BACKGROUND
influenza is a contagious disease that represents a serious health threat to humans and other animals worldwide.
influenza a viruses, in particular, can infect a variety of species.
a global reservoir for these viruses exists in wild waterfowl and shorebirds, from which novel viruses can emerge to infect mammalian species.
influenza is therefore a potential threat to humans, pigs, horses, sea mammals, ferrets, mink as well as many terrestrial bird species
<cit> .
in the past decades, several dramatic episodes of large-scale mortality have occurred in domestic birds, humans, and other species.
to date, the main approach to control influenza epidemics and pandemics in human and other animal populations is through vaccination  and biosecurity, although other approaches may help prevent the transmission of highly pathogenic influenza in some species
<cit> .
strategies for regenerating rna viral genomes from plasmid dna clones  have enabled the powerful technique of reverse genetics to be applied to many different rna viruses including, among others, influenza, rabies, coronavirus, rift valley fever virus or fish rna viruses
<cit> .
this technique allows clonal virus stocks with a defined genotype to be engineered.
it therefore has major medical applications as it enables vaccines to be rationally designed by inserting attenuating mutations or chosen antigens into a defined viral background, enhancing our control options for pathogens such as influenza virus
<cit> .
in fundamental virus studies, it also enables researchers to test the consequences of defined genetic differences on phenotype, such as growth properties, cell-virus interaction, replication cycle characteristics, pathogenicity, or others - the approach known as “reverse genetics”.
various solutions have been developed for satisfying the requirements for the transcribed rnas to become infectious when produced inside a permissive cell.
among the most complex and demanding systems is that required for the segmented negative stranded rna viruses such as influenza virus.
the basic strategy for the most commonly used influenza virus rescue systems comprises a set of plasmids that each drives the expression of one viral genome-sense transcript corresponding to each of the eight viral segments.
accurately tailored 5' and 3' termini are achieved by positioning rna polymerase i  transcription initiation and termination sites respectively .
because the rna is negative sense, the virus polymerase must be produced before replication can ensue.
this can be achieved by co-transfecting a further four plasmids that produce the replication proteins  in the so-called “12-plasmid system”
<cit> .
a derivative of this system uses tandem opposing rna pol i and cytomegalovirus  promoters to drive expression of viral genome rnas and mrnas respectively encoding all the viral proteins, commonly referred to as the “8-plasmid system”
<cit> .
another development has been the production of vectors carrying multiple genome segment expression cassettes
<cit> .
these strategies can be effective and have proven very useful for a number of purposes.
however regenerating field strains and some mutants and reassortants is often less efficient than for well-adapted laboratory strains and it is not uncommon for several attempts to be needed before a particular viral construct is successfully rescued.
this suggests the existence of one or more limiting factors in the process and prompted us to test whether some of these could be overcome to increase rescue efficiency.
several factors may impact virus rescue efficiency.
examples include the replicative ability of a field-derived avian virus in mammalian cells or the balance of the virus’ haemagglutinin and neuraminidase activities
<cit> .
we hypothesized that one limitation might be the ability to successfully transfect and simultaneously express eight different plasmids within an individual cell.
we therefore reasoned that cell fusion might maximize the likelihood of all eight influenza gene-bearing plasmids being present in the same polykaryon and thereby enhance rescue efficiency.
the maedi-visna virus  is a lentivirus causing chronic pneumonia or a progressive demyelinating disease in sheep.
its envelop glycoprotein  is a major target for virus neutralization and can cause substantial host cell fusion.
receptor to this protein has not yet been identified but is known to be expressed on cells from a wide range of species including primates, avians and rodents
<cit> .
we therefore examined co-transfecting an env expression vector along with the eight plasmid rescue system into hek 293t/ <dig> cells to determine its effect on virus rescue efficiency .
