Resultados

Recovery of several NSVs from cDNA clones has been reported (Table1.3). The first attempt towards rescue of NSVs was achieved by Luytjes et al. (1989), using a plasmid encoding influenza A minigenome (CAT gene negative-sense, flanked by influenza A UTRs) which can be linearized and transcribed in vitro. Combination of purified influenza A virus NP, PB1, PB2 and PA proteins with minigenome transcripts enables the formation of RNPs invitro. This complex was then transfected into cells pre-infected with helper virus. Minigenome RNA was thus replicated and incorporated into some of the new progeny virions (Figure 1.14). However, it was not sustained in the genome beyond three passages. This obstacle was overcome using selective pressures, which enabled the isolation of recombinant viruses from helper viruses. Selection methods used include: neutralizing antibody selection (Enami and Palese, 1991; Horimoto and Kawaoka, 1994), host-range restriction (Enami et al., 1990; Subbarao et al., 1993), drug resistance (Castrucci and Kawaoko, 1995) and temperature sensitivity (Enami et al., 1991; Li et al., 1995; Yasuda et al., 1994).These methods allowed the generation of influenza viruses carrying a single cDNA-derived genome segment. Neumann et al. (1994) recovered influenza A viral RNPs using an RNA polymerase I expression system. Virus-like RNA (CAT gene negative sense flanked by viral untranslated region) was cloned into a plasmid contains RNA pol I promoter and terminator. Transfection of the latter plasmid into mammalian cells lead to generation of minigenome in the nucleus. Cells were then infected with helper virus to allow the encapsidation, transcription and packaging of the minigenome.

Further improvement was reported when the minigenome RNA was produced intracelluarly by using a T7 RNA polymerase–driven expression system. Importantly, the recombinant RNA must contain the authenic viral 3’ end. This was achieved by using hepatitis δ ribozyme which allows self-cleavage at the ribozyme sequence, producing authentic viral 3’ ends (Pattnaiket al., 1992).

Bridgen and Elliott (1996) reported the first segmented negative sense RNA virus to be rescued entirely from cDNA. In this system, HeLa cells were first infected with recombinant vaccinia virus vTF7-3 to express T7 RNA polymerase. Three viral

protein expression plasmids under control of T7 promoter were then transfected, followed by a second transfection of three ribozyme-based plasmids encoding the antigenome RNAs. In the latter constructs, BUNV sequences were flanked by the T7 promoter at the 5’ end and a hepatitisδribozyme followed by the T7 terminator at the 3’ end. Recoverd BUNV was purified by passaging the supernatant on C6/36Aedes albopictus cells allowing BUNV growth but not that of vaccinia virus (Figure1.15). However, the yield was very poor with transfection of 107cells only producing 10-100 infectious virus particles.

Recently, an improved and highly efficient BUNV rescue system has been developed (Lowen et al., 2004). BSR-T7/5 cells constitutively expressing T7 polymerase were transfected with three ribozyme-based plasmids expressing antigenome L, M and S RNAs using DAC-30 (transfection reagent) for 5 h at 37˚C. Fresh medium (4ml) was added and cells were incubated 4-5 days at 37˚C and supernatant was then used in plaque assay (Figure1.16).

Using a different protocol from that of BUNV rescue system, the recovery of influenza virus from cDNA has been reported (Fodor et al., 1999; Neumann et al., 1999). In this system, RNA Pol I promoter and terminator sequences were used for synthesis of viral RNAs while RNA Pol II promoters were also used for producing viral mRNA. Moreover, genome expression plasmids were used instead of using antigenome expression plasmids (Figure1.17). There are two reasons to explain the success of this system. First, lack of processing of Pol I transcripts and secondly, influenza virus replication takes place in the nucleus so no hybridization between the mRNAs and the viral genome would occur. In the latter system, influenza virus was recovered from transfection of either 12 or 17 plasmids. Cells were cotransfected with eight Pol I based plasmids encoding the genome segments and either four or nine protein expression plasmids. The yields was significantly higher (105 pfu/ml) when 17 plasmids were used compared to 103 pfu/ml obtained with 12 plasmids. Moreover, Hoffmannet al. (2000a) simplified the system by using Pol I/Pol II expression system which enables recovery of influenza virus from only eight plasmids. To produce a cDNA which can be used for transcription of both negative sense genomic RNA and mRNA, viral coding sequence was cloned in the positive sense between a pol II

Table 1.3.Negative-strand RNA viruses recovered from cDNA clones (adapted from Neumannet al., 2002).

