C*I
发帖数: 4736 | 1 一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须
经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人
民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过
其它受体/宿体，而直接传染给人。 他们在发paper 时明确强调的核心就是：中国的
bats corona virus 可以直接传染给人，不需要经过其它中间动物/宿体。
Preliminary in vitro testing indicates that WIV1 also has a broad species
tropism. Our results provide the strongest evidence to date that Chinese
horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate
hosts may not be necessary for direct human infection by some bat SL-CoVs.
请看他们自己在2013年11月号发表在nature上的研究成果时怎么说的吧！
Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the
ACE2 receptor
Xing-Yi Ge, Jia-Lu Li, Xing-Lou Yang, Aleksei A. Chmura, Guangjian Zhu,
Jonathan H. Epstein, Jonna K. Mazet, Ben Hu, Wei Zhang, Cheng Peng, Yu-Ji
Zhang, Chu-Ming Luo, Bing Tan, Ning Wang, Yan Zhu, Gary Crameri, Shu-Yi
Zhang, Lin-Fa Wang, Peter Daszak & Zheng-Li Shi
Nature volume 503, pages535–538(2013)Cite this article
Abstract
The 2002–3 pandemic caused by severe acute respiratory syndrome coronavirus
(SARS-CoV) was one of the most significant public health events in recent
history1. An ongoing outbreak of Middle East respiratory syndrome
coronavirus2 suggests that this group of viruses remains a key threat and
that their distribution is wider than previously recognized. Although bats
have been suggested to be the natural reservoirs of both viruses3,4,5,
attempts to isolate the progenitor virus of SARS-CoV from bats have been
unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been
reported from bats in China, Europe and Africa5,6,7,8, but none is
considered a direct progenitor of SARS-CoV because of their phylogenetic
disparity from this virus and the inability of their spike proteins to use
the SARS-CoV cellular receptor molecule, the human angiotensin converting
enzyme II (ACE2)9,10. Here we report whole-genome sequences of two novel bat
coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan
, China: RsSHC014 and Rs3367. These viruses are far more closely related to
SARS-CoV than any previously identified bat coronaviruses, particularly in
the receptor binding domain of the spike protein. Most importantly, we
report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from
bat faecal samples in Vero E6 cells, which has typical coronavirus
morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans,
civets and Chinese horseshoe bats for cell entry. Preliminary in vitro
testing indicates that WIV1 also has a broad species tropism. Our results
provide the strongest evidence to date that Chinese horseshoe bats are
natural reservoirs of SARS-CoV, and that intermediate hosts may not be
necessary for direct human infection by some bat SL-CoVs. They also
highlight the importance of pathogen-discovery programs targeting high-risk
wildlife groups in emerging disease hotspots as a strategy for pandemic
preparedness.
Main
The 2002–3 pandemic of SARS1 and the ongoing emergence of the Middle East
respiratory syndrome coronavirus (MERS-CoV)2 demonstrate that CoVs are a
significant public health threat. SARS-CoV was shown to use the human ACE2
molecule as its entry receptor, and this is considered a hallmark of its
cross-species transmissibility11. The receptor binding domain (RBD) located
in the amino-terminal region (amino acids 318–510) of the SARS-CoV spike (S
) protein is directly involved in binding to ACE2 (ref. 12). However,
despite phylogenetic evidence that SARS-CoV evolved from bat SL-CoVs, all
previously identified SL-CoVs have major sequence differences from SARS-CoV
in the RBD of their S proteins, including one or two deletions6,9. Replacing
the RBD of one SL-CoV S protein with SARS-CoV S conferred the ability to
use human ACE2 and replicate efficiently in mice9,13. However, to date, no
SL-CoVs have been isolated from bats, and no wild-type SL-CoV of bat origin
has been shown to use ACE2.
We conducted a 12-month longitudinal survey (April 2011–September 2012) of
SL-CoVs in a colony of Rhinolophus sinicus at a single location in Kunming,
Yunnan Province, China (Extended Data Table 1). A total of 117 anal swabs or
faecal samples were collected from individual bats using a previously
published method5,14. A one-step reverse transcription (RT)-nested PCR was
conducted to amplify the RNA-dependent RNA polymerase (RdRP) motifs A and C,
which are conserved among alphacoronaviruses and betacoronaviruses15.
