 1970s it became clear that both the glycoproteins and the internal proteins play a role in pathogenicity. In many experiments segments from a human or animal origin virus were introduced into a pathogenic avian influenza virus and pathogenicity tested in chickens. Often the pathogenicity was reduced [67;68]. However, increased monitoring of avian influenza viruses in Hong Kong indicated that most naturally occurring reassortant H5N1 viruses were lethal to birds [69]. Serial passage of pathogenic avian influenza in MDCK cells and selection of large plaques resulted in reduced pathogenicity in mice suggesting that differences in the host cell type that the virus is propagated in play a role in pathogenicity [70]. Interestingly, the attenuated variants all had common mutations in the polymerase genes and grew to higher titers on MDCK cells than virus purified from small plaques. This suggests that an equilibrium between replication efficiency and pathogenicity is being altered when a virus is adapting to a new host. This is in contrast to the increased pathogenicity seen when faster growing viruses are compared to slower growing viruses in the same host; for example, avian viruses grown

There are other examples of changes in the replication machinery of the influenza virus affecting pathogenicity. In one study it was found that reassortant avian H5N1 viruses containing the PB2 gene from a human H3N2 virus had increased pathogenicity in mice [73]. It was also shown that the introduction of a human PB1 segment alone did not enhance pathogenicity but had a cooperative effect when the human PB2 was present. As noted above, the segments containing the polymerase often cosegregate, which could be troubling if this

Pathogenicity is not only dependent on the virus, it also depends on the host species and even where in the host the virus replicates [74]. The pathogenicity caused by a virus is often due to the host response to the virus and is perhaps best exemplified by the cytokine storm. In such instances an excessive amount of proinflammatory cytokines are released and inflammation spreads from the site of infection. Acute lung injury, or the more severe acute respiratory distress syndrome, is associated with influenza infections (reviewed in [75]). As the NS-1 protein encoded on segment 8 has a role in modulating the host immune response, one would expect that segregation of segment 8 during reassortment might alter virus pathogenicity. However, while addition of an H3N2 NS segment to a 2009 H1N1 virus increased replication efficiency, the virus was not as pathogenic in mice as the parental H1N1 strain [54]. Also, the

A high growth phenotype in a virus seed strain is beneficial for vaccine production. There are many proteins that might have an effect on the virus growth rate. The envelope proteins determine the infectivity through effects on attachment, entry and budding. The replicase proteins affect the speed of transcription and replication. And finally, NS1 protein modulates the host response. Thus, differing combinations can result in changes in growth rates. Many vaccine seed strains have been made using PR/8 as the backbone, that is, replacement of the HA and NA segments whilst retaining the six remaining PR/8 segments. Usually the intro‐ duced HA and NA segments come from the same virus so one would assume that the encoded proteins are compatible with each other. Thus, any reduction in growth rate is due to the change in interactions between the HA and NA combination and other proteins. Increased growth rates are often achieved by passaging the virus but the enhanced growth sometimes results in antigenic changes to HA rather than adaptation of the other proteins.

Many insights about virus growth have come from analysis of changes on one segment. It has been observed that culture of many influenza viruses in eggs results in amino acid changes in HA as the virus adapts to the new host. Similar observations have been made when viruses are grown in different cell types. For example, most viruses have an asparagine residue in the haemagglutinin at position 117 (H1 subtype) or 116 (H3 subtype). Substitution of this residue with aspartic acid does not alter the growth in MDCK cells but enhances growth in Vero cells [81]. This mutation was shown to alter the pH range for virus membrane fusion indicating that this is an important factor for optimal growth [81]. However, HA does not act in isolation and the best growth of vaccine seed strains will depend on how differences in HA affect interactions with other proteins, in particular, the other major envelope proteins NA and M.

recognized respectively by the host protein G-rich RNA sequence binding factor 1 (GRSF-1) and the viral NS1 protein may also be present in the non-coding regions (reviewed in [1]). Both of these proteins have been shown to stimulate translation [91;92]. In addition, there are changes in translation that most likely due to changes in the RNA structure. Single nucleotide changes in the 5' and 3' non-coding regions of PA were shown to have no effect on translation individually, but together these changes almost completely abolished protein expression [90]. It is not known if the changes altered mRNA production or affected translation itself, but one would expect that the resultant loss of viral protein would affect virus replication and possibly virulence. Indeed, the mutations that resulted in low protein production were based on the

