How China’s ‘Bat Woman’ Hunted Down Viruses from SARS to the New Coronavirus
Wuhan-based virologist Shi Zhengli has identified dozens of deadly SARS-like viruses in bat caves, and she warns there are more out there
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How China’s ‘Bat Woman’ Hunted Down Viruses from SARS to the New Coronavirus
Wuhan-based virologist Shi Zhengli has identified dozens of deadly SARS-like viruses in bat caves, and she warns there are more out there
Credit: Richard Borge
Read more from this special report:
The New Coronavirus Outbreak: What We Know So Far
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Editor’s Note (4/24/20): This article was originally published online on March 11. It has been updated for inclusion in the June 2020 issue of Scientific American
and to address rumors that SARS-CoV-2 emerged from Shi Zhengli’s lab in China.
The mysterious patient samples arrived at the Wuhan Institute of Virology at 7 P.M. on December 30, 2019. Moments later Shi Zhengli’s cell phone rang. It was her boss, the institute’s director. The Wuhan Center for Disease Control and Prevention had detected a novel coronavirus in two hospital patients with atypical pneumonia, and it wanted Shi’s renowned laboratory to investigate. If the finding was confirmed, the new pathogen could pose a serious public health threat—because it belonged to the same family of viruses as the one that caused severe acute respiratory syndrome (SARS), a disease that plagued 8,100 people and killed nearly 800 of them between 2002 and 2003. “Drop whatever you are doing and deal with it now,” she recalls the director saying.
Shi, a virologist who is often called China’s “bat woman” by her colleagues because of her virus-hunting expeditions in bat caves over the past 16 years, walked out of the conference she was attending in Shanghai and hopped on the next train back to Wuhan. “I wondered if [the municipal health authority] got it wrong,” she says. “I had never expected this kind of thing to happen in Wuhan, in central China.” Her studies had shown that the southern, subtropical provinces of Guangdong, Guangxi and Yunnan have the greatest risk of coronaviruses jumping to humans from animals—particularly bats, a known reservoir. If coronaviruses were the culprit, she remembers thinking, “Could they have come from our lab?”
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While Shi’s team at the Wuhan institute, an affiliate of the Chinese Academy of Sciences, raced to uncover the identity of the contagion—over the following week they connected the illness to the novel coronavirus that become known as SARS-CoV-2—the disease spread like wildfire. By April 20 more than 84,000 people in China had been infected. About 80 percent of them lived in the province of Hubei, of which Wuhan is the capital, and more than 4,600 had died. Outside of China, about 2.4 million people across 210 or so countries and territories had caught the virus, and more than 169,000 had perished from the disease it caused, COVID-19.
Read more from this special report:The New Coronavirus Outbreak: What We Know So Far
Scientists
have long warned that the rate of emergence of new infectious diseases is accelerating—especially in developing countries where high densities of people and animals increasingly mingle and move about. “It’s incredibly important to pinpoint the source of infection and the chain of cross-species transmission,” says disease ecologist Peter Daszak, president of EcoHealth Alliance, a New York City–based nonprofit research organization that collaborates with researchers, such as Shi, in 30 countries in Asia, Africa and the Middle East to discover new viruses in wildlife. An equally important task, he adds, is to hunt down other pathogens to “prevent similar incidents from happening again.”
OUTSIDE A BAT CAVE in China's Guangxi province in 2004, Shi Zhengli releases a fruit bat after taking a blood sample. Credit: Shuyi Zhang
THE CAVES
To Shi, her first virus-discovery expedition felt like a vacation. On a breezy, sunny spring day in 2004, she joined an international team of researchers to collect samples from bat colonies in caves near Nanning, the capital of Guangxi. Her inaugural cave was typical of the region: large, rich in limestone columns and—as a popular tourist destination—easily accessible. “It was spellbinding,” Shi recalls. Milky-white stalactites hung from the ceiling like icicles, glistening with moisture.