Twenty-seven of the 117 samples (23%) were classed as positive by PCR and
subsequently confirmed by sequencing. The species origin of all positive
samples was confirmed to be R. sinicus by cytochrome b sequence analysis, as
described previously16. A higher prevalence was observed in samples
collected in October (30% in 2011 and 48.7% in 2012) than those in April (7.
1% in 2011) or May (7.4% in 2012) (Extended Data Table 1). Analysis of the S
protein RBD sequences indicated the presence of seven different strains of
SL-CoVs (Fig. 1a and Extended Data Figs 1 and 2). In addition to RBD
sequences, which closely matched previously described SL-CoVs (Rs672, Rf1
and HKU3)5,8,17,18, two novel strains (designated SL-CoV RsSHC014 and Rs3367
) were discovered. Their full-length genome sequences were determined, and
both were found to be 29,787 base pairs in size (excluding the poly(A) tail)
. The overall nucleotide sequence identity of these two genomes with human
SARS-CoV (Tor2 strain) is 95%, higher than that observed previously for bat
SL-CoVs in China (88–92%)5,8,17,18 or Europe (76%)6 (Extended Data Table 2
and Extended Data Figs 3 and 4). Higher sequence identities were observed at
the protein level between these new SL-CoVs and SARS-CoVs (Extended Data
Tables 3 and 4). To understand the evolutionary origin of these two novel SL
-CoV strains, we conducted recombination analysis with the Recombination
Detection Program 4.0 package19 using available genome sequences of bat SL-
CoV strains (Rf1, Rp3, Rs672, Rm1, HKU3 and BM48-31) and human and civet
representative SARS-CoV strains (BJ01, SZ3, Tor2 and GZ02). Three
breakpoints were detected with strong P values (<10−20) and supported
by similarity plot and bootscan analysis (Extended Data Fig. 5a, b).
Breakpoints were located at nucleotides 20,827, 26,553 and 28,685 in the
Rs3367 (and RsSHC014) genome, and generated recombination fragments covering
nucleotides 20,827–26,533 (5,727 nucleotides) (including partial open
reading frame (ORF) 1b, full-length S, ORF3, E and partial M gene) and
nucleotides 26,534–28,685 (2,133 nucleotides) (including partial ORF M,
full-length ORF6, ORF7, ORF8 and partial N gene). Phylogenetic analysis
using the major and minor parental regions suggested that Rs3367, or
RsSHC014, is the descendent of a recombination of lineages that ultimately
lead to SARS-CoV and SL-CoV Rs672 (Fig. 1b).
Figure 1: Phylogenetic tree based on amino acid sequences of the S RBD
region and the two parental regions of bat SL-CoV Rs3367 or RsSHC014.
figure1
a, SARS-CoV S protein amino acid residues 310–520 were aligned with
homologous regions of bat SL-CoVs using the ClustalW software. A maximum-
likelihood phylogenetic tree was constructed using a Poisson model with
bootstrap values determined by 1,000 replicates in the MEGA5 software
package. The RBD sequences identified in this study are in bold and named by
the sample numbers. The key amino acid residues involved in interacting
with the human ACE2 molecule are indicated on the right of the tree. SARS-
CoV GZ02, BJ01 and Tor2 were isolated from patients in the early, middle and
late phase, respectively, of the SARS outbreak in 2003. SARS-CoV SZ3 was
identified from Paguma larvata in 2003 collected in Guangdong, China. SL-CoV
Rp3, Rs672 and HKU3-1 were identified from R. sinicus collected in China (
respectively: Guangxi, 2004; Guizhou, 2006; Hong Kong, 2005). Rf1 and Rm1
were identified from R. ferrumequinum and R. macrotis, respectively,
collected in Hubei, China, in 2004. Bat SARS-related CoV BM48-31 was
identified from R. blasii collected in Bulgaria in 2008. Bat CoV HKU9-1 was
identified from Rousettus leschenaultii collected in Guangdong, China in
2005/2006 and used as an outgroup. All sequences in bold and italics were
identified in the current study. Filled triangles, circles and diamonds
indicate samples with co-infection by two different SL-CoVs. ‘–’
indicates the amino acid deletion. b, Phylogenetic origins of the two
parental regions of Rs3367 or RsSHC014. Maximum likelihood phylogenetic
trees were constructed from alignments of two fragments covering nucleotides
20,827–26,533 (5,727 nucleotides) and 26,534 –28,685 (2,133 nucleotides)
of the Rs3367 genome, respectively. For display purposes, the trees were
midpoint rooted. The taxa were annotated according to strain names: SARS-CoV
, SARS coronavirus; SARS-like CoV, bat SARS-like coronavirus. The two novel
SL-CoVs, Rs3367 and RsSHC014, are in bold and italics.
