Muth, Doreen: Assessment of virulence factors of a reservoir-borne SARS-related coronavirus by reverse genetics. - Bonn, 2013. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Doreen Muth}},
title = {Assessment of virulence factors of a reservoir-borne SARS-related coronavirus by reverse genetics},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = jan,

note = {The pandemic of the „Severe acute respiratory syndrome“ (SARS) at the end of 2002 caused more than 8000 infections and led to more than 800 deaths. The disease was soon associated with a new zoonotic coronavirus, the SARS-CoV. At first, civet cats were thought to be the natural reservoir of SARS-CoV, but further studies revealed that SARS-related CoVs are present at high diversities in Asian bats of the genus Rhinolophus. Emerging viruses are a constant threat to public health and it is of utmost importance to evaluate risk factors that facilitate host transitions.
SARS-CoV encodes several accessory proteins with partially known functions in immune evasion. Minor relevance was assigned to those proteins in cell culture models, but it is generally assumed that they might have important functions in the natural reservoir.
Using the full-length genome sequence of a Bulgarian SARS-related bat-CoV (BG-CoV) identified by us in Rhinolophus blasii functionalities of two selected accessory proteins were investigated.
To this end, a Rhinolophus bat embryonic lung cell line carrying the human SARS-CoV receptor angiotensin converting enzyme 2 (hACE2) was established (RhiLu-hACE2) to resemble the SARS-CoV reservoir. Type I interferon assays were established for cells from different hosts and extensive reverse genetic studies on SARS-CoV were conducted to project the zoonotic risk of the bat-borne SARS-related CoV.
(i) Protein 6, encoded by open reading frame 6 (ORF6), interacts directly with Karyopherin (KPN) α2 thereby inhibiting interferon (IFN) signaling. BG-CoV protein 6 (BG-p6) shares 78.1% amino acid similarity with human SARS-CoV protein 6 (SA-p6). Overexpressed BG-p6 was able to inhibit nuclear translocation of co-transfected KPN α2 like SA-p6. Moreover, the IFN antagonistic ability of both proteins was determined by the inhibition of nuclear translocation of overexpressed STAT1 due to the effect described above. It was shown that overexpressed BG-p6 inhibited STAT1 translocation as efficiently as SA-p6.
With the help of the SARS-CoV reverse genetics system a chimeric recombinant SARS-CoV carrying the BG-CoV ORF6 (BGO6-rSCV) was generated for studies in a in the context of viral replication in primate and RhiLu-hACE2 cells. In primate as well as in RhiLu-hACE2 cells BGO6-rSCV replicated less efficiently than wild type rSCV. Interestingly, when cells were preincubated with universal type I IFN BGO6-rSCV exhibited an increased IFN sensitivity on RhiLu-hACE2 cells only. This suggests a host-independent attenuating effect of BG-p6. (ii) The genomic region around ORF8 was subject to an unusual high mutation rate throughout the SARS pandemic. SARS-related CoVs found in Asian Rhinolophus bats, civet cats and early human isolates of SARS-CoV carried a single full-length ORF8. The human pandemic SARS-CoV acquired a 29 nucleotide deletion leading to the disruption of ORF8 into two ORFs, ORF8a and 8b. In the Bulgarian SARS-related bat-CoV we found that ORF8 was entirely missing. Analysis of more Rhinolophid bats from Spain, Italy and Slovenia revealed that most likely all European SARS-related bat-CoVs lack ORF8.
The influence of ORF8 integrity on virus replication was investigated with the help of rSCVs carrying these three ORF8 variants. On primate and bat cell culture rSCV carrying the full-length ORF8 grew to the highest titers, while the ORF8 deletion variant grew the worst. IFN pretreatment of the tested cell lines resulted in an overall decrease in virus replication of all ORF8 variants suggesting an IFN-independent mechanism for ORF8 influenced virus replication.
In summary, the present study showed that BG-p6 was able to antagonize type I IFN signaling in primate cells with wild type efficiency. In the full virus context a SARS-CoV expressing BG-p6 was attenuated in primate and bat cell culture. Furthermore, the absence of ORF8 greatly reduced virus replication efficiency. Taken together both findings suggest potentially reduced virulence of the European SARS-related bat-CoVs. For the first time these data indicate a feasible approach to assessing zoonotic risks emanating from bat-borne SARS-related CoVs.},

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