But the holidaylike atmosphere soon dissipated. Many bats—including several insect-eating species of horseshoe bats that are abundant in southern Asia—roost in deep, narrow caves on steep terrain. Often guided by tips from local villagers, Shi and her colleagues had to hike for hours to potential sites and inch through tight rock crevasses on their stomachs. And the flying mammals can be elusive. In one frustrating week, the team explored more than 30 caves and saw only a dozen bats.
These expeditions were part of the effort to catch the culprit in the SARS outbreak, the first major epidemic of the 21st century. A Hong Kong team had
reported that wildlife traders in Guangdong first caught the SARS coronavirus from civets, mongooselike mammals that are native to tropical and subtropical Asia and Africa.
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Before SARS, the world had only an inkling of coronaviruses—so named because their spiky surface resembles a crown when seen under a microscope, says Linfa Wang, who directs the emerging infectious diseases program at Singapore’s Duke-NUS Medical School. Coronaviruses were mostly known for causing common colds. “The SARS outbreak was a game changer,” Wang says. It was the first emergence of a deadly coronavirus with pandemic potential. The incident helped to jump-start a global search for animal viruses that could find their way into humans. Shi was an early recruit of that effort, and both Daszak and Wang have been her long-term collaborators.
With the SARS virus, just how the civets got it remained a mystery. Two previous incidents were telling: Australia’s 1994 Hendra virus infections, in which the contagion jumped from horses to humans, and Malaysia’s 1998 Nipah virus outbreak, in which it moved from pigs to people. Wang found that both diseases were caused by pathogens that originated in fruit-eating bats. Horses and pigs were merely the intermediate hosts. Bats in the Guangdong market also contained traces of the SARS virus, but many scientists dismissed this as contamination. Wang, however, thought bats might be the source.
In those first virus-hunting months in 2004, whenever Shi’s team located a bat cave, it would put a net at the opening before dusk and then wait for the nocturnal creatures to venture out to feed for the night. Once the bats were trapped, the researchers took blood and saliva samples, as well as fecal swabs, often working into the small hours. After catching up on some sleep, they would return to the cave in the morning to collect urine and fecal pellets.
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But sample after sample turned up no trace of genetic material from coronaviruses. It was a heavy blow. “Eight months of hard work seemed to have gone down the drain,” Shi says. “We thought maybe bats had nothing to do with SARS.” The scientists were about to give up when a research group in a neighboring lab handed them a diagnostic kit for testing antibodies produced by people with SARS.
There was no guarantee that the test would work for bat antibodies, but Shi gave it a go anyway. “What did we have to lose?” she says.
The results exceeded her expectations. Samples from three horseshoe bat species contained antibodies to the SARS virus. “It was a turning point for the project,” Shi says. The researchers learned that the presence of the coronavirus in bats was ephemeral and seasonal—but an antibody reaction could last from weeks to years. The diagnostic kit, therefore, offered a valuable pointer as to how to hunt down viral genomic sequences.
Shi’s team used the antibody test to narrow down the list of locations and bat species to pursue in the quest for genomic clues. After roaming mountainous terrain in most of China’s dozens of provinces, the researchers turned their attention to one spot:
Shitou Cave, on the outskirts of Kunming, the capital of Yunnan, where they conducted intense sampling during different seasons over five consecutive years.
The efforts paid off. The pathogen hunters discovered hundreds of bat-borne coronaviruses with incredible genetic diversity. “The majority of them are harmless,” Shi says. But dozens belong to the
same group as SARS. They can infect human lung cells in a petri dish and cause SARS-like diseases in mice.
In Shitou Cave—where painstaking scrutiny has yielded a natural genetic library of bat-borne viruses—the team discovered a coronavirus strain that came
from horseshoe bats with a genomic sequence nearly 97 percent identical to the one found in civets in Guangdong. The finding concluded a decade-long search for the natural reservoir of the SARS coronavirus.
ON THE SAME 2004 trip, a group of researchers prepare bat blood samples that they will screen for viruses and other pathogens. Credit: Shuyi Zhang
A DANGEROUS MIX
In many bat dwellings Shi has sampled, including Shitou Cave, “constant mixing of different viruses creates a great opportunity for dangerous new pathogens to emerge,” says Ralph Baric, a virologist at the University of North Carolina at Chapel Hill. In the vicinity of such viral melting pots, Shi says, “you don’t need to be a wildlife trader to be infected.”