Family Genus Species Abbreviation Reference

Vesiculovirus Vesicular stomatitis virus VSV Lawsonet al. (1995) Whelanet al. (1995) Rhabdoviridae

Lyssavirus Rabies virus RV Schnellet al. (1994)

Henipavirus Nipah virus NiV Yonedaet al., 2006

Measles virus MV Radeckeet al. (1995)

Rinderpest virus

RPV

Baron & Barrett (1997)

Morbillivirus

Canine distemper

virus CDV Gassenetal.(2000)

Sendai virus SeV

Gassenet al. (1995) Katoet al.(1996) Human parainfluenza virus type 3 hPIV3 Durbinet al. (1997a)

Hoffman & Baneriee (1997) Respirovirus Bovine parainfluenza virus type 3 bPIV3 Halleret al. (2000)

Simian virus type 5

SV5

Heet al(1997)

Mumps virus Clarkeet al. (2000)

Human parainfluenza virus type 2 hPIV2 Kawanoet al. (2001) Rubulavirus Newcastle disease virus NDV Peeterset al.(1999) Romer-Oberdorferet al. (1999) Krishnamurthyet al. (2000) Human respiratory

syncytial virus hRSV Collinset al. (1999)

Paramyxoviridae

Peneumovirus

Bovine respiratory

syncytial virus bRSV Buchholzet al. (1999)

Filoviridae

Ebola-like viruses

Ebola virus EBoV

Volchkovet al. (2000)

Neumannet al. (2002)

Orthobunyavirus Bunyamwera,

LaCrosse BUNV, LACV

Bridgen & Elliott (1996) Blakqori & Weber (2005)

Bunyaviridae

Phlebovirus

Rift valley fever

RVFV Ikegamiet al(2006)

Influenzavirus A Influenza A virus Neumannet al. (1999)

Fodoret al.(1999)

Orthomyxoviridae

RNP Mini-genome RNA N PA PB1 PB2 Helper virus

Figure 1.14.Influenza A virus RNPin vitro transfection method for recovery of

viable virus. Influenza mini-genome RNA is transcribedin vitro. The influenza- like RNA is then combined with purified, virion-derived, NP, PB1, PB2 and PA proteins to allow RNP formation. The latter is transfected into cells pre-infected with helper virus. Some of the off-spring contain the heterologous segment (adapted from Luytjeset al., 1989).

N Protein expression plasmids Antigenome expression plasmids vTF7-3 T7 Hela L L M S C6/36 T7 promoter

Hepatitis & ribozyme T7 terminator

δ

Figure 1.15.Bunyamwera virus rescue system.

HeLa cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase followed by transfection of three supported plasmids encoding the viral L, Gn, Gc and N proteins. Second transfection includes three ribozyme- based plasmids encoding the antigenome RNAs supernatant from transfected cells. This was passaged onto C6/36 mosquito cells to isolate BUNV (adapted from Bridgen and Elliott, 1996).

N L Golgi L M S T7 promoter

Hepatitis & ribozyme T7 teminator BSRT7 Antigenome expression Plasmids T7 Promoter Hepatitisδribozyme T7 terminator BSR-T7/5 cells

Figure 1.16.BUNV rescue system.

BSR-T7/5 cells which stably express T7 polymerase, are cotransfected with antigenomic expressing plasmids result in recovery of a viable virus.

12 Protein expression plasmids Under control pol II promoters 8 Genome expressing plasmids

Under control pol I promoters

PB1 PB2 PA Hemagglutinin Nucleocapsid Neuraminidase Matrix/M2

Nuclear export protein and M2

Figure 1.17.Influenza A virus rescue system utilizing 12 or 17 plasmids.

Cells are transfected with eight genome expressing plasmids followed by transfection of cells with either four (RNP) proteins or all nine structural proteins (adapted from Neumannet al., 1999).

promoter and polyadenylation signal and the latter cassette was then inserted in a negative sense between Pol I promoter and terminator sequences.

1.17. Rescue of non-segmented negative strand RNA