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The most notable sequence differences between these two new SL-CoVs and
previously identified SL-CoVs is in the RBD regions of their S proteins.
First, they have higher amino acid sequence identity to SARS-CoV (85% and 96
% for RsSHC014 and Rs3367, respectively). Second, there are no deletions and
they have perfect sequence alignment with the SARS-CoV RBD region (Extended
Data Figs 1 and 2). Structural and mutagenesis studies have previously
identified five key residues (amino acids 442, 472, 479, 487 and 491) in the
RBD of the SARS-CoV S protein that have a pivotal role in receptor
binding20,21. Although all five residues in the RsSHC014 S protein were
found to be different from those of SARS-CoV, two of the five residues in
the Rs3367 RBD were conserved (Fig. 1 and Extended Data Fig. 1).
Despite the rapid accumulation of bat CoV sequences in the last decade,
there has been no report of successful virus isolation6,22,23. We attempted
isolation from SL-CoV PCR-positive samples. Using an optimized protocol and
Vero E6 cells, we obtained one isolate which caused cytopathic effect during
the second blind passage. Purified virions displayed typical coronavirus
morphology under electron microscopy (Fig. 2). Sequence analysis using a
sequence-independent amplification method14 to avoid PCR-introduced
contamination indicated that the isolate was almost identical to Rs3367,
with 99.9% nucleotide genome sequence identity and 100% amino acid sequence
identity for the S1 region. The new isolate was named SL-CoV-WIV1.
Figure 2: Electron micrograph of purified virions.
figure2
Virions from a 10-ml culture were collected, fixed and concentrated/purified
by sucrose gradient centrifugation. The pelleted viral particles were
suspended in 100 μl PBS, stained with 2% phosphotungstic acid (pH&#
8201;7.0) and examined directly using a Tecnai transmission electron
microscope (FEI) at 200 kV.
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To determine whether WIV1 can use ACE2 as a cellular entry receptor, we
conducted virus infectivity studies using HeLa cells expressing or not
expressing ACE2 from humans, civets or Chinese horseshoe bats. We found that
WIV1 is able to use ACE2 of different origins as an entry receptor and
replicated efficiently in the ACE2-expressing cells (Fig. 3). This is, to
our knowledge, the first identification of a wild-type bat SL-CoV capable of
using ACE2 as an entry receptor.
Figure 3: Analysis of receptor usage of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR.
figure3
Determination of virus infectivity in HeLa cells with and without the
expression of ACE2. b, bat; c, civet; h, human. ACE2 expression was detected
with goat anti-humanACE2 antibody followed by fluorescein isothiocyanate (
FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with
rabbit antibody against the SL-CoV Rp3 nucleocapsid protein followed by
cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with
DAPI (4′,6-diamidino-2-phenylindole). The columns (from left to right) show
staining of nuclei (blue), ACE2 expression (green), virus replication (red)
, merged triple-stained images and real-time PCR results, respectively. (n =
3); error bars represent standard deviation.
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To assess its cross-species transmission potential, we conducted infectivity
assays in cell lines from a range of species. Our results (Fig. 4 and
Extended Data Table 5) indicate that bat SL-CoV-WIV1 can grow in human
alveolar basal epithelial (A549), pig kidney 15 (PK-15) and Rhinolophus
sinicus kidney (RSKT) cell lines, but not in human cervix (HeLa), Syrian
golden hamster kidney (BHK21), Myotis davidii kidney (BK), Myotis chinensis
kidney (MCKT), Rousettus leschenaulti kidney (RLK) or Pteropus alecto kidney
(PaKi) cell lines. Real-time RT–PCR indicated that WIV1 replicated much
less efficiently in A549, PK-15 and RSKT cells than in Vero E6 cells (Fig. 4
).
Figure 4: Analysis of host range of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR.
figure4
Virus infection in A549, RSKT, Vero E6 and PK-15 cells. Virus replication
was detected as described for Fig. 3. The columns (from left to right) show
staining of nuclei (blue), virus replication (red), merged double-stained
images and real-time PCR results, respectively. n = 3; error bars represent
s.d.
