Published: 05 February 2021
SARS-CoV-2 evolution during treatment of chronic infection

Abstract
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (?H69/?V70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ?H69/?V70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ?H69/?V70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

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The priority should be to “follow the animals”, starting at the Huanan market, says Eddie Holmes, a virologist at the University of Sydney in Australia. Given the large number of animal species that SARS-CoV-2 can infect, that sampling should be as expansive as possible, say researchers.

And it should definitely include bats. The closest known relative of SARS-CoV-2 is a bat coronavirus called RaTG13, isolated from a bat in a mine in Mojiang, southern China. But it shares only 96% of its genome with SARS-CoV-2, meaning that it is only distantly related. Courtier says that more bats should be sampled from that mine, and that researchers should share the sequences of other coronaviruses isolated there.

But Linfa Wang, a virologist at Duke–National University of Singapore Medical School, doubts whether closer relatives will be found, given the exhaustive sampling done in the cave by researchers over the past decade. “If you gave me a billion dollars, I would not sample in Mojiang cave. I would sample in Southeast Asia,” adds Wang, who says that sampling should extend to lesser-sampled regions such as Thailand and Cambodia, where other relatives of SARS-CoV-2 have recently been isolated.

More: https://www.nature.com/articles/d41586-021-00877-4

The Governments of Australia, Canada, Czechia, Denmark, Estonia, Israel, Japan, Latvia, Lithuania, Norway, the Republic of Korea, Slovenia, the United Kingdom, and the United States of America remain steadfast in our commitment to working with the World Health Organization (WHO), international experts who have a vital mission, and the global community to understand the origins of this pandemic in order to improve our collective global health security and response. Together, we support a transparent and independent analysis and evaluation, free from interference and undue influence, of the origins of the COVID-19 pandemic. In this regard, we join in expressing shared concerns regarding the recent WHO-convened study in China, while at the same time reinforcing the importance of working together toward the development and use of a swift, effective, transparent, science-based, and independent process for international evaluations of such outbreaks of unknown origin in the future.

The mission of the WHO is critical to advancing global health and health security, and we fully support its experts and staff and recognize their tireless work to bring an end to the COVID-19 pandemic, including understanding how the pandemic started and spread. With such an important mandate, it is equally essential that we voice our shared concerns that the international expert study on the source of the SARS-CoV-2 virus was significantly delayed and lacked access to complete, original data and samples. Scientific missions like these should be able to do their work under conditions that produce independent and objective recommendations and findings. We share these concerns not only for the benefit of learning all we can about the origins of this pandemic, but also to lay a pathway to a timely, transparent, evidence-based process for the next phase of this study as well as for the next health crises.

We note the findings and recommendations, including the need for further studies of animals to find the means of introduction into humans, and urge momentum for expert-driven phase 2 studies. Going forward, there must now be a renewed commitment by WHO and all Member States to access, transparency, and timeliness. In a serious outbreak of an unknown pathogen with pandemic potential, a rapid, independent, expert-led, and unimpeded evaluation of the origins is critical to better prepare our people, our public health institutions, our industries, and our governments to respond successfully to such an outbreak and prevent future pandemics. It is critical for independent experts to have full access to all pertinent human, animal, and environmental data, research, and personnel involved in the early stages of the outbreak relevant to determining how this pandemic emerged. With all data in hand, the international community may independently assess COVID-19 origins, learn valuable lessons from this pandemic, and prevent future devastating consequences from outbreaks of disease.

We underscore the need for a robust, comprehensive, and expert-led mechanism for expeditiously investigating outbreaks of unknown origin that is conducted with full and open collaboration among all stakeholders and in accordance with the principles of transparency, respect for privacy, and scientific and research integrity. We will work collaboratively and with the WHO to strengthen capacity, improve global health security, and inspire public confidence and trust in the world’s ability to detect, prepare for, and respond to future outbreaks.

https://pubmed.ncbi.nlm.nih.gov/33200842/

The genetic structure of SARS-CoV-2 does not rule out a laboratory origin: SARS-COV-2 chimeric structure and furin cleavage site might be the result of genetic manipulation

Severe acute respiratory syndrome-coronavirus (SARS-CoV)-2's origin is still controversial. Genomic analyses show SARS-CoV-2 likely to be chimeric, most of its sequence closest to bat CoV RaTG13, whereas its receptor binding domain (RBD) is almost identical to that of a pangolin CoV. Chimeric viruses can arise via natural recombination or human intervention. The furin cleavage site in the spike protein of SARS-CoV-2 confers to the virus the ability to cross species and tissue barriers, but was previously unseen in other SARS-like CoVs. Might genetic manipulations have been performed in order to evaluate pangolins as possible intermediate hosts for bat-derived CoVs that were originally unable to bind to human receptors? Both cleavage site and specific RBD could result from site-directed mutagenesis, a procedure that does not leave a trace. Considering the devastating impact of SARS-CoV-2 and importance of preventing future pandemics, researchers have a responsibility to carry out a thorough analysis of all possible SARS-CoV-2 origins.

