Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins

Abstract

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

FIG. 1.

Generation of full-length AMPV/CO plasmid. Full-length AMPV/CO cDNA was assembled in pBR 322 from five subgenomic cDNA fragments that were generated by high-fidelity RT-PCR. The fragments were inserted in between the T7 RNA polymerase promoter sequence and the hepatitis delta ribozyme autocatalytic sequence, which was followed by the T7 terminator sequence. The names of the restriction enzymes used for the assembly are shown at the top, and the order in which the fragments were assembled is shown on each of them. The XhoI, PvuI, and MluI sites were introduced to facilitate construction and serve as markers (sequence changes shown in lowercase and underlined).

FIG. 2.

Construction of plasmid pAMPV/CO-GFP expressing the complete antigenomic RNA of AMPV/CO and GFP as a foreign gene. GFP was inserted in the putative 5′ noncoding region of the G gene as a separate transcription cassette. The cassette contained the GFP ORF (gray rectangle) that was flanked on the upstream side by an MluI site (underlined), the G gene end motif (italicized), an intergenic region comprised of two C residues, and the N gene start motif (bold). The GFP ORF was flanked on the downstream side by an MluI site. The translational initiation and termination codons of the GFP ORF are in bold and underlined. le, leader; tr, trailer.

FIG. 3.

Replication kinetics of wild-type AMPV/CO, rAMPV/CO, and rAMPV/CO-GFP. Vero cells were infected at a multiplicity of infection of 0.01 with wild-type AMPV/CO (♦), rAMPV/CO (▪), or rAMPV/CO-GFP (▴). Supernatants (0.2 ml out of a total volume of 3 ml per well) were taken at the indicated time points postinoculation and replaced by an equivalent amount of fresh medium. The samples were flash frozen and analyzed later for virus titers by plaque assay and immunostaining. Each time point was represented by two wells, and each titration was performed in duplicate. The mean virus titers are shown. Bars indicate standard deviations.

FIG. 4.

(A) Cytopathic effect of recombinant AMPV/CO in Vero cells. Vero cells were either infected with wild-type (wt) AMPV/CO, rAMPV/CO, or rAMPV/CO-GFP or mock infected with phosphate-buffered saline and observed for the appearance of CPE characteristic of AMPV. The recovered recombinant viruses induced syncytia indistinguishable from those of wild-type AMPV/CO, at 72 h postinfection and at an MOI of 0.1. (B) Immunostaining of AMPV/CO-infected Vero cells. Vero cells were either infected with 0.1 MOI of wt AMPV/CO, rAMPV/CO, or rAMPV/CO-GFP or mock infected with phosphate-buffered saline, and the infected monolayer was grown in 2% Eagle's minimal essential medium and 0.8% methyl cellulose overlay. After 72 h of infection, the overlay was removed and the monolayers were subjected to immunoperoxidase staining. An antipeptide antibody raised against the N protein of AMPV/CO was utilized for the staining. The recovered recombinant virus-induced plaques were indistinguishable in morphology as well as staining characteristics from those induced by wild-type AMPV/CO.