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To assess the cross-neutralization activity of human SARS-CoV sera against
WIV1, we conducted serum-neutralization assays using nine convalescent sera
from SARS patients collected in 2003. The results showed that seven of these
were able to completely neutralize 100 tissue culture infectious dose 50 (
TCID50) WIV1 at dilutions of 1:10 to 1:40, further confirming the close
relationship between WIV1 and SARS-CoV.
Our findings have important implications for public health. First, they
provide the clearest evidence yet that SARS-CoV originated in bats. Our
previous work provided phylogenetic evidence of this5, but the lack of an
isolate or evidence that bat SL-CoVs can naturally infect human cells, until
now, had cast doubt on this hypothesis. Second, the lack of capacity of SL-
CoVs to use of ACE2 receptors has previously been considered as the key
barrier for their direct spillover into humans, supporting the suggestion
that civets were intermediate hosts for SARS-CoV adaptation to human
transmission during the SARS outbreak24. However, the ability of SL-CoV-WIV1
to use human ACE2 argues against the necessity of this step for SL-CoV-WIV1
and suggests that direct bat-to-human infection is a plausible scenario for
some bat SL-CoVs. This has implications for public health control measures
in the face of potential spillover of a diverse and growing pool of recently
discovered SARS-like CoVs with a wide geographic distribution.
Our findings suggest that the diversity of bat CoVs is substantially higher
than that previously reported. In this study we were able to demonstrate the
circulation of at least seven different strains of SL-CoVs within a single
colony of R. sinicus during a 12-month period. The high genetic diversity of
SL-CoVs within this colony was mirrored by high phenotypic diversity in the
differential use of ACE2 by different strains. It would therefore not be
surprising if further surveillance reveals a broad diversity of bat SL-CoVs
that are able to use ACE2, some of which may have even closer homology to
SARS-CoV than SL-CoV-WIV1. Our results—in addition to the recent
demonstration of MERS-CoV in a Saudi Arabian bat25, and of bat CoVs closely
related to MERS-CoV in China, Africa, Europe and North America3,26,27—
suggest that bat coronaviruses remain a substantial global threat to public
health.
Finally, this study demonstrates the public health importance of pathogen
discovery programs targeting wildlife that aim to identify the ‘known
unknowns’—previously unknown viral strains closely related to known
pathogens. These programs, focused on specific high-risk wildlife groups and
hotspots of disease emergence, may be a critical part of future global
strategies to predict, prepare for, and prevent pandemic emergence28.
Methods Summary
Throat and faecal swabs or fresh faecal samples were collected in viral
transport medium as described previously14. All PCR was conducted with the
One-Step RT–PCR kit (Invitrogen). Primers targeting the highly conserved
regions of the RdRP gene were used for detection of all alphacoronaviruses
and betacoronaviruses as described previously15. Degenerate primers were
designed on the basis of all available genomic sequences of SARS-CoVs and SL
-CoVs and used for amplification of the RBD sequences of S genes or full-
length genomic sequences. Degenerate primers were used for amplification of
the bat ACE2 gene as described previously29. PCR products were gel purified
and cloned into pGEM-T Easy Vector (Promega). At least four independent
clones were sequenced to obtain a consensus sequence. PCR-positive faecal
samples (in 200 μl buffer) were gradient centrifuged at 3,000–12,
000g and supernatant diluted at 1:10 in DMEM before being added to Vero E6
cells. After incubation at 37 °C for 1 h, inocula were removed
and replaced with fresh DMEM with 2% FCS. Cells were incubated at 37
°C and checked daily for cytopathic effect. Cell lines from different
origins were grown on coverslips in 24-well plates and inoculated with the
novel SL-CoV at a multiplicity of infection of 10. Virus replication
was detected at 24 h after infection using rabbit antibodies against
the SL-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated goat anti-
rabbit IgG.
Online Methods
Sampling
Bats were trapped in their natural habitat as described previously5. Throat
and faecal swab samples were collected in viral transport medium (VTM)
composed of Hank’s balanced salt solution, pH 7.4, containing BSA (1%
), amphotericin (15 μg ml−1), penicillin G (100 U&#
8201;ml−1) and streptomycin (50 μg ml−1). To
collect fresh faecal samples, clean plastic sheets measuring 2.0 by 2.0&#
8201;m were placed under known bat roosting sites at about 18:00 h
each evening. Relatively fresh faecal samples were collected from sheets at
approximately 05:30–06:00 the next morning and placed in VTM. Samples were
transported to the laboratory and stored at −80 °C until use.