RESULTS
cell fusion with the maedi-visna virus envelope protein  in  <dig> t
additional file
<dig>  and additional file
<dig>  show  <dig> t cells transfected with  <dig>  μg of a green fluorescent protein-expressing plasmid plus  <dig>  μg of an inert plamid , or  <dig>  μg of the gfp plasmid plus  <dig>  μg of the env-expressing plasmid .
cells were kept in fcs-containing medium for the whole duration of the footage.
the video shows a marked fusion process, including visible cell-to-cell gfp transfer, in cells transfected with the env plasmid, while cells keep dividing over time .
enhanced yields with the maedi-visna virus envelope protein
for virus rescues, we initially worked with a fixed total mass of  <dig>  μg of dna per well in a 24-well plate format, which had previously been determined to be the optimal amount of dna for best transfection efficiency in our lab.
we varied the proportion of viral genes-containing  and env-expressing  plasmids comprising the  <dig>  μg total in  <dig>  μg  increments.
results of this titration for the lab-adapted strain a/puerto rico/8/ <dig>  are shown in figure
<dig>  transfecting  <dig>  μg of the viral rescue plasmids alone resulted in an average yield of  <dig> x103 ±  <dig> x <dig>  plaque forming units  per millilitre.
reducing the amount of viral rescue plasmids by replacing them proportionately with the b <dig> control plasmid exhibited an initial plateau but reduced the yield sharply at the  <dig>  μg level.
co-transfecting the env plasmid tended to increase the yield at all levels and had a particularly marked effect when the viral rescue plasmids were limiting  p.f.u./ml when as little as  <dig>  μg viral rescue plasmids were included; t4 = − <dig> , p =  <dig> ).
increasing the proportion of viral rescue plasmids while decreasing the proportion of env-expressing plasmid gave higher yields until an apparent optimum was reached for  <dig>  μg  of env-expressing plasmid and  <dig>  μg  of viral rescue plasmids.
dispersion of rescue yield values was however generally high and statistical significance was not detected for the differences observed at the  <dig>  μg and  <dig>  μg levels.
application to the rescue of a strain derived from a field sample
we tested whether using the env-expressing plasmid in the proportions determined above would enhance the practical rescue of a viral strain derived from a low pathogenicity avian influenza  field sample .
such strains are usually more challenging to rescue than laboratory adapted strains such as pr <dig>  giving low yields or failing to rescue in all attempts.
in this experiment, lipofectamine ltx  was used instead of fugene  <dig>  as transfection reagent as the latter had been discontinued by the manufacturer.
figure
<dig> shows the comparison of yields when using 100% of viral rescue plasmids versus using 75% of viral rescue plasmids plus 25% of env-expressing plasmid.
this experiment showed an average yield increase of between one and two log in 6-well plates  and in 24-well plates  when using fusion.
a  <dig>  μg total dna mass was used per well in 24-well plates whereas a  <dig> μg total dna mass was used per well in 6-well plates.
in 6-well plates, one out of three replicates where fusion was not used was negative .
fusion rescue of incomplete combinations of viral segments
to test whether virus could be rescued from cells containing incomplete complements of viral segments by fusing with cells carrying the missing components, we transfected cells in separate batches with partial complements of pr <dig> rescue plasmids and then co-cultured them.
in an experiment named “4 + 4”,  <dig>  μg of each one of segments  <dig>   <dig>   <dig>  and  <dig> as well as  <dig>  μg of either b <dig> or env-expressing plasmids were transfected into one batch of cells, and identical amounts of segments  <dig>   <dig>   <dig>  and  <dig> as well as  <dig>  μg of either b <dig> or env-expressing plasmids were transfected into another batch of cells.
in another experiment named “7 + 1”,  <dig>  μg of each one of segments  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> as well as  <dig>  μg of either b <dig> or env-expressing plasmids were transfected into one batch of cells, and  <dig>  μg of segment  <dig> as well as  <dig>  μg of either b <dig> or env-expressing plasmids were transfected into another batch of cells.
