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. 2016 Apr 5:6:23887.
doi: 10.1038/srep23887.

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

T Nolden  1 , F Pfaff  2 , S Nemitz  1 , C M Freuling  1 , D Höper  2 , T Müller  1 , Stefan Finke  1
Affiliations

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

T Nolden et al. Sci Rep. .

Abstract

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

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Figures

Figure 1
Figure 1. Comparison of Linear-to-circular (LCHR) with Linear-to-linear (LLHR) homologous recombination and adapted LLHR for RABV reverse genetics.
While LCHR (A) depends on active replication, LLHR (B) uses a different mechanism and can be adapted to RABV reverse genetics vector cloning (C). The linear vector codes for an ampicillin resistance gene (ampR) and contains an origin of replication for plasmid propagation. Additionally, the minimal linear vector contains T7 and CMV promotors (in blue) and a T7 termination signal (in orange) for transcription termination and polyadenylation. Ribozyme sequences flank the RABV virus cDNA after LLHR recombination (Hammerhead ribozyme, Ha; Hepatitis δ ribozyme, Hd). Homologous sequences required for recombination between target molecules and vector sequences are specified by red and green arrows.
Figure 2
Figure 2. LLHR of virus genomic cDNA and pHaHdmin linear vector DNA.
Schematic presentation of the performed LLHR experiment. Linear full length genome PCR products and PCR-amplified linear vector DNA were electroporated into into arabinose induce or non-induced E. coli Gb05-Dir. (A). Agarose gels showing full genome PCR products, gel purified 12 kb PCR products and PCR-amplified linear minimal cloning vector pHaHdmin (B). The effectiveness of LLHR in the presence of arabinose was quantified by colony counting after selection on ampicillin containing LB-plates (LB amp+) (C). The mean of absolute colony numbers from three independent recombination experiments are shown with indicated standard deviation by error bars (D).
Figure 3
Figure 3. SacI digest of ampicillin resistant recombinants revealed that LLHR of full length RABV cDNAs occurs at high frequency.
(A) Exemplary restriction pattern of pSADL16, pRABV-Dog and pRABV-Fox full length cDNA plasmids after SacI digest. A virus cDNA clone was considered positive, when the sum of all fragment sizes corresponded to the expected 14.5 kb plasmid (11.9 kb virus genome plus 2.7 kb vector fragment). (B) Correctly recombined full length RABV genomes were averaged from two independent LLHR experiments and expressed as percentage of analyzed recombinants.
Figure 4
Figure 4. Red fluorescent protein expression from recombinant dog and fox viruses.
(A) Genome organization recombinant viruses. The red fluorescent Katushka reporter gene was inserted between the G and L genes. (B) Red fluorescence in virus infected cells and indirect immunofluorescences against RABV N (green) in N42/13 cells at 48 hrs post infection. Left: rRABV-Dog-Katushka (top) and rRABV-Fox-Katushka (bottom). Right: rRABV-Dog clone 14 (top) and rRABV-Fox clone 9 (bottom). Shown are maximum- projections of confocal z-stacks.
Figure 5
Figure 5. Growth kinetics of recombinant RABVs and wild type field isolates from fox and dog.
N42/13 neuroblastoma and BSR T7/5 cells were infected with a MOI of 0.01 ffu/ml and samples were taken at 0, 16, 24, 48, 72 and 96 hours post infection (hpi) and infectious virus titers were determined on N42/13 cells. (A) wt RABV-Dog, rRABV-dog clone 14 and rRABV-Dog Katushka on N42/13 neuroblastoma cells. (B) wt RABV-Dog, rRABV-dog clone 14 and rRABV-Dog Katushka on BSR T7/5 baby hamster kidney cells. (C) wt Fox-Dog, rRABV-fox clone 9 and rRABV-Fox Katushka on N42/13 neuroblastoma cells. (D) wt Fox-Dog, rRABV-fox clone 9 and rRABV-Fox Katushka on BSR T7/5 baby hamster kidney cells. Error bars indicate min/max values from two indepentend experiments.
Figure 6
Figure 6. Virulence of progenitor fox and dog virus isolates and selected recombinant clones rRABV-dog clone 14 and rRABV-fox clone 9 after intracranial (i.c.) and intramuscular (i.m.) inoculation of 3 to 4 week old BALB/c mice.
Groups of 5 animals were inoculated with the indicated viruses either by the i.c. (50 ffu/animal) or by the i.m. (5000 or 50 ffu/animal) route of infection. Survival of infected mice was monitored daily. (A) Survival of mice after i.c. inoculation with wt rRABV-Fox virus and recombinant rRABV-Fox clone 9. (B) Survival of mice after i.m. inoculation with wt rRABV-Fox virus and recombinant rRABV-Fox clone 9 at doses of 5000 ffu/animal (blue lines) and 50 ffu/animal (grey/black lines). (C) Survival of mice after i.c. inoculation with wt rRABV-Fox virus and recombinant rRABV-Fox clone 9. (D) Survival of mice after i.m. inoculation with wt rRABV-Fox virus and recombinant rRABV-Fox clone 9 at doses of 5000 ffu/animal (blue lines) and 50 ffu/animal (grey/black lines).
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