All animals trapped for this study were released back to their habitat after
sample collection. All sampling processes were performed by veterinarians
with approval from Animal Ethics Committee of the Wuhan Institute of
Virology (WIVH05210201) and EcoHealth Alliance under an inter-institutional
agreement with University of California, Davis (UC Davis protocol no. 16048).
RNA extraction, PCR and sequencing
RNA was extracted from 140 μl of swab or faecal samples with a Viral
RNA Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was
eluted in 60 μl RNAse-free buffer (buffer AVE, Qiagen), then
aliquoted and stored at −80 °C. One-step RT–PCR (Invitrogen)
was used to detect coronavirus sequences as described previously15. First
round PCR was conducted in a 25-μl reaction mix containing 12.5 μl
PCR 2× reaction mix buffer, 10 pmol of each primer, 2.5 mM
MgSO4, 20 U RNase inhibitor, 1 μl SuperScript III/ Platinum Taq
Enzyme Mix and 5 μl RNA. Amplification of the RdRP-gene fragment was
performed as follows: 50 °C for 30 min, 94 °C for 2&#
8201;min, followed by 40 cycles consisting of 94 °C for 15 s,
62 °C for 15 s, 68 °C for 40 s, and a final
extension of 68 °C for 5 min. Second round PCR was conducted in
a 25-μl reaction mix containing 2.5 μl PCR reaction buffer, 5
;pmol of each primer, 50 mM MgCl2, 0.5 mM dNTP, 0.1 μl
Platinum Taq Enzyme (Invitrogen) and 1 μl first round PCR product. The
amplification of RdRP-gene fragment was performed as follows: 94 °C
for 5 min followed by 35 cycles consisting of 94 °C for 30&#
8201;s, 52 °C for 30 s, 72 °C for 40 s, and a final
extension of 72 °C for 5 min.
To amplify the RBD region, one-step RT–PCR was performed with primers
designed based on available SARS-CoV or bat SL-CoVs (first round PCR primers
; F, forward; R, reverse: CoVS931F-5′-VWGADGTTGTKAGRTTYCCT-3′ and
CoVS1909R-5′-TAARACAVCCWGCYTGWGT-3′; second PCR primers: CoVS951F-5′-
TGTKAGRTTYCCTAAYATTAC-3′ and CoVS1805R-5′-ACATCYTGATANARAACAGC-3′). First
-round PCR was conducted in a 25-μl reaction mix as described above except
primers specific for the S gene were used. The amplification of the RBD
region of the S gene was performed as follows: 50 °C for 30 min
, 94 °C for 2 min, followed by 35 cycles consisting of 94
;°C for 15 s, 43 °C for 15 s, 68 °C for 90
s, and a final extension of 68 °C for 5 min. Second-round PCR
was conducted in a 25-μl reaction mix containing 2.5 μl PCR reaction
buffer, 5 pmol of each primer, 50 mM MgCl2, 0.5 mM dNTP,
0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl first round
PCR product. Amplification was performed as follows: 94 °C for 5&#
8201;min followed by 40 cycles consisting of 94 °C for 30 s, 41
°C for 30 s, 72 °C for 60 s, and a final
extension of 72 °C for 5 min.
PCR products were gel purified and cloned into pGEM-T Easy Vector (Promega).
At least four independent clones were sequenced to obtain a consensus
sequence for each of the amplified regions.
Sequencing full-length genomes
Degenerate coronavirus primers were designed based on all available SARS-CoV
and bat SL-CoV sequences in GenBank and specific primers were designed from
genome sequences generated from previous rounds of sequencing in this study
(primer sequences will be provided upon request). All PCRs were conducted
using the One-Step RT–PCR kit (Invitrogen). The 5′ and 3′ genomic ends
were determined using the 5′ or 3′ RACE kit (Roche), respectively. PCR
products were gel purified and sequenced directly or following cloning into
pGEM-T Easy Vector (Promega). At least four independent clones were
sequenced to obtain a consensus sequence for each of the amplified regions
and each region was sequenced at least twice.
Sequence analysis and databank accession numbers
Routine sequence management and analysis was carried out using DNAStar or
Geneious. Sequence alignment and editing was conducted using ClustalW,
BioEdit or GeneDoc. Maximum Likelihood phylogenetic trees based on the
protein sequences were constructed using a Poisson model with bootstrap
values determined by 1,000 replicates in the MEGA5 software package.