on the day after transfection, media were removed, cells were washed once with pbs and then pooled, and on day  <dig> treated with trypsin.
infectious yields were assessed by plaque formation assay.
table
<dig> shows successful rescue of live virus in all attempts where cells transfected separately with complementary sets of influenza rescue plasmids were allowed to fuse together at day  <dig> post transfection.
no live virus was recovered when the empty b <dig> plasmid was used instead of the env-expressing plasmid, except for one  <dig> μl aliquot from the “7 + 1” experiment which yielded one p.f.u.
this table shows the viral yields  from cells transfected with the eight pr <dig> rescue plasmids split into separate batches at the time of transfection and brought together on the next day.
<dig> in the "4 + 4" experiment, one batch of cells was transfected with plasmids encoding segments  <dig>   <dig>   <dig>   <dig>  while a complementary batch of cells was transfected with plasmids encoding segments  <dig>   <dig>   <dig>   <dig>  in the "7 + 1" experiment, one batch was transfected with plasmids encoding segments  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and the complementary batch was transfected with segment  <dig>  the cells from complementary batches were dislodged and brought together at day  <dig> post transfection.
these experiments consisted of triplicate transfections for each condition .
<dig> in addition to the transfection with influenza plasmids, cells were transfected with either the env-expressing or the empty b <dig> plasmid.
addition of extra env-expressing plasmid
we tested whether adding env-expressing plasmid beyond a total amount of dna of  <dig>  μg per well in a 24-well plate would be detrimental or beneficial to the rescue.
figure
<dig> shows the result of a titration where a fixed amount of  <dig>  μg of pr <dig> rescue plasmids was used in addition to which env-expressing plasmid was added in quantities varying from  <dig>  μg to  <dig>  μg.
the total amount of dna transfected was therefore  <dig>  μg to  <dig>  μg per well.
transfection reagent was adjusted to maintain the appropriate ratio to dna quantity.
in order to look solely at primary yield, trypsin was not added during this rescue and viruses were harvested at day  <dig> post transfection.
this experiment confirmed that in the absence of fusion, adding dna beyond  <dig>  μg per well is detrimental to the rescue, as shown by decreasing yields when adding increasing amounts of the inert plasmid b <dig>  probably due to toxicity or origin competition.
however, adding env-expressing plasmid to pr <dig> rescue plasmids beyond a total of  <dig>  μg of dna per well gave a modest increase in yields, with a maximum for  <dig>  μg of env-expressing plasmid added to  <dig>  μg of pr <dig> rescue plasmids.
discussion
increased rescue efficiency with the env-expressing plasmid
virus rescue has major applications in the rational design of influenza a vaccines
<cit> , which are to date the main control options in use against this disease.
it is also a powerful technique in fundamental research on influenza a, b and c
<cit>  and many other rna viruses
<cit> .
it is useful in this context to enhance the technique and improve our ability to rescue viruses bearing a variety of gene combinations.
this report shows that cell fusion mediated by the maedi-visna virus envelope protein can be an effective tool in overcoming the challenge of having eight different plasmids expressed together in a single cell for the purpose of influenza a virus rescue.
virus rescue yields remain highly variable as shown by yield variability between replicate transfections observed also when using fusion.
however, using fusion increased yields which can help maximize the chances of rescuing some constructs bearing relevant specific gene combinations that make them difficult to rescue.
we showed here that it is possible to use up to  <dig>  μg env-expressing dna together with  <dig>  μg of viral rescue dna in a 24-well plate format to optimize rescue efficiency.
we have found that these conditions improved our ability to rescue some artificially reassorted strains bearing specific gene combinations that we had previously not been able to rescue .
the difference in transfected dna concentration between 24- and 6-well plate formats may explain the apparently better virus yield observed in 24-well plates .
cell fusion also allows virus rescue using only a fraction of the amount of cloned viral genes that would normally be required , a potential advantage when large numbers of different constructs have to be made or where some genes that are challenging to clone are available in limiting quantity.
