Covid reseach

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https://doi.org/10.1371/

Evolution analysis

Full-length genome sequences of the 15 SARSr-CoVs detected from bats in the cave surveyed

in this study were aligned with those of selected SARS-CoVs using MUSCLE [40]. The aligned

sequences were scanned for recombination events by Recombination Detection Program

(RDP) [41]. The potential recombination events suggested by strong P values (<10−20) were

further confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1

[42]. Phylogenetic trees based on nucleotide sequences were constructed using the Maximum

Likelihood algorithm under the LG model with bootstrap values determined by 1000 replicates

in the PhyML (version 3.0) software package [43].

Virus isolation

The Vero E6 cell line was kindly provided by Australian Animal Health Laboratory, CSIRO

(Geelong, Australia). Vero E6 monolayer was maintained in DMEM medium supplemented

with 10% fetal calf serum (FCS). Fecal samples (in 200 μl buffer) were gradient centrifuged at

3,000–12,000 g, and the supernatant was diluted 1:10 in DMEM before being added to Vero

E6 cells. After incubation at 37˚C for 1 h, the inoculum was removed and replaced with fresh

DMEM medium with 2% FCS. The cells were incubated at 37˚C and checked daily for cyto-

pathic effect. All tissue culture media were supplemented with triple antibiotics penicillin/

streptomycin/amphotericin (Gibco) (penicillin 200 IU/ml, streptomycin 0.2 mg/ml, ampho-

tericin 0.5 μg/ml). Three blind passages were carried out for each sample. After each passage,

both the culture supernatant and cell pellet were examined for presence of SARSr-CoV by

RT-PCR using specific primers targeting the RdRp or S gene. The viruses which caused obvi-

ous cytopathic effect and could be detected in three blind passages by RT-PCR were further

confirmed by electron microscopy.

Construction of recombinant viruses

Recombinant viruses with the S gene of the novel bat SARSr-CoVs and the backbone of the

infectious clone of SARSr-CoV WIV1 were constructed using the reverse genetic system

described previously [23] (S9 Fig). The fragments E and F were re-amplified with primer pairs

(FE, 5’-AGGGCCCACCTGGCACTGGTAAGAGTCATTTTGC-3’, R-EsBsaI, 5’-ACTGGT

CTCTTCGTTTAGTTATTAACTAAAATATCACTAGACACC-3’) and (F-FsBsaI, 5’-TGA

GGTCTCCGAACTTATGGATTTGTTTATGAG-3’, RF, 5’-AGGTAGGCCTCTAGGGCA

GCTAAC-3’), respectively. The products were named as fragment Es and Fs, which leave the

spike gene coding region as an independent fragment. BsaI sites (5’-GGTCTCN|NNNN-3’)

were introduced into the 3’ terminal of the Es fragment and the 5’ terminal of the Fs fragment,

respectively. The spike sequence of Rs4231 was amplified with the primer pair (F-Rs4231-

BsmBI, 5’-AGTCGTCTCAACGAACATGTTTATTTTCTTATTCTTTCTCACTCTCAC-3’

and R-Rs4231-BsmBI, 5’-TCACGTCTCAGTTCGTTTATGTGTAATGTAATTTGACAC

CCTTG-3’). The S gene sequence of Rs7327 was amplified with primer pair (F-Rs7327-BsaI,

5’-AGTGGTCTCAACGAACATGAAATTGTTAGTTTTAGTTTTTGCTAC-3’ and R-

A gene pool of bat SARS-related coronaviruses

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.100... 30, 2017 

Rs7327-BsaI, 5’- TCAGGTCTCAGTTCGTTTATGTGTAATGTAATTTAACACCCTTG-3’).

The fragment Es and Fs were both digested with BglI (NEB) and BsaI (NEB). The Rs4231 S

gene was digested with BsmBI. The Rs7327 S gene was digested with BsaI. The other fragments

and bacterial artificial chromosome (BAC) were prepared as described previously. Then the

two prepared spike DNA fragments were separately inserted into BAC with Es, Fs and other

fragments. The correct infectious BAC clones were screened. The chimeric viruses were res-

cued as described previously [23].

Determination of virus infectivity by immunofluorescence assay

The HeLa cell line was kindly provided by Australian Animal Health Laboratory, CSIRO (Gee-

long, Australia). HeLa cells expressing human ACE2 were constructed as described previously

[17]. HeLa cells expressing human ACE2 and Vero E6 cells were cultured on coverslips in

24-well plates (Corning) incubated with the newly isolated or recombinant bat SARSr-CoVs at

a multiplicity of infection (MOI) = 1.0 for 1h. The inoculum was removed and the cells were

washed twice with PBS and supplemented with medium. Vero E6 cells without virus inocula-

tion and HeLa cells without ACE2 were used as negative control. Twenty-four hours after

infection, cells were rinsed with PBS and fixed with 4% formaldehyde in PBS (pH7.4) at 4˚C

for 20 min. ACE2 expression was detected by using goat anti-human ACE2 immunoglobulin

followed by FITC-labelled donkey anti-goat immunoglobulin (PTGLab). Virus replication was

detected by using rabbit antibody against the nucleocapsid protein of bat SARSr-CoV Rp3 fol-

lowed by Cy3-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI. Staining pat-

terns were observed under an FV1200 confocal microscope (Olympus).

glen jenvey www.glen-jenvey.co.uk