Sequences obtained in this study have been deposited in GenBank as follows (
accession numbers given in parenthesis): full-length genome sequence of SL-
CoV RsSHC014 and Rs3367 (KC881005, KC881006); full-length sequence of WIV1 S
(KC881007); RBD (KC880984-KC881003); ACE2 (KC8810040). SARS-CoV sequences
used in this study: human SARS-CoV strains Tor2 (AY274119), BJ01 (AY278488),
GZ02 (AY390556) and civet SARS-CoV strain SZ3 (AY304486). Bat coronavirus
sequences used in this study: Rs672 (FJ588686), Rp3 (DQ071615), Rf1 (
DQ412042), Rm1 (DQ412043), HKU3-1 (DQ022305), BM48-31 (NC_014470), HKU9-1 (
NC_009021), HKU4 (NC_009019), HKU5 (NC_009020), HKU8 (DQ249228), HKU2 (
EF203067), BtCoV512 (NC_009657), 1A (NC_010437). Other coronavirus sequences
used in this study: HCoV-229E (AF304460), HCoV-OC43 (AY391777), HCoV-NL63 (
AY567487), HKU1 (NC_006577), EMC (JX869059), FIPV (NC_002306), PRCV (
DQ811787), BWCoV (NC_010646), MHV (AY700211), IBV (AY851295).
Amplification, cloning and expression of the bat ACE2 gene
Construction of expression clones for human and civet ACE2 in pcDNA3.1 has
been described previously29. Bat ACE2 was amplified from a R. sinicus (
sample no. 3357). In brief, total RNA was extracted from bat rectal tissue
using the RNeasy Mini Kit (Qiagen). First-strand complementary DNA was
synthesized from total RNA by reverse transcription with random hexamers.
Full-length bat ACE2 fragments were amplified using forward primer bAF2 and
reverse primer bAR2 (ref. 29). The ACE2 gene was cloned into pCDNA3.1 with
KpnI and XhoI, and verified by sequencing. Purified ACE2 plasmids were
transfected to HeLa cells. After 24 h, lysates of HeLa cells expressing
human, civet, or bat ACE2 were confirmed by western blot or
immunofluorescence assay.
Western blot analysis
Lysates of cells or filtered supernatants containing pseudoviruses were
separated by SDS–PAGE, followed by transfer to a nitrocellulose membrane (
Millipore). For detection of S protein, the membrane was incubated with
rabbit anti-Rp3 S fragment (amino acids 561–666) polyantibodies (1:200),
and the bound antibodies were detected by alkaline phosphatase (AP)-
conjugated goat anti-rabbit IgG (1:1,000). For detection of HIV-1 p24 in
supernatants, monoclonal antibody against HIV p24 (p24 MAb) was used as the
primary antibody at a dilution of 1:1,000, followed by incubation with AP-
conjugated goat anti-mouse IgG at the same dilution. To detect the
expression of ACE2 in HeLa cells, goat antibody against the human ACE2
ectodomain (1:500) was used as the first antibody, followed by incubation
with horseradish peroxidase-conjugated donkey anti-goat IgG (1:1,000).
Virus isolation
Vero E6 cell monolayers were maintained in DMEM supplemented with 10% FCS.
PCR-positive samples (in 200 μl buffer) were gradient centrifuged at
3,000–12,000g, and supernatant were diluted 1:10 in DMEM before being added
to Vero E6 cells. After incubation at 37 °C for 1 h, inocula
were removed and replaced with fresh DMEM with 2% FCS. Cells were incubated
at 37 °C for 3 days and checked daily for cytopathic effect.
Double-dose triple antibiotics penicillin/streptomycin/amphotericin (Gibco)
were included in all tissue culture media (penicillin 200 IU ml&
#8722;1, streptomycin 0.2 mg ml−1, amphotericin 0.5
μg ml−1). Three blind passages were carried out for each sample
. After each passage, both the culture supernatant and cell pellet were
examined for presence of virus by RT–PCR using primers targeting the RdRP
or S gene. Virions in supernatant (10 ml) were collected and fixed
using 0.1% formaldehyde for 4 h, then concentrated by
ultracentrifugation through a 20% sucrose cushion (5 ml) at 80,000g
for 90 min using a Ty90 rotor (Beckman). The pelleted viral particles
were suspended in 100 μl PBS, stained with 2% phosphotungstic acid (
pH 7.0) and examined using a Tecnai transmission electron microscope (
FEI) at 200 kV.
