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| A perfect bio-weapon: Bat Virus with HIV mutation insertion. |
The new coronavirus has an HIV-like mutation that means its ability to bind with human cells could be up to 1,000 times as strong as the Sars virus, according to new research by scientists in China and Europe. The discovery could help to explain not only how the infection has spread but also where it came from and how best to fight it.
Scientists showed that Sars (severe acute respiratory syndrome) entered the human body by binding with a receptor protein called ACE2 on a cell membrane. And some early studies suggested that the new coronavirus, which shares about 80 per cent of the genetic structure of Sars, might follow a similar path.
But the ACE2 protein does not exist in large quantities in healthy people, and this partly helped to limit the scale of the Sars outbreak of 2002-03, in which infected about 8,000 people around the world.
Other highly contagious viruses, including HIV and Ebola, target an enzyme called furin, which works as a protein activator in the human body. Many proteins are inactive or dormant when they are produced and have to be “cut” at specific points to activate their various functions.
When looking at the genome sequence of the new coronavirus, Professor Ruan Jishou and his team at Nankai University in Tianjin found a section of mutated genes that did not exist in Sars, but were similar to those found in HIV and Ebola.
“This finding suggests that 2019-nCoV [the new coronavirus] may be significantly different from the Sars coronavirus in the infection pathway,” the scientists said in a paper published this month on Chinaxiv.org, a platform used by the Chinese Academy of Sciences to release scientific research papers before they have been peer-reviewed. “This virus may use the packing mechanisms of other viruses such as HIV.”
Spiky invader Makes Virus Easily Spread Between People
To infect a cell, coronaviruses use a ‘spike’ protein that binds to the cell membrane, a process that's activated by specific cell enzymes. Genomic analyses of the new coronavirus have revealed that its spike protein differs from those of close relatives, and suggest that the protein has a site on it which is activated by a host-cell enzyme called furin.
This is significant because furin is found in lots of human tissues, including the lungs, liver and small intestines, which means that the virus has the potential to attack multiple organs, says Li Hua, a structural biologist at Huazhong University of Science and Technology in Wuhan, China, where the outbreak began.
The finding could explain some of the symptoms observed in people with the coronavirus, such as liver failure, says Li, who co-authored a genetic analysis of the virus that was posted on the ChinaXiv preprint server on 23 February2. SARS and other coronaviruses in the same genus as the new virus don't have furin activation sites, he says.
The furin activation site “sets the virus up very differently to SARS in terms of its entry into cells, and possibly affects virus stability and hence transmission”, says Gary Whittaker, a virologist at Cornell University in Ithaca, New York. His team published another structural analysis of the coronavirus’s spike protein on bioRxiv on 18 February3.
Several other groups have also identified the activation site as possibly enabling the virus to spread efficiently between humans4. They note that these sites are also found in other viruses that spread easily between people, including severe strains of the influenza virus. On these viruses, the activation site is found on a protein called haemagglutinin, not on the spike protein.
(Blogger's Notes: What are the odds that a SARS-like
coronavirus with overlapping genetics from HIV mutated and crossed over into
humans, next door to a laboratory which had been enhancing coronavirus with HIV
for over a decade?
Following is the so-called scientific research paper (or a shoddy cover-up) from Dr Zhou "Wuhan Virus" Peng - published on 22 January 2020 - supposedly discovering the new coronavirus in Wuhan with the definite proof that it originated from the Wuhan Seafood Market in mid-December 2019.
But on 23 February 2020 China officially admits the virus did not originate from the Wuhan Seafood Market. And by then it was very clear that the so-called COVID-19 the deadly Wuhan Virus was created in the Wuhan Institute off Virology and from there the virus somehow escaped into the nearby populace.)
Following is the so-called scientific research paper (or a shoddy cover-up) from Dr Zhou "Wuhan Virus" Peng - published on 22 January 2020 - supposedly discovering the new coronavirus in Wuhan with the definite proof that it originated from the Wuhan Seafood Market in mid-December 2019.
But on 23 February 2020 China officially admits the virus did not originate from the Wuhan Seafood Market. And by then it was very clear that the so-called COVID-19 the deadly Wuhan Virus was created in the Wuhan Institute off Virology and from there the virus somehow escaped into the nearby populace.)
Discovery of a novel coronavirusassociated with the recent pneumonia outbreak in humans and its potential bat origin:
(Abstract: Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans.
Here
we report the identification and characterization of a novel coronavirus
(nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans,
in Wuhan, China. The epidemic, started from December 12th, 2019, has caused 198
laboratory confirmed infections with three fatal cases by January 20th, 2020.