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Very similar viruses may produce quite different amounts of viral protein and several groups have tried to find the underlying reasons. It is common for several reassortants to be made using different seasonal isolates in an effort to create a suitable high yield vaccine seed virus. As noted previously the PB1 segment from the seasonal strain was found in many high yield reassortants made for vaccine manufacture [18]. Analysis of protein production from reassortants with or without the seasonal PB1 segment and the 2009 H1N1 HA and NA as been performed. One group found that the presence of the PB1 from the 2009 strain increased protein yield while another found that it decreased protein yield [85;93]. Both groups used PR/8 as the high yield donor strain but they each used differ‐ ent 2009 pandemic isolates. This highlights one difficulty in predicting what gene constel‐ lations may be beneficial for protein production; minor strain variations may have major translational effects. Comparison of both the PR/8 strains and both 2009 pandemic strains

The in vitro production of ressortant viruses using the classical reassortant method involves high multiplicities of infection to increase the chance of both viruses infecting cell. Early studies demonstrated that passage of influenza at high multiplicities of infection could result in the production of non-infectious particles [94;95]. These particles are now known as defective interfering, or DI, particles. Subsequently it was discovered that the polymer‐ ase genes frequently contained deletions [96;97]. Thus, although eight genomic segments were packaged, the viruses could not make proteins essential for replication. In addition, non-infectious particles lacking the glycoproteins have also been described [98]. This explains the loss of infectivity even though many particles are detected by haemagglutina‐ tion [99]. Kaverin et al., [99] were able to show that one fraction of a DI population could complement another fraction of the DI population. More recently, Odagiri and Tashiro [100] were able to show that non-coding sequences were responsible for the preferential packaging of DI RNA rather than the full length RNA segment. It is not clear when or how the portions of the RNA are deleted but it is likely that the structure and the sequence of the RNA play a significant role. Using a bioinformatics approach, Priore et al. [101] analyzed the extensive base pairing that exists throughout the genomic segments of avian, swine and human influenza A viruses. The results indicated that there were significant

sequence of a low pathogenic avian influenza virus.

used in these studies may provide interesting information.

**3.4. Incomplete genomes and RNA structure**

The introduction of HA from a seasonal H1N1 virus into the 2009 pandemic strain backbone resulted in larger plaque size and higher viral titers in cell culture [82]. It was further shown that, in contrast to the predominantly filamentous parental strains, the reassortant virus was predominantly spherical and enhanced yields could be obtained by introducing the same seasonal HA into other swine-origin backbones [82]. By using the opposite approach, introduc‐ ing swine-origin segments into the seasonal virus backbone Octaviani et al. were able to show that the high yield was primarily due to the presence of swine-origin HA and M segments [82].

The HA and NA segments from A/Vietnam/1194/2004 and the PR/8 backbone were combined to make a reassortant prepandemic vaccine seed virus but it gave low antigen yield and did not grow well. Incorporation of the M gene segment from the A/Panama/2007/1999 H3N2 strain or from the A/Vietnam/1203/2004 H5N1 strain enhanced growth [83]. The M1 proteins differed at positions T167A, R174K, I219V, A227T and A239K, while the M2 proteins differed at positions N31S, R54L, Y57H, S82N and G89S from the PR/8 segment 7 proteins. It is unknown how the synergy between these segments works but, with structures available for the major envelope proteins and advances in electron tomography, these interactions may be revealed in the near future [84].

Several vaccine seed strains have included the seasonal PB1 gene [18;19]. Growth rates were also shown to improve when the indigenous PB1 was included in the 2009 H1N1 reassortant [85] and with a H5 reassortant [86]. However, this result did not extend to a different H5 reassortant [83]. We postulated that certain residues within the PB1 protein might be important for growth and yield. Rather than target those amino acids known to be involved enzyme activity or protein interactions with the other polymerase proteins, we made changes based on sequence similarity between diverse PB1 proteins that reassorted *in ovo* during the pro‐ duction of influenza vaccine seed viruses. Using this impartial approach, we made changes to the PR/8 PB1 gene that, when combined with the HA and NA from the low yield H3N2 Wyoming/03 strain, resulted in faster growth in both egg and cell culture [87].