Lethal Pneumonia Cases in Mojiang Miners (2012) and the Mineshaft Could Provide Important Clues to the Origin of SARS-CoV-2

With the COVID-19 pandemic reaching its worst heights, people are interested in the origin of SARS-CoV-2. This study started with two important questions: first, were there any similar atypical pneumonia outbreaks, even on a smaller level, reported between SARS in 2004 and COVID-19 in 2019/20 in China. Second, examining the beta-coronavirus most closely related to date with SARS-CoV-2 at the genome sequence level, strain RaTG13 (CoV4991), which was sampled from a horseshoe bat in Yunnan province, we asked where exactly did it come from. It was found that RaTG13/CoV4991 was collected from Tongguan mineshaft in Mojiang, Yunnan, China, in 2013. Surprisingly, the same mineshaft was also associated with a severe pneumonia-like illness in miners in 2012 killing three of the six miners. A Master's thesis (in the Chinese language) was found on the cnki.net website which described in detail the severe illness in miners. The thesis concluded that a SARS-like CoV originating from Chinese horseshoe bats (Rhinolophus) was the predicted causative agent. The cases were remotely monitored by a prominent pulmonologist in China. Retrospective analysis of the pneumonia cases shows striking similarities with COVID-19. Bilateral pneumonia, vascular complications like pulmonary thromboembolism, and secondary infections are the main similarities. The treatment regimes were similar to the current treatments for COVID-19. We propose that the Mojiang mineshaft miners' illness could provide important clues to the origin of SARS-CoV-2. These cases should be studied by various academicians, researchers, and medical professionals as many important questions are raised in this context.

More: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606707/

Addendum: A pneumonia outbreak associated with a new coronavirus of probable bat origin

Peng Zhou, Xing-Lou Yang, […] Zheng-Li Shi
Nature volume 588, pageE6 (2020)

Here we provide further information about the bat SARS-related coronavirus (SARSr-CoV) strain RaTG13 reported in our Article. Between 1 July and 1 October 2012, we received 13 serum samples collected from 4 patients (one of whom was deceased) who showed severe respiratory disease. These patients had visited a mine cave in Tongguan town, Mojiang County, Yunnan Province, China, to clean bat faeces in order to mine copper before being admitted to the First Affiliated Hospital of Kunming Medical University on 26–27 April 2012. The samples we received were collected by the hospital staff in June, July, August and September 2012. To investigate the cause of the respiratory disease, we tested the samples using PCR methods developed in our laboratory targeting the RNA-dependent RNA polymerases (RdRp) of Ebola virus, Nipah virus and bat SARSr-CoV Rp3, and all of the samples were negative for the presence of these viruses. We also tested the serum samples for the presence of antibodies against the nucleocapsid proteins of these three viruses, and none of the samples gave a positive result. Recently, we retested the samples with our validated enzyme-linked immunosorbent assay (ELISA) against the SARS coronavirus 2 (SARS-CoV-2) nucleocapsid protein—which has greater than 90% amino acid sequence identity with bat SARSr-CoV Rp3—and confirmed that these patients were not infected by SARS-CoV-2.

We suspected that the patients had been infected by an unknown virus. Therefore, we and other groups sampled animals including bats, rats and musk shrews in or around the cave, and found some alphacoronaviruses1 and paramyxoviruses2. Between 2012 and 2015, our group sampled bats once or twice a year in this cave and collected a total of 1,322 samples. From these samples, we detected 293 highly diverse coronaviruses, of which 284 were designated alphacoronaviruses and 9 were designated betacoronaviruses on the basis of partial RdRp sequences. All of the nine betacoronaviruses are SARSr-CoVs, one of which (sample ID4991; renamed RaTG13 in our Article to reflect the bat species, the location and the sampling year) was described in a 2016 publication1. The partial RdRp sequence (370 bp) of ID4991 was deposited in GenBank in 2016 under accession number KP876546. All of the identified bat SARSr-CoVs are distantly related to SARS-CoV based on partial RdRp sequences. In 2018, as the next-generation sequencing technology and capability in our laboratory had improved, we performed further sequencing of these bat viruses and obtained almost the full-length genome sequence (without the 5? and 3? ends) of RaTG13. In 2020, we compared the sequence of SARS-CoV-2 with our unpublished bat coronavirus sequences and found that it shared a 96.2% identity with RaTG13.

https://www.nature.com/articles/s41586-020-2951-z

China clamps down in hidden hunt for coronavirus origins

By DAKE KANG, MARIA CHENG and SAM MCNEIL
December 30, 2020

MOJIANG, China (AP) — Deep in the lush mountain valleys of southern China lies the entrance to a mine shaft that once harbored bats with the closest known relative of the COVID-19 virus.

The area is of intense scientific interest because it may hold clues to the origins of the coronavirus that has killed more than 1.7 million people worldwide. Yet for scientists and journalists, it has become a black hole of no information because of political sensitivity and secrecy.

A bat research team visiting recently managed to take samples but had them confiscated, two people familiar with the matter said. Specialists in coronaviruses have been ordered not to speak to the press. And a team of Associated Press journalists was tailed by plainclothes police in multiple cars who blocked access to roads and sites in late November.

More than a year since the first known person was infected with the coronavirus, an AP investigation shows the Chinese government is strictly controlling all research into its origins, clamping down on some while actively promoting fringe theories that it could have come from outside China.

More: https://apnews.com/article/united-nations-coronavirus-pandemic-china-only-on-ap-bats-24fbadc58cee3a40bca2ddf7a14d2955