Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo

Abstract

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

FIG. 1.

Assembly of full-length MHV-A59 cDNA by in vitro ligation. (A) The structural relationship of the MHV-A59 genome, MHV-A59-derived cDNA fragments, and full-length cDNA is shown. Four cDNA fragments (F1 to F4), derived from p5′Sac (F1), RT-PCR from MHV-A59 RNA (F2), pSap2ex (F3), and pMHe-link (F4), are assembled by in vitro ligation using appropriate restriction sites as indicated. Also shown is the position of the bacteriophage T7 RNA polymerase promoter (prom.) sequence in fragment F1. The full-length MHV-A59 cDNA is inserted into the vaccinia virus vNotI/tk genome by ligation of full-length MHV-A59 cDNA containing EagI-cleaved DNA ends with NotI-cleaved vNotI/tk genomic DNA (VV). The positions of nucleotide changes introduced by RT-PCR fragment F2 are indicated (*). (B) Pulsed-field gel electrophoresis analysis of the ligation reaction mixture containing fragments F1 to F4. Reaction products corresponding to the full-length MHV-A59 cDNA fragment, intermediate reaction products, and inserted cDNA fragments are indicated.

FIG. 2.

Repair of cloned full-length MHV-A59 cDNA. The positions of nucleotide changes within the cloned MHV-A59 cDNA of recombinant vaccinia virus (VV) vMHV-E6 are shown (*). The sequence corresponding to MHV-A59 nucleotides 4779 to 15035 containing these changes has been replaced by the MHV-A59 consensus sequence using four rounds of vaccinia virus-mediated homologous recombination with gpt-positive or -negative selection conditions as indicated. Also shown are plasmid DNAs, RT-PCR products, and in vitro ligation products that were used for homologous recombination. The numbers depicted on DNA fragments correspond to the positions of the MHV-A59 genome sequence in kilobases.

FIG. 3.

Analysis of MHV replication in tissue culture. Plaque morphology (A), growth curves (B), and analysis of intracellular [³H]uridine-labeled RNA of parental MHV-A59 and recombinant MHV-inf-1 (C) are shown. p.i., postinfection.

FIG. 4.

Virus replication in mice. Shown are viral titers in the brains and livers of 4-week-old C57BL/6 mice infected with either parental MHV-A59 or recombinant MHV-inf-1. Viral titers were examined by plaque assays on L2 cells. Each time point represents the average titer of the organs of two mice. p.i., postinfection.

FIG. 5.

Histopathology of mouse brain, spinal cord, and liver following infection with recombinant MHV-inf-1. Brain sections (A to C) at 7 days postinfection show acute encephalitis with microglial nodules (A), perivascular mononuclear cellular infiltration in brain parenchyma (B) and meninges, and microglial proliferation (C). (D) Liver sections at 5 days postinfection show numerous foci of necrosis and inflammation throughout the liver parenchyma. (E) Sections of spinal cord at 30 days postinfection show multiple demyelinating lesions (arrow) in the white matter. Panels A to C are HE stained at ×400 magnification; plate D is an HE stain at ×200 magnification; panel E is a Luxol fast blue stain for myelin at ×50 magnification.