cell fusion may enhance influenza virus rescue through different mechanisms.
we assumed that one limiting factor was the requirement to successfully deliver eight separate plasmids to the same cell and anticipated that this could be enhanced by using cell fusion.
this is supported by the “4 + 4” and “7 + 1” experiment results  where the complement of plasmids was deliberately incomplete.
it is also supported by experiments where the concentration of viral genes-carrying plasmids was reduced to limiting levels, and where inclusion of the env plasmid restored rescue efficiency .
another hypothesis to account for enhanced yield is that the mvv envelope protein from the host cell might be incorporated into the primary progeny virus envelope and facilitate the first round of infection and hence amplification.
however we believe the latter to be unlikely as in order for viral rnps to uncoat from the matrix protein, they must be exposed to the acid environment of the endosome .
direct membrane fusion mediated via mvv env is expected to release viral cores directly into the cytoplasm without prior acid exposure and thus non-infectious.
other applications of the fusion methodology
in one experiment , we used the trypsin dependence of the pr <dig> virus in cell culture to eliminate the contribution of amplified virus to the overall yield.
firstly, if it is possible to generate sufficient quantities of primary progeny, one can examine the effect of lethal mutations that may otherwise be unable to undergo amplification .
viral progeny can be analysed directly for genome complement and ability to initiate a first round of infection.
our method can generate about  <dig> p.f.u./ml of primary progeny in a 24-well plate format, which could be sufficient for such purposes.
a second application results from the potential offered by fusing cells transfected with different complements of virus segments.
it may be possible to establish partial infections in two cell populations, then allow fusion and assay the completion of the virus life cycle.
this would be a novel tool studying interactions between different segments during the packaging process and trafficking of viral components.
CONCLUSIONS
the simple modification described herein has the potential to improve the efficiency of the virus rescue process and expand the potential application for reverse genetic studies.

 influenza is a contagious disease that represents a serious health threat to humans and other animals worldwide. influenza a viruses, in particular, can infect a variety of species. a global reservoir for these viruses exists in wild waterfowl and shorebirds, from which novel viruses can emerge to infect mammalian species. influenza is therefore a potential threat to humans, pigs, horses, sea mammals, ferrets, mink as well as many terrestrial bird species. in the past decades, several dramatic episodes of large-scale mortality have occurred in domestic birds, humans, and other species. to date, the main approach to control influenza epidemics and pandemics in human and other animal populations is through vaccination and biosecurity, although other approaches may help prevent the transmission of highly pathogenic influenza in some species. strategies for regenerating rna viral genomes from plasmid dna clones have enabled the powerful technique of reverse genetics to be applied to many different rna viruses including, among others, influenza, rabies, coronavirus, rift valley fever virus or fish rna viruses. this technique allows clonal virus stocks with a defined genotype to be engineered. it therefore has major medical applications as it enables vaccines to be rationally designed by inserting attenuating mutations or chosen antigens into a defined viral background, enhancing our control options for pathogens such as influenza virus. in fundamental virus studies, it also enables researchers to test the consequences of defined genetic differences on phenotype, such as growth properties, cell-virus interaction, replication cycle characteristics, pathogenicity, or others - the approach known as “reverse genetics”. various solutions have been developed for satisfying the requirements for the transcribed rnas to become infectious when produced inside a permissive cell. among the most complex and demanding systems is that required for the segmented negative stranded rna viruses such as influenza virus. the basic strategy for the most commonly used influenza virus rescue systems comprises a set of plasmids that each drives the expression of one viral genome-sense transcript corresponding to each of the eight viral segments. accurately tailored 5' and 3' termini are achieved by positioning rna polymerase i transcription initiation and termination sites respectively. because the rna is negative sense, the virus polymerase must be produced before replication can ensue. this can be achieved by co-transfecting a further four plasmids that produce the replication proteins in the