Rescue of Influenza C Virus from Recombinant DNA

Abstract

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3 and 5 noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10 3 and 10 4 PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.

A seven plasmid-based system for the rescue of influenza C virus

“…Regarding the viral 5'ends, the first twelve nucleotides as well as nucleotide 15' are conserved among the segments, with exception of the NS segment, where nucleotide 6' differs from the others (see Table 1). Concerning the two variable positions 13' and 14', PB2 and PB1 harbour an A at position 13', while the other segments have a G, and NP has an A at position 14', where all other segments have a G. Our results coincide with recent sequence analyses from strain C/JHB/1/66 (Crescenzo-Chaigne and van der Werf, 2007) and strain C/AnnArbor/1/50 (Muraki et al, 2007). In all segments, nucleotides 10-14 are complementary to nucleotides 11'-15', which supports the idea that influenza C virus RNAs form a double-stranded structure of five base pairs at this region (Crescenzo-Chaigne and van der Werf, 2001).…”

“…1), as initially established by Hoffmann et al (2000). We decided to transfect our plasmids into Vero cells instead of 293T cells, which were used to produce recombinant influenza C viruses by two other groups (Crescenzo-Chaigne and van der Werf, 2007; Muraki et al, 2007), as we observed that Vero cells promote growth of influenza C viruses and are well transfectable. Although they cannot compete with 293T cells in transfection efficiency, 293T cells on the other hand are very sensitive to trypsin and therefore hard to handle.…”

“…In comparison, Muraki et al (2007) recovered recombinant influenza C viruses with maximum titres of 2,5 X 10 3 EID 50 /ml by transfection of 11 to 16 plasmids into 293T cells and inoculation of transfection supernatants after 48 to 60 hours in chicken eggs. Crescenzo-Chaigne and van der Werf (2007) transfected 11 plasmids into 293T cells and measured 10 3 to 10 4 p.f.u./ml eight to ten days post transfection. Transferring the supernatants into chicken eggs led to titres of 10 9 p.f.u./ml, whereas further growth on SK 93/2 cells yielded 10 5 to 10 6 p.f.u./ml.…”

“…Subsequently, bidirectional vector systems were established for influenza A and B viruses to restrict the number of plasmids to be transfected to eight: Full-length cDNAs were inserted between RNA polymerase I promoter and terminator as well as RNA polymerase II promoter and termination signal in opposite directions to yield all eight vRNAs and all viral mRNAs simultaneously (reviewed in: Palese and Shaw, 2007). Since then, two groups have developed a system for the generation of recombinant influenza C viruses (Crescenzo-Chaigne and van der Werf, 2007; Muraki et al, 2007), using seven plasmids for production of vRNAs plus protein expression plasmids. Here, we describe the first reverse-genetics system for influenza C virus, which is based on tranfection of only seven bidirectional plasmids.…”

A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF

“…Methods to rescue all three types of influenza viruses, A, B, and C, have been successfully established (5,8,14,15,24,27). Reverse genetics has been utilized to study the influenza viral replication cycle, to characterize the function of influenza viral proteins, and to understand the high pathogenicity of the 1918 and H5N1 viruses (4,11,16,39,44).…”