Virus infectivity detected by immunofluorescence assay
Cell lines used for this study and their culture conditions are summarized
in Extended Data Table 5. Virus titre was determined in Vero E6 cells by
cytopathic effect (CPE) counts. Cell lines from different origins and HeLa
cells expressing ACE2 from human, civet or Chinese horseshoe bat were grown
on coverslips in 24-well plates (Corning) incubated with bat SL-CoV-WIV1 at
a multiplicity of infection = 10 for 1 h. The inoculum was removed and
washed twice with PBS and supplemented with medium. HeLa cells without ACE2
expression and Vero E6 cells were used as negative and positive controls,
respectively. At 24 h after infection, cells were washed with PBS and
fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at 4
;°C. ACE2 expression was detected using goat anti-human ACE2 immunoglobulin
(R&D Systems) followed by FITC-labelled donkey anti-goat immunoglobulin (
PTGLab). Virus replication was detected using rabbit antibody against the SL
-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated mouse anti-rabbit
IgG. Nuclei were stained with DAPI. Staining patterns were examined using a
FV1200 confocal microscope (Olympus).
Virus infectivity detected by real-time RT–PCR
Vero E6, A549, PK15, RSKT and HeLa cells with or without expression of ACE2
of different origins were inoculated with 0.1 TCID50 WIV-1 and incubated for
1 h at 37 °C. After removing the inoculum, the cells were
cultured with medium containing 1% FBS. Supernatants were collected at 0, 12
, 24 and 48 h. RNA from 140 μl of each supernatant was
extracted with the Viral RNA Mini Kit (Qiagen) following manufacturer’s
instructions and eluted in 60 μl buffer AVE (Qiagen). RNA was
quantified on the ABI StepOne system, with the TaqMan AgPath-ID One-Step RT
–PCR Kit (Applied Biosystems) in a 25 μl reaction mix containing 4&#
8201;μl RNA, 1 × RT–PCR enzyme mix, 1 × RT–PCR
buffer, 40 pmol forward primer (5′-GTGGTGGTGACGGCAAAATG-3′), 40&#
8201;pmol reverse primer (5′-AAGTGAAGCTTCTGGGCCAG-3′) and 12 pmol
probe (5′-FAM-AAAGAGCTCAGCCCCAGATG-BHQ1-3′). Amplification parameters were
10 min at 50 °C, 10 min at 95 °C and 50 cycles of
15 s at 95 °C and 20 s at 60 °C. RNA dilutions
from purified WIV-1 stock were used as a standard.
Serum neutralization test
SARS patient sera were inactivated at 56 °C for 30 min and then
used for virus neutralization testing. Sera were diluted starting with 1:10
and then serially twofold diluted in 96-well cell plates to 1:40. Each 100&
#8201;μl serum dilution was mixed with 100 μl viral supernatant
containing 100 TCID50of WIV1 and incubated at 37 °C for 1
;h. The mixture was added in triplicate wells of 96-well cell plates with
plated monolayers of Vero E6 cells and further incubated at 37 °C for
2 days. Serum from a healthy blood donor was used as a negative
control in each experiment. CPE was observed using an inverted microscope 2&
#8201;days after inoculation. The neutralizing antibody titre was read as
the highest dilution of serum which completely suppressed CPE in infected
wells. The neutralization test was repeated twice.
Recombination analysis
Full-length genomic sequences of SL-CoV Rs3367 or RsSHC014 were aligned with
those of selected SARS-CoVs and bat SL-CoVs using Clustal X. The aligned
sequences were preliminarily scanned for recombination events using
Recombination Detection Program (RDP) 4.0 (ref. 19). The potential
recombination events suggested by RDP owing to their strong P values (
<10–20) were investigated further by similarity plot and bootscan analyses
implemented in Simplot 3.5.1. Phylogenetic origin of the major and minor
parental regions of Rs3367 or RsSHC014 were constructed from the
concatenated sequences of the essential ORFs of the major and minor parental
regions of selected SARS-CoV and SL-CoVs. Two genome regions between three
estimated breakpoints (20,827–26,553 and 26,554–28,685) were aligned
independently using ClustalX and generated two alignments of 5,727
base pairs and 2,133 base pairs. The two alignments were used to
construct maximum likelihood trees to better infer the fragment parents. All
nucleotide numberings in this study are based on Rs3367 genome position.
https://www.nature.com/articles/nature12711 |
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