so-called “12-plasmid system”. a derivative of this system uses tandem opposing rna pol i and cytomegalovirus promoters to drive expression of viral genome rnas and mrnas respectively encoding all the viral proteins, commonly referred to as the “8-plasmid system”. another development has been the production of vectors carrying multiple genome segment expression cassettes. these strategies can be effective and have proven very useful for a number of purposes. however regenerating field strains and some mutants and reassortants is often less efficient than for well-adapted laboratory strains and it is not uncommon for several attempts to be needed before a particular viral construct is successfully rescued. this suggests the existence of one or more limiting factors in the process and prompted us to test whether some of these could be overcome to increase rescue efficiency. several factors may impact virus rescue efficiency. examples include the replicative ability of a field-derived avian virus in mammalian cells or the balance of the virus’ haemagglutinin and neuraminidase activities. we hypothesized that one limitation might be the ability to successfully transfect and simultaneously express eight different plasmids within an individual cell. we therefore reasoned that cell fusion might maximize the likelihood of all eight influenza gene-bearing plasmids being present in the same polykaryon and thereby enhance rescue efficiency. the maedi-visna virus is a lentivirus causing chronic pneumonia or a progressive demyelinating disease in sheep. its envelop glycoprotein is a major target for virus neutralization and can cause substantial host cell fusion. receptor to this protein has not yet been identified but is known to be expressed on cells from a wide range of species including primates, avians and rodents. we therefore examined co-transfecting an env expression vector along with the eight plasmid rescue system into hek 293t/ cells to determine its effect on virus rescue efficiency.  cell fusion with the maedi-visna virus envelope protein in t additional file and additional file show t cells transfected with μg of a green fluorescent protein-expressing plasmid plus μg of an inert plamid, or μg of the gfp plasmid plus μg of the env-expressing plasmid. cells were kept in fcs-containing medium for the whole duration of the footage. the video shows a marked fusion process, including visible cell-to-cell gfp transfer, in cells transfected with the env plasmid, while cells keep dividing over time. enhanced yields with the maedi-visna virus envelope protein for virus rescues, we initially worked with a fixed total mass of μg of dna per well in a 24-well plate format, which had previously been determined to be the optimal amount of dna for best transfection efficiency in our lab. we varied the proportion of viral genes-containing and env-expressing plasmids comprising the μg total in μg increments. results of this titration for the lab-adapted strain a/puerto rico/8/ are shown in figure transfecting μg of the viral rescue plasmids alone resulted in an average yield of x103 ±  x plaque forming units per millilitre. reducing the amount of viral rescue plasmids by replacing them proportionately with the b control plasmid exhibited an initial plateau but reduced the yield sharply at the μg level. co-transfecting the env plasmid tended to increase the yield at all levels and had a particularly marked effect when the viral rescue plasmids were limiting p.f.u./ml when as little as μg viral rescue plasmids were included; t4 = −, p =  ). increasing the proportion of viral rescue plasmids while decreasing the proportion of env-expressing plasmid gave higher yields until an apparent optimum was reached for μg of env-expressing plasmid and μg of viral rescue plasmids. dispersion of rescue yield values was however generally high and statistical significance was not detected for the differences observed at the μg and μg levels. application to the rescue of a strain derived from a field sample we tested whether using the env-expressing plasmid in the proportions determined above would enhance the practical rescue of a viral strain derived from a low pathogenicity avian influenza field sample. such strains are usually more challenging to rescue than laboratory adapted strains such as pr giving low yields or failing to rescue in all attempts. in this experiment, lipofectamine ltx was used instead of fugene as transfection reagent as the latter had been discontinued by the manufacturer. figure shows the comparison of yields when using 100% of viral rescue plasmids versus using 75% of viral rescue plasmids plus 25% of env-expressing plasmid. this experiment showed an average yield increase of between one and two log in 6-well plates and in 24-well plates when using fusion. a μg total dna mass was used per well in 24-well plates whereas a μg total dna mass was used per well in 6-well plates. in 6-well plates, one out of three replicates