#### **3.3. Altered protein production**

The 5' untranslated regions of the influenza A genomic segments contain signals that stimulate translation. These signals regulate the amount of protein produced from each segment and differ between segments. The non-coding regions also differ in length among the different segments and virus types [88-90]. The sequence motifs AGGGU and GGUAGAUA that are recognized respectively by the host protein G-rich RNA sequence binding factor 1 (GRSF-1) and the viral NS1 protein may also be present in the non-coding regions (reviewed in [1]). Both of these proteins have been shown to stimulate translation [91;92]. In addition, there are changes in translation that most likely due to changes in the RNA structure. Single nucleotide changes in the 5' and 3' non-coding regions of PA were shown to have no effect on translation individually, but together these changes almost completely abolished protein expression [90]. It is not known if the changes altered mRNA production or affected translation itself, but one would expect that the resultant loss of viral protein would affect virus replication and possibly virulence. Indeed, the mutations that resulted in low protein production were based on the sequence of a low pathogenic avian influenza virus.

Very similar viruses may produce quite different amounts of viral protein and several groups have tried to find the underlying reasons. It is common for several reassortants to be made using different seasonal isolates in an effort to create a suitable high yield vaccine seed virus. As noted previously the PB1 segment from the seasonal strain was found in many high yield reassortants made for vaccine manufacture [18]. Analysis of protein production from reassortants with or without the seasonal PB1 segment and the 2009 H1N1 HA and NA as been performed. One group found that the presence of the PB1 from the 2009 strain increased protein yield while another found that it decreased protein yield [85;93]. Both groups used PR/8 as the high yield donor strain but they each used differ‐ ent 2009 pandemic isolates. This highlights one difficulty in predicting what gene constel‐ lations may be beneficial for protein production; minor strain variations may have major translational effects. Comparison of both the PR/8 strains and both 2009 pandemic strains used in these studies may provide interesting information.

#### **3.4. Incomplete genomes and RNA structure**

Many insights about virus growth have come from analysis of changes on one segment. It has been observed that culture of many influenza viruses in eggs results in amino acid changes in HA as the virus adapts to the new host. Similar observations have been made when viruses are grown in different cell types. For example, most viruses have an asparagine residue in the haemagglutinin at position 117 (H1 subtype) or 116 (H3 subtype). Substitution of this residue with aspartic acid does not alter the growth in MDCK cells but enhances growth in Vero cells [81]. This mutation was shown to alter the pH range for virus membrane fusion indicating that this is an important factor for optimal growth [81]. However, HA does not act in isolation and the best growth of vaccine seed strains will depend on how differences in HA affect interactions

The introduction of HA from a seasonal H1N1 virus into the 2009 pandemic strain backbone resulted in larger plaque size and higher viral titers in cell culture [82]. It was further shown that, in contrast to the predominantly filamentous parental strains, the reassortant virus was predominantly spherical and enhanced yields could be obtained by introducing the same seasonal HA into other swine-origin backbones [82]. By using the opposite approach, introduc‐ ing swine-origin segments into the seasonal virus backbone Octaviani et al. were able to show that the high yield was primarily due to the presence of swine-origin HA and M segments [82]. The HA and NA segments from A/Vietnam/1194/2004 and the PR/8 backbone were combined to make a reassortant prepandemic vaccine seed virus but it gave low antigen yield and did not grow well. Incorporation of the M gene segment from the A/Panama/2007/1999 H3N2 strain or from the A/Vietnam/1203/2004 H5N1 strain enhanced growth [83]. The M1 proteins differed at positions T167A, R174K, I219V, A227T and A239K, while the M2 proteins differed at positions N31S, R54L, Y57H, S82N and G89S from the PR/8 segment 7 proteins. It is unknown how the synergy between these segments works but, with structures available for the major envelope proteins and advances in electron tomography, these interactions may be revealed

Several vaccine seed strains have included the seasonal PB1 gene [18;19]. Growth rates were also shown to improve when the indigenous PB1 was included in the 2009 H1N1 reassortant [85] and with a H5 reassortant [86]. However, this result did not extend to a different H5 reassortant [83]. We postulated that certain residues within the PB1 protein might be important for growth and yield. Rather than target those amino acids known to be involved enzyme activity or protein interactions with the other polymerase proteins, we made changes based on sequence similarity between diverse PB1 proteins that reassorted *in ovo* during the pro‐ duction of influenza vaccine seed viruses. Using this impartial approach, we made changes to the PR/8 PB1 gene that, when combined with the HA and NA from the low yield H3N2

The 5' untranslated regions of the influenza A genomic segments contain signals that stimulate translation. These signals regulate the amount of protein produced from each segment and differ between segments. The non-coding regions also differ in length among the different segments and virus types [88-90]. The sequence motifs AGGGU and GGUAGAUA that are

Wyoming/03 strain, resulted in faster growth in both egg and cell culture [87].

with other proteins, in particular, the other major envelope proteins NA and M.