Full-length
genome sequences were obtained from five patients at the early stage of the
outbreak. They are almost identical to each other and share 79.5% sequence
identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical
at the whole genome level to a bat coronavirus.
The
pairwise protein sequence analysis of seven conserved non-structural proteins
show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus
was then isolated from the bronchoalveolar lavage fluid of a critically ill
patient, which can be neutralized by sera from several patients. Importantly,
we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as
SARS-CoV.)
Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans. Here we report the identification and characterization of a novel coronavirus (nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans, in Wuhan, China.
The epidemic, started from December 12th 29 , 2019, has caused 198 laboratory confirmed infections with three fatal cases by January 20th 30 , 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical at the whole genome level to a bat coronavirus.
The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients.
Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV. Coronavirus has caused two large-scale pandemic in the last two decades, SARS an MERS (Middle East respiratory syndrome). It was generally believed that SARSrCoV, mainly found in bats, might cause future disease outbreak.
Disease onset may result in progressive respiratory failure due to alveolar damage and even death. The disease was determined as viral induced pneumonia by clinicians according to clinical symptoms and other criteria including body temperature rising, lymphocytes and white blood cells decreasing (sometimes normal for the later), new pulmonary infiltrates on chest radiography, and no obvious improvement upon three days antibiotics treatment.
It appears most of the early cases had contact history with the original seafood market, and no large scale of human-to-human transmission was observed so far. Samples from seven patients with severe pneumonia (six are seafood market peddlers or delivers), who were enrolled in intensive unit cares at the beginning of the outbreak, were sent to WIV laboratory for pathogen diagnosis.
As a CoV lab, we first used pan-CoV PCR primers to test these samples , considering the outbreak happened in winter and in a market, same environment as SARS. We found five PCR positive. A sample (WIV04) collected from bronchoalveolar lavage fluid (BALF) was analysed by metagenomics analysis using next-generation sequencing (NGS) to identify potential etiological agents.
Of the 1582 total reads obtained after human genome filtering, 1378 (87.1%) matched sequences of SARSr-CoV. By de novo assembly and targeted PCR, we obtained a 29,891-bp CoV genome that shared 79.5% sequence identity to SARS-CoV BJ01 (GenBank accession number AY278488.2).
This sequence has been submitted to GISAID (accession no. EPI_ISL_402124). Following the name by WHO, we tentatively call it novel coronavirus 2019 (nCoV-2019). Four more full-length genome sequences of nCoV71 2019 (WIV02, WIV05, WIV06, and WIV07) GISAID accession nos. EPI_ISL_402127-402130) that were above 99.9% identical to each other were subsequently obtained from other four patients.
The virus genome consists of six major open reading frames (ORFs) common to coronaviruses and a number of other accessory genes (Fig. 1b). Further analysis indicates that some of the nCoV-2019 genes shared less than 80% nt sequence identity to SARS-CoV. However, the seven conserved replicase domains in ORF1ab that were used for CoV species classification, are 94.6% aa sequence identical between nCoV-2019 and SARS-CoV, implying the two belong to same species.
We then found a short RdRp region from a bat coronavirus termed BatCoV RaTG13 which we previously detected in Rhinolophus affinis from Yunnan Province showed high sequence identity to nCoV-2019. We did full-length sequencing to this RNA sample. Simplot analysis showed that nCoV-2019 was highly similar throughout the genome to RaTG13, with 96.2% overall genome sequence identity.
The phylogenetic analysis also showed that RaTG13 is the closest relative of the nCoV89 2019 and form a distinct lineage from other SARSr-CoVs. The receptor binding protein spike (S) gene was highly divergent to other CoVs, with less than 75% nt sequence identity to all previously described SARSr92 CoVs except a 93.1% nt identity to RaTG13.
The S genes of nCoV-2019 and RaTG13 S gene are longer than other SARSr-CoVs. The major differences in nCoV-2019 are the three short insertions in the N-terminal domain, and four out of five key residues changes in the receptor-binding motif, in comparison with SARS-CoV. The close phylogenetic relationship to RaTG13 provides evidence for a bat origin of nCoV-2019.
We rapidly developed a qPCR detection based on the receptor-binding domain of spike gene, the most variable region among genome. Our data show the primers could differentiate nCoV-2019 with all other human coronaviruses including bat SARSr-CoV WIV1, which is 95% identity to SARS-CoV.