Near Shitou Cave, for example, many villages sprawl among the lush hillsides in a region known for its roses, oranges, walnuts and hawthorn berries. In October 2015 Shi’s team collected blood samples from more than 200 residents in four of those villages. It
found that six people, or nearly 3 percent, carried antibodies against SARS-like coronaviruses from bats—even though none of them had handled wildlife or reported SARS-like or other pneumonialike symptoms. Only one had traveled outside of Yunnan prior to the sampling, and all said they had seen bats flying in their village.
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Three years earlier Shi’s team had been called in to investigate the virus profile of a mine shaft in Yunnan’s mountainous Mojiang County—famous for its fermented Pu’er tea—where six miners suffered from pneumonialike diseases and two died. After sampling the cave for a year, the researchers discovered a
diverse group of coronaviruses in six bat species. In many cases, multiple viral strains had infected a single animal, turning it into a flying factory for new viruses.
“The mine shaft stunk like hell,” says Shi, who, like her colleagues, went in wearing a protective mask and clothing. “Bat guano, covered in fungus, littered the cave.” Although the fungus turned out to be the pathogen that had sickened the miners, she says it would have been only a matter of time before they caught the coronaviruses if the mine had not been promptly shut.
With growing human populations increasingly encroaching on wildlife habitats, with unprecedented changes in land use, with wildlife and livestock transported across countries and their products around the world, and with sharp increases in both domestic and international travel, pandemics of new diseases are a mathematical near certainty. This had been keeping Shi and many other researchers awake at night long before the mysterious samples landed at the Wuhan Institute of Virology on that ominous evening last December.
More than a year ago Shi’s team published two comprehensive reviews about coronaviruses in
Viruses and
Nature Reviews Microbiology. Drawing evidence from her own studies—many of which were published in top academic journals—and from others, Shi and her co-authors warned of the risk of future outbreaks of bat-borne coronaviruses.
NIGHTMARE SCENARIO
On the train back to Wuhan on December 30 last year, Shi and her colleagues discussed ways to immediately start testing the patients’ samples. In the following weeks—the most intense and the most stressful time of her life—China’s bat woman felt she was fighting a battle in her worst nightmare, even though it was one she had been preparing for over the past 16 years. Using a technique called polymerase chain reaction, which can detect a virus by amplifying its genetic material, the team found that samples from five of seven patients had genetic sequences present in all coronaviruses.
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Shi instructed her group to repeat the tests and, at the same time, sent the samples to another facility to sequence the full viral genomes. Meanwhile she frantically went through her own lab’s records from the past few years to check for any mishandling of experimental materials, especially during disposal. Shi breathed a sigh of relief when the results came back: none of the sequences matched those of the viruses her team had sampled from bat caves. “That really took a load off my mind,” she says. “I had not slept a wink for days.”
By January 7 the Wuhan team had determined that the new virus had indeed caused the disease those patients suffered—a conclusion based on results from analyses using polymerase chain reaction, full genome sequencing, antibody tests of blood samples and the virus’s ability to infect human lung cells in a petri dish. The genomic sequence of the virus, eventually named SARS-CoV-2, was 96 percent identical to that of a coronavirus the researchers had identified in horseshoe bats in Yunnan. Their results appeared in a
paper published online on February 3 in
Nature. “It’s crystal clear that bats, once again, are the natural reservoir,” says Daszak, who was not involved in the study.
Since then, researchers have published more than 4,500 genomic sequences of the virus, showing that samples around the world appear to “share a common ancestor,” Baric says. The data also point to a single introduction into humans followed by sustained human-to-human transmission, researchers say.
Given that the virus seems fairly stable initially and that many infected individuals appear to have mild symptoms, scientists suspect that the pathogen might have been around for weeks or even months before severe cases raised the alarm. “There might have been mini outbreaks, but the viruses either burned out or maintained low-level transmission before causing havoc,” Baric says. Most animal-borne viruses reemerge periodically, he adds, so “the Wuhan outbreak is by no means incidental.”
