Synthetic Reconstruction of Zoonotic and Early Human Severe Acute Respiratory Syndrome Coronavirus Isolates That Produce Fatal Disease in Aged Mice

Abstract

The severe acute respiratory syndrome (SARS) epidemic was characterized by high mortality rates in the elderly. The molecular mechanisms that govern enhanced susceptibility of elderly populations are not known, and robust animal models are needed that recapitulate the increased pathogenic phenotype noted with increasing age. Using synthetic biology and reverse genetics, we describe the construction of a panel of isogenic SARS coronavirus (SARS-CoV) strains bearing variant spike glycoproteins that are representative of zoonotic strains found in palm civets and raccoon dogs, as well as isolates spanning the early, middle, and late phases of the SARS-CoV epidemic. The recombinant viruses replicated efficiently in cell culture and demonstrated variable sensitivities to neutralization with antibodies. The human but not the zoonotic variants replicated efficiently in human airway epithelial cultures, supporting earlier hypotheses that zoonotic isolates are less pathogenic in humans but can evolve into highly pathogenic strains. All viruses replicated efficiently, but none produced clinical disease or death in young animals. In contrast, severe clinical disease, diffuse alveolar damage, hyaline membrane formation, alveolitis, and death were noted in 12-month-old mice inoculated with the palm civet HC/SZ/61/03 strain or early-human-phase GZ02 variants but not with related middle-and late-phase epidemic or raccoon dog strains. This panel of SARS-CoV recombinants bearing zoonotic and human epidemic spike glycoproteins will provide heterologous challenge models for testing vaccine efficacy against zoonotic reintroductions as well as provide the appropriate model system for elucidating the complex virus-host interactions that contribute to more-severe and fatal SARS-CoV disease and acute respiratory distress in the elderly.

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

“…Perhaps this highlights an important intrinsic difference in the species response to virus infection. In future, a heterologous challenge with different SARS-CoV strains is also an important consideration (Rockx et al, 2007). In conclusion, this report adds to others (ter Meulen et al, 2004) that have suggested that a protective SARS vaccine should be possible; however, protection in ferrets (and humans) may be more difficult than the mouse models suggest.…”

“…The presence of defective interfering particles common in CoVs might have attenuated the infection in other published challenge studies. Alternatively, the ferrets used in this study were older, and age has been shown to be an important susceptibility factor in the mouse model (Rockx et al, 2007). No other animal, including cynomolgus macaques, has been reported to experience fever, which is the most common sign in human SARS-CoV infection (above 99 %) (Skowronski et al, 2005;Lawler et al, 2006).…”

“…Furthermore, the WKV vaccine was more potent than the Ad S/N vaccine in reducing viral replication and in inducing neutralizing antibodies (See et al, 2006). One limitation of mouse models is that they do not reproduce the clinical signs and severe disease of SARS in humans (Glass et al, 2004;Subbarao et al, 2004) unless aged mice are used (Rockx et al, 2007). Ferrets have been used widely for the study of influenza and are susceptible to SARS-CoV infection, exhibiting lung pathology and virus shedding (Martina et al, 2003;ter Meulen et al, 2004).…”

Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice

“…Similarly, the segmented genome of the 1918 strain of influenza was reconstructed, in part, by using synthetic design (39). Human endogenous retroviruses were assembled by PCR-directed assembly of synthetic oligonucleotides into a consensus provirus (40), and recombinant SARS-CoVs bearing synthetic zoonotic Spike sequences were derived by our group (22,32). Last, the cloning of the 580-kb microbial genome of Mycoplasma genitalium was reported, although this work has not yet yielded a replicating organism (41).…”

“…We have previously identified zoonotic SARS-CoV variants that replicated efficiently in Vero cells, but not in HAE cultures (22,32). Therefore, HAE cultures provide a more relevant and stringent measure of the replicative potential of chimeric recombinant zoonotic SARS-CoV viruses in the human host.…”