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

in the near future [84].

**3.3. Altered protein production**

The in vitro production of ressortant viruses using the classical reassortant method involves high multiplicities of infection to increase the chance of both viruses infecting cell. Early studies demonstrated that passage of influenza at high multiplicities of infection could result in the production of non-infectious particles [94;95]. These particles are now known as defective interfering, or DI, particles. Subsequently it was discovered that the polymer‐ ase genes frequently contained deletions [96;97]. Thus, although eight genomic segments were packaged, the viruses could not make proteins essential for replication. In addition, non-infectious particles lacking the glycoproteins have also been described [98]. This explains the loss of infectivity even though many particles are detected by haemagglutina‐ tion [99]. Kaverin et al., [99] were able to show that one fraction of a DI population could complement another fraction of the DI population. More recently, Odagiri and Tashiro [100] were able to show that non-coding sequences were responsible for the preferential packaging of DI RNA rather than the full length RNA segment. It is not clear when or how the portions of the RNA are deleted but it is likely that the structure and the sequence of the RNA play a significant role. Using a bioinformatics approach, Priore et al. [101] analyzed the extensive base pairing that exists throughout the genomic segments of avian, swine and human influenza A viruses. The results indicated that there were significant differences between species in the PB2, NP, M and NS-containing segments. These differences were only on the positive strand which could indicate a role in either protein production or negative strand synthesis. Given that these segments do not reassort as frequently as the other polymerase segments or glycoprotein segments, it would seem that genome wide RNA structural organization does not contribute to reassortment. Al‐ though, it is postulated that global organizational RNA structure could be a mechanism by which the virus adapts to the host environment [101] leaving open the possibility that the RNA structure of a particular segment affects the chances of it being involved in reassortment.

or the replicase proteins independently, some of the work described here demonstrates that these two groups of proteins have an effect on each other. The interaction between

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While several groups have analyzed the genomes of reassortant viruses there is still a great need for better understanding what features contribute to genomic reassortment. With more whole virus genome sequences available for analysis there is a better chance that the features important for reassortment can be determined. Retrospective analysis of reassor‐ tant viruses can illuminate which genomic features are compatible. In vitro construction of reassortant viruses can highlight which segments, or parts of segments, are not compati‐ ble. After a reassortment event is detected there needs to be more analyses of the muta‐ tions that occurred in each segment as they may have facilitated the reassortment event. Mutations necessary for reassortment would occur prior to reassortment and perhaps be present in bottleneck viruses. Mutations that occur with passage are those that increase the fitness of the reassortant. Description of both types of mutations would enhance our understanding of the network of interactions between viral proteins. In addition to the changes in coding sequences, analysis of the untranslated regions of the genomes is also important. There is no available information about the compatibility of segments with the replication and translation machinery, or how this contributes to the gene constellation. Finally, an understanding of the gene constellation effect will allow for the selection of better reassortant viruses for vaccine production. Currently both the *in ovo* and reverse genetic methods use an impirical approach to get the best viruses that express the desired HA and NA proteins. Knowing how the different segments contribute to the network of interactions that result in high yield will enable researchers to produce strains that will provide the best backbone for an influenza vaccine seed virus. The optimal backbones may be universal, or differ for the different virus subtypes, or differ according to the host species that the virus providing the HA and NA infects. But without optimal virus backbones, the production of

these two groups of proteins at a functional level needs to be elucidated.

high yield reassortant influenza vaccine seed viruses will remain inefficient.

The findings and conclusions in this chapter have not been formally disseminated by the Food and Drug Administration and should not be construed to represent an Agency determination

**Acknowledgements**

or policy.

**Author details**

Ewan P. Plant and Zhiping Ye

Food and Drug Administration, USA

\*Address all correspondence to: ewan.plant@fda.hhs.gov

DI particles represent an evolutionary dead end with regard to a natural infection. However, particles that lack a complete genome could be either detrimental or beneficial in vaccine production. Particles that are defective in the polymerase will alter growth characteristics and would not function in a live attenuated vaccine. In contrast, particles with incomplete genomes represent an abundance of antigen with no pathogenicity, which could be viewed as desirable in an inactivated vaccine.