IN YUNNAN PROVINCE, CHINA, scientists from EcoHealth Alliance, an international group that searches for diseases that can jump from animals to people, hunt for pathogens in a bat cave. Credit: EcoHealth Alliance
MARKET FORCES
To many, the region’s burgeoning wildlife markets—which sell a wide range of animals such as bats, civets, pangolins, badgers and crocodiles—are perfect viral melting pots. Although humans could have caught the deadly virus from bats directly (according to several studies, including
those by Shi and her colleagues), independent teams have suggested that
pangolins may have been an intermediate host. These teams have reportedly uncovered
SARS-CoV-2-like coronaviruses in pangolins that were seized in antismuggling operations in southern China.
On February 24 China announced a
permanent ban on wildlife consumption and trade except for research, medicinal or display purposes—which will stamp out an industry worth $76 billion and put approximately 14 million people out of jobs, according to a 2017 report commissioned by the Chinese Academy of Engineering. Some welcome the initiative, whereas others, such as Daszak, worry that without efforts to change people’s traditional beliefs or to provide alternative livelihoods, a blanket ban may simply push the business underground. This could make disease detection even more challenging. “Eating wildlife has been part of the cultural tradition” in China for thousands of years, Daszak says. “It won’t change overnight.”
In any case, Shi says, “wildlife trade and consumption are only part of problem.” In late 2016 pigs across four farms in Qingyuan County in Guangdong—60 miles from the site where the SARS outbreak originated—suffered from acute vomiting and diarrhea, and nearly 25,000 of the animals died. Local veterinarians could not detect any known pathogen and called Shi for help. The cause of the illness—swine acute diarrhea syndrome (SADS)—turned out to be a virus whose genomic sequence was
98 percent identical to that of a coronavirus found in horseshoe bats in a nearby cave.
“This is a serious cause for concern,” says Gregory Gray, an infectious disease epidemiologist at Duke University. Pigs and humans have very similar immune systems, making it easy for viruses to cross between the two species. Moreover, a team at Zhejiang University in the Chinese city of Hangzhou
found that the SADS virus could infect cells from many organisms in a petri dish, including rodents, chickens, nonhuman primates and humans. Given the scale of swine farming in many countries, such as China and the U.S., Gray says, looking for novel coronaviruses in pigs should be a top priority.
The current outbreak follows several others during the past three decades that have been caused by six different bat-borne viruses: Hendra, Nipah, Marburg, SARS-CoV, MERS-CoV (Middle East respiratory syndrome) and Ebola. But “the animals [themselves] are not the problem,” Wang says. In fact, bats promote biodiversity and ecosystem health by eating insects and pollinating plants. “The problem arises when we get in contact with them,” he says.
TOWARD PREVENTION
When I spoke to Shi in late February—two months into the epidemic and one month after the government imposed severe movement restrictions in Wuhan, a megacity of 11 million—she said, laughing, that life felt almost normal. “Maybe we are getting used to it. The worst days are certainly over.” The institute staffers had a special pass to travel from home to their lab, but they could not go anywhere else. They had to subsist on instant noodles during their long hours at work because the institute’s canteen was closed.
New revelations about the coronavirus kept coming to light. The researchers discovered, for instance, that the pathogen enters human lung cells by using a receptor called angiotensin-converting enzyme 2, and they and other groups have since been screening for drugs that can block it. Scientists are also racing to develop vaccines. In the long run, the Wuhan team plans to develop
broad-spectrum vaccines and drugs against coronaviruses deemed risky to humans. “The Wuhan outbreak is a wake-up call,” Shi says.
Many scientists say the world should move beyond merely responding to deadly pathogens when they arise. “The best way forward is prevention,” Daszak says. Because 70 percent of emerging infectious diseases of animal origins
come from wildlife, a top priority should be identifying them and developing better diagnostic tests, he adds. Doing so would essentially mean continuing on a
much larger scale what researchers such as Daszak and Shi had been doing before their
funding ended this year.