From the seven patients, we found nCoV-2019 positive in six BALF and five oral swab samples during the first sampling by qPCR and conventional PCR. However, we can no longer find viral positive in oral swabs, anal swabs, and blood from these patients during the second sampling. Based on these findings, we conclude that the disease should be transmitted through airway, yet we can’t rule out other possibilities if the investigation extended to include more patients.
For serological detection of nCoV-2019, we used previously developed bat SARSr112 CoV Rp3 nucleocapsid protein (NP) as antigen in IgG and IgM ELISA test, which showed no cross-reactivity against other human coronaviruses except SARSr-CoV7 113.
As a research lab, we were only able to get five serum samples from the seven viral infected patients. We monitored viral antibody levels in one patient (ICU-06) at seven, eight, nine, and eighteen days after disease onset. A clear trend of IgG and IgM titre (decreased at the last day) increase was observed.
For a second investigation, we tested viral antibody for five of the seven viral positive patients around twenty days after disease onset. All patient samples, but not samples from healthy people, showed strong viral IgG positive. We also found three IgM positive, indicating acute infection.
We then successfully isolated the virus (named nCoV-2019 BetaCoV/Wuhan/WIV04/2019), in Vero and Huh7 cells using BALF sample from ICU-06 patient. Clear cytopathogenic effects were observed in cells after three days incubation. The identity of the strain WIV04 was verified in Vero E6 cells by immunofluorescence microscopy using cross-reactive viral NP antibody (Extended Data Figure 5c and 5d), and by metagenomic sequencing, from which most of the reads mapped to nCoV-2019.
We demonstrate that all samples were able to neutralize 120 TCID50 nCoV-2019 at a dilution of 1:40-1:80. We also show that this virus could be cross-neutralized by horse anti-SARS-CoV serum at dilutions 1:80, further confirming the relationship of the two viruses. Angiotensin converting enzyme II (ACE2) was known as cell receptor for SARSCoV14 140 .
To determine whether nCoV-2019 also use ACE2 as a cellular entry receptor, we conducted virus infectivity studies using HeLa cells expressing or not expressing ACE2 proteins from humans, Chinese horseshoe bats, civet, pig, and mouse.
We show that nCoV-2019 is able to use all but mouse ACE2 as an entry receptor in the ACE2-expressing cells, but not cells without ACE2, indicating which is likely the cell receptor of nCoV-2019. We also proved that nCoV-2019 does not use other coronavirus receptors, aminopeptidase N and dipeptidyl peptidase 4.
The study provides the first detailed report on nCoV-2019, the likely etiology agent responsible for ongoing acute respiratory syndrome epidemic in Wuhan, central China. Viral specific nucleotide positive and viral protein seroconversion observed in all patients tested provides evidence of an association between the disease and the presence of this virus.
However, there are still many urgent questions to be answered. We need more clinical data and samples to confirm if this virus is indeed the etiology agent for this epidemic. In addition, we still don’t know if this virus continue evolving and become more transmissible between human-to-human.
Moreover, we don’t know the transmission routine of this virus among hosts yet. We showed viral positive in oral swabs, implying nCoV-2019 may be transmitted through airway. However, this needs to be confirmed by extending detection range.
Finally, based on our results, it should be expected and worth to test if ACE2 targeting or SARS-CoV targeting drugs can be used for nCoV-2019 patients. At this stage, we know very little about the virus, including basic biology, animal source or any specific treatment.
The almost identical sequences of this virus in different patients imply a probably recent introduction in humans, thus future surveillance on viral mutation and transmission ability and further global research attention are urgently needed.
Authors:
Affiliations:
(1)
CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center
for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, People’s
Republic of China.
(2) Wuhan
Jinyintan hospital, Wuhan, China.
(3) University
of Chinese Academy of Sciences, Beijing, People’s Republic of China.
(4) Hubei
Provincial Center for Disease Control and Prevention, Wuhan, People’s Republic
of China.
These authors contributed equally.
 |
| Only a genetically-engineered virus could spread and kill this rapidly: not a naturally evolving one. |
(From the Global Times: The study published
on ChinaXiv, a Chinese open repository for scientific researchers, reveals the
new coronavirus was introduced to the seafood market from another location - which was not disclosed by China - and
then spread rapidly from market to market.
The findings were the result of analyses of
genome-wide data, sources of infection and the route of spread of 93 samples of
the novel coronavirus collected from 12 countries across four continents.
The
study believes that the patient zero - whose identity was still not disclosed
by the Chinese -transmitted the virus to workers or sellers at the Huanan
seafood market. The crowded market facilitated the further transmission of the
virus to buyers, which caused a wider spread in early December 2019.)
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