where fusion was not used was negative. fusion rescue of incomplete combinations of viral segments to test whether virus could be rescued from cells containing incomplete complements of viral segments by fusing with cells carrying the missing components, we transfected cells in separate batches with partial complements of pr rescue plasmids and then co-cultured them. in an experiment named “4 + 4”, μg of each one of segments and as well as μg of either b or env-expressing plasmids were transfected into one batch of cells, and identical amounts of segments and as well as μg of either b or env-expressing plasmids were transfected into another batch of cells. in another experiment named “7 + 1”, μg of each one of segments and as well as μg of either b or env-expressing plasmids were transfected into one batch of cells, and μg of segment as well as μg of either b or env-expressing plasmids were transfected into another batch of cells. on the day after transfection, media were removed, cells were washed once with pbs and then pooled, and on day treated with trypsin. infectious yields were assessed by plaque formation assay. table shows successful rescue of live virus in all attempts where cells transfected separately with complementary sets of influenza rescue plasmids were allowed to fuse together at day post transfection. no live virus was recovered when the empty b plasmid was used instead of the env-expressing plasmid, except for one μl aliquot from the “7 + 1” experiment which yielded one p.f.u. this table shows the viral yields from cells transfected with the eight pr rescue plasmids split into separate batches at the time of transfection and brought together on the next day. in the "4 + 4" experiment, one batch of cells was transfected with plasmids encoding segments while a complementary batch of cells was transfected with plasmids encoding segments in the "7 + 1" experiment, one batch was transfected with plasmids encoding segments and the complementary batch was transfected with segment the cells from complementary batches were dislodged and brought together at day post transfection. these experiments consisted of triplicate transfections for each condition. in addition to the transfection with influenza plasmids, cells were transfected with either the env-expressing or the empty b plasmid. addition of extra env-expressing plasmid we tested whether adding env-expressing plasmid beyond a total amount of dna of μg per well in a 24-well plate would be detrimental or beneficial to the rescue. figure shows the result of a titration where a fixed amount of μg of pr rescue plasmids was used in addition to which env-expressing plasmid was added in quantities varying from μg to μg. the total amount of dna transfected was therefore μg to μg per well. transfection reagent was adjusted to maintain the appropriate ratio to dna quantity. in order to look solely at primary yield, trypsin was not added during this rescue and viruses were harvested at day post transfection. this experiment confirmed that in the absence of fusion, adding dna beyond μg per well is detrimental to the rescue, as shown by decreasing yields when adding increasing amounts of the inert plasmid b probably due to toxicity or origin competition. however, adding env-expressing plasmid to pr rescue plasmids beyond a total of μg of dna per well gave a modest increase in yields, with a maximum for μg of env-expressing plasmid added to μg of pr rescue plasmids. discussion increased rescue efficiency with the env-expressing plasmid virus rescue has major applications in the rational design of influenza a vaccines, which are to date the main control options in use against this disease. it is also a powerful technique in fundamental research on influenza a, b and c and many other rna viruses. it is useful in this context to enhance the technique and improve our ability to rescue viruses bearing a variety of gene combinations. this report shows that cell fusion mediated by the maedi-visna virus envelope protein can be an effective tool in overcoming the challenge of having eight different plasmids expressed together in a single cell for the purpose of influenza a virus rescue. virus rescue yields remain highly variable as shown by yield variability between replicate transfections observed also when using fusion. however, using fusion increased yields which can help maximize the chances of rescuing some constructs bearing relevant specific gene combinations that make them difficult to rescue. we showed here that it is possible to use up to μg env-expressing dna together with μg of viral rescue dna in a 24-well plate format to optimize rescue efficiency. we have found that these conditions improved our ability to rescue some artificially reassorted strains bearing specific gene combinations that we had previously not been able to rescue. the difference in transfected dna concentration between 24- and 6-well plate formats may explain the apparently better virus yield