Such efforts should focus on high-risk viral groups in mammals prone to coronavirus infections, such as bats, rodents, badgers, civets, pangolins and nonhuman primates, Daszak says. He adds that developing countries in the tropics, where wildlife diversity is greatest, should be the
front line of this battle against viruses.
Daszak and his colleagues have analyzed approximately 500 human infectious diseases from the past century. They found that the emergence of new pathogens tends to happen in places where a dense population has been
changing the landscape—by building roads and mines, cutting down forests and intensifying agriculture. “China is not the only hotspot,” he says, noting that other major emerging economies, such as India, Nigeria and Brazil, are also at great risk.
Once potential pathogens are mapped out, scientists and public health officials can regularly check for possible infections by analyzing blood and swab samples from livestock, from wild animals that are farmed and traded, and from high-risk human populations such as farmers, miners, villagers who live near bats, and people who hunt or handle wildlife, Gray says. This approach, known as “
One Health,” aims to integrate the health management of wildlife, livestock and people. “Only then can we catch an outbreak before it turns into an epidemic,” he says, adding that the strategy could potentially save the hundreds of billions of dollars such an epidemic can cost.
Back in Wuhan, where the lockdown was finally lifted on April 8, China’s bat woman is not in a celebratory mood. She is distressed because stories from the Internet and major media have repeated a tenuous suggestion that SARS-CoV-2 accidentally leaked from her lab—despite the fact that its genetic sequence does not match any her lab had previously studied. Other scientists are quick to dismiss the allegation. “Shi leads a world-class lab of the highest standards,” Daszak says.
Despite the disturbance, Shi is determined to continue her work. “The mission must go on,” she says. “What we have uncovered is just the tip of an iceberg.” She is planning to lead a national project to systematically sample viruses in bat caves, with much wider scope and intensity than previous attempts. Daszak’s team has estimated that there are
more than 5,000 coronavirus strains waiting to be discovered in bats globally.
“Bat-borne coronaviruses will cause more outbreaks,” Shi says with a tone of brooding certainty. “We must find them before they find us.”
#21
Discovery of a Novel Coronavirus, China Rattus Coronavirus HKU24, from Norway Rats Supports the Murine Origin of Betacoronavirus 1 and Has Implications for the Ancestor of Betacoronavirus Lineage A
We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative...
jvi.asm.org
More coronaviruses which were published. Enough of their lies.
Genetic Diversity and Evolution
Discovery of a Novel Coronavirus, China Rattus Coronavirus HKU24, from Norway Rats Supports the Murine Origin of Betacoronavirus 1 and Has Implications for the Ancestor of Betacoronavirus Lineage A
Susanna K. P. Lau, Patrick C. Y. Woo, Kenneth S. M. Li, Alan K. L. Tsang, Rachel Y. Y. Fan, Hayes K. H. Luk, Jian-Piao Cai, Kwok-Hung Chan, Bo-Jian Zheng, Ming Wang, Kwok-Yung Yuen
R. M. Sandri-Goldin, Editor
DOI: 10.1128/JVI.02420-14
ABSTRACT
We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (βCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B βCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by βCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A βCoVs.
IMPORTANCE While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A βCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A βCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A βCoVs.
INTRODUCTION
Coronaviruses (CoVs) infect a wide variety of animals, including humans, causing respiratory, enteric, hepatic, and neurological diseases of various severities. On the basis of genotypic and serological characterization, CoVs were traditionally classified into three distinct groups (
1,
2). Recently, the Coronavirus Study Group of the International Committee on Taxonomy of Viruses (ICTV) has revised the nomenclature and taxonomy to reclassify the three CoV groups into three genera, Alphacoronavirus, Betacoronavirus, and Gammacoronavirus (
3). Novel CoVs, which represent a novel genus, Deltacoronavirus, have also been identified (
4–6). As a result of the ability to use a variety of host receptors and evolve rapidly through mutation and recombination, CoVs are able to adapt to new hosts and ecological niches, causing a wide spectrum of diseases (
2,
7–12).