observed in 24-well plates. cell fusion also allows virus rescue using only a fraction of the amount of cloned viral genes that would normally be required, a potential advantage when large numbers of different constructs have to be made or where some genes that are challenging to clone are available in limiting quantity. cell fusion may enhance influenza virus rescue through different mechanisms. we assumed that one limiting factor was the requirement to successfully deliver eight separate plasmids to the same cell and anticipated that this could be enhanced by using cell fusion. this is supported by the “4 + 4” and “7 + 1” experiment results where the complement of plasmids was deliberately incomplete. it is also supported by experiments where the concentration of viral genes-carrying plasmids was reduced to limiting levels, and where inclusion of the env plasmid restored rescue efficiency. another hypothesis to account for enhanced yield is that the mvv envelope protein from the host cell might be incorporated into the primary progeny virus envelope and facilitate the first round of infection and hence amplification. however we believe the latter to be unlikely as in order for viral rnps to uncoat from the matrix protein, they must be exposed to the acid environment of the endosome. direct membrane fusion mediated via mvv env is expected to release viral cores directly into the cytoplasm without prior acid exposure and thus non-infectious. other applications of the fusion methodology in one experiment, we used the trypsin dependence of the pr virus in cell culture to eliminate the contribution of amplified virus to the overall yield. firstly, if it is possible to generate sufficient quantities of primary progeny, one can examine the effect of lethal mutations that may otherwise be unable to undergo amplification. viral progeny can be analysed directly for genome complement and ability to initiate a first round of infection. our method can generate about p.f.u./ml of primary progeny in a 24-well plate format, which could be sufficient for such purposes. a second application results from the potential offered by fusing cells transfected with different complements of virus segments. it may be possible to establish partial infections in two cell populations, then allow fusion and assay the completion of the virus life cycle. this would be a novel tool studying interactions between different segments during the packaging process and trafficking of viral components.  the simple modification described herein has the potential to improve the efficiency of the virus rescue process and expand the potential application for reverse genetic studies. 
the simple modification described here has the potential to improve the efficiency of the virus rescue process and expand the potential applications for reverse genetic studies. we investigated the possibility of enhancing influenza virus rescue by eliciting cell fusion to increase the chances of having all necessary plasmids expressed within the same polykaryon. co-transfecting cells with the eight plasmids necessary to rescue influenza virus plus a plasmid expressing the maedi-visna virus envelope protein resulted in increased rescue efficiency. to this end we used the maedi-visna virus envelope protein which has potent fusion activity in cells from a wide range of different species. virus rescue from transfected cells is an extremely useful technique that allows defined viral clones to be engineered for the purpose of rational vaccine design or fundamental reverse genetics studies. in addition, partial complements of the 8-plasmid rescue system could be transfected into two separate populations of cells, which upon fusion led to live virus rescue.
 virus rescue from transfected cells is an extremely useful technique that allows defined viral clones to be engineered for the purpose of rational vaccine design or fundamental reverse genetics studies. however, it is often hindered by low primary rescue success rates or yields, especially with field-derived viral strains. approach we investigated the possibility of enhancing influenza virus rescue by eliciting cell fusion to increase the chances of having all necessary plasmids expressed within the same polykaryon. to this end we used the maedi-visna virus envelope protein which has potent fusion activity in cells from a wide range of different species.  co-transfecting cells with the eight plasmids necessary to rescue influenza virus plus a plasmid expressing the maedi-visna virus envelope protein resulted in increased rescue efficiency. in addition, partial complements of the 8-plasmid rescue system could be transfected into two separate populations of cells, which upon fusion led to live virus rescue.  the simple modification described here has the potential to improve the efficiency of the virus rescue process and expand the potential applications for reverse genetic studies. influenzavirus rescuecell fusionreverse genetics 