The severe acute respiratory syndrome (SARS) epidemic and identification of SARS-CoV-like viruses from palm civets and horseshoe bats in China have boosted interest in the discovery of novel CoVs in both humans and animals (
13–20). It is now known that CoVs from all four genera can be found in mammals. Historically, alphacoronaviruses (αCoVs) and betacoronaviruses (βCoVs) have been found in mammals, while gammacoronaviruses (γCoVs) have been found in birds. However, recent findings also suggested the presence of γCoVs in mammals (
5,
21,
22). Although deltacoronaviruses (δCoVs) are also mainly found in birds, potential mammalian δCoVs have been reported (
4,
23). In particular, a δCoV closely related to sparrow CoV HKU17, porcine CoV HKU15, has been identified in pigs, which suggested bird-to-mammal transmission (
4). On the basis of current findings, a model for CoV evolution was proposed, where bat CoVs are likely the gene source of Alphacoronavirus and Betacoronavirus and avian CoVs are the gene source of Gammacoronavirus and Deltacoronavirus (
4). However, one notable exception to this model is Betacoronavirus lineage A.
The genus Betacoronavirus consists of four lineages, A to D. While human coronavirus (HCoV) OC43 and HCoV HKU1 belong to Betacoronavirus lineage A (
20,
24–27), SARS-CoV belongs to Betacoronavirus lineage B and the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to Betacoronavirus lineage C. No human CoV has yet been identified from Betacoronavirus lineage D. On the other hand, besides Alphacoronavirus, diverse bat CoVs have been found in Betacoronavirus lineage B (e.g., SARS-related Rhinolophus bat CoVs), lineage C (e.g., Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5), and lineage D (e.g., Rousettus bat CoV HKU9) (
8,
14,
15,
28–37), supporting the suggestion that bat CoVs are likely the ancestral origin of other mammalian CoVs in these lineages. However, no bat CoVs belonging to Betacoronavirus lineage A have yet been identified, despite the numerous surveillance studies on bat CoVs conducted in various countries over the years (
38). Therefore, the ancestral origin of the mammalian lineage A βCoVs, such as HCoV OC43 and HCoV HKU1, remains obscure.
While HCoV OC43 is likely to have originated from zoonotic transmission, sharing a common ancestor with bovine coronavirus (BCoV) that dates back to 1890 (
27,
30,
39), closely related CoVs belonging to the same species, Betacoronavirus 1, have also been found in various mammals, including pigs, horses, dogs, waterbucks, sable antelope, deer, giraffes, alpaca, and dromedary camels, suggesting a common ancestor in mammals with subsequent frequent interspecies transmission (
40–47). Although no zoonotic origin of HCoV HKU1 has been identified, the virus is most closely related to mouse hepatitis virus (MHV) and rat coronavirus (RCoV), which together are now classified as murine coronavirus (
3,
20,
42). We therefore hypothesize that rodent CoVs are the ancestral origin of Betacoronavirus lineage A. In this study, we tested samples from various rodent species in Hong Kong and southern China for the presence of lineage A βCoVs. A novel CoV, China Rattus coronavirus (ChRCoV) HKU24, was discovered from Norway rats in Guangzhou, China. Complete genome analysis showed that ChRCoV HKU24 represents a novel species within Betacoronavirus lineage A but possesses features that resemble those of both Betacoronavirus 1 and murine coronavirus. A high seroprevalence was also demonstrated among Norway rats from Guangzhou using Western blot analysis against ChRCoV HKU24 recombinant N protein and spike polypeptide. The present results suggest that ChRCoV HKU24 likely represents the murine origin of Betacoronavirus 1 and provides insights into the ancestor of Betacoronavirus lineage A.
MATERIALS AND METHODS
Sample collection.All rodent samples were collected from January 2010 to August 2012 using procedures described previously (
5,
14). Samples from southern China were collected from animal markets or restaurants. Samples from Hong Kong were collected from wild and street rodents by the Agriculture, Fisheries and Conservation Department and the Food and Environmental Hygiene Department of the Hong Kong Special Administrative Region (HKSAR), respectively. Alimentary tract samples were placed in viral transport medium containing Earle's balanced salt solution (Invitrogen, NY, USA), 20% glucose, 4.4% NaHCO3, 5% bovine albumin, 50,000 μg/ml vancomycin, 50,000 μg/ml amikacin, and 10,000 units/ml nystatin, before transportation to the laboratory for RNA extraction. The study was approved by the Committee on the Use of Live Animals for Teaching and Research, The University of Hong Kong, and the Institutional Review Board, The University of Hong Kong/Hospital Authority.
RNA extraction.Viral RNA was extracted from the samples using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany). The RNA was eluted in 60 μl of buffer AVE and was used as the template for reverse transcription-PCR (RT-PCR).
RT-PCR of the RdRp gene of CoVs using conserved primers and DNA sequencing.Initial CoV screening was performed by amplifying a 440-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene of CoVs using conserved primers (5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-CCATCATCAGATAGAATCATCATA-3′) designed by the use of multiple-sequence alignments of the nucleotide sequences of available RdRp genes of known CoVs (
14,
20). Reverse transcription was performed using a SuperScript III kit (Invitrogen, San Diego, CA, USA). The PCR mixture (25 μl) contained cDNA, PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.01% gelatin), 200 μM each deoxynucleoside triphosphate, and 1.0 U Taq polymerase (Applied Biosystems, Foster City, CA, USA). The mixtures were amplified by 60 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystems, Foster City, CA, USA). Standard precautions were taken to avoid PCR contamination, and no false-positive result was observed for the negative controls.
PCR products were gel purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA), using the two PCR primers. The sequences of the PCR products were compared with known sequences of the RdRp genes of CoVs in the GenBank database.
Viral culture.The three rodent samples positive for ChRCoV HKU24 by RT-PCR were subject to virus isolation in Huh-7.5 (human hepatoma), Vero E6 (African green monkey kidney), HRT-18G (human rectum epithelial), BSC-1 (African green monkey renal epithelial), RK13 (rabbit kidney), MDBK (bovine kidney), NIH 3T3 (mouse embryonic fibroblast), J774 (mouse macrophage), BHK-21 (baby hamster kidney), RK3E (rat kidney), RMC (rat kidney mesangial), RAW 264.7 (mouse macrophage), and primary SD rat lung cells as described previously (
48,
49).
Real-time RT-PCR quantitation.Real-time RT-PCR was performed on rodent samples positive for ChRCoV HKU24 by RT-PCR using previously described procedures (
14). Reverse transcription was performed using the SuperScript III kit with random primers (Invitrogen, San Diego, CA, USA). cDNA was amplified in a LightCycler instrument with a FastStart DNA Master SYBR green I mix reagent kit (Roche Diagnostics GmbH, Mannheim, Germany) using specific primers (5′-ACAGGTTCTCCCTTTATAGATGAT-3′ and 5′-TCTCCTGTATAGTAGCAGAAGCAT-3′) targeting the RdRp gene of ChRCoV HKU24 using procedures described previously (
14,
50). For quantitation, a reference standard was prepared using the pCRII-TOPO vector (Invitrogen, San Diego, CA, USA) containing the target sequence. Tenfold dilutions equivalent to 3.77 to 3.77 × 109 copies per reaction were prepared to generate concomitant calibration curves. At the end of the assay, PCR products (a 133-bp fragment of RdRp) were subjected to melting curve analysis (65 to 95°C, 0.1°C/s) to confirm the specificity of the assay. The detection limit of this assay was 3.77 copies per reaction.
Complete genome sequencing.Three complete genomes of ChRCoV HKU24 were amplified and sequenced using the RNA extracted from the original alimentary tract samples as the templates. The RNA was converted to cDNA by a combined random priming and oligo(dT) priming strategy. The cDNA was amplified by degenerate primers designed by multiple-sequence alignments of the genomes of other CoVs for which complete genomes are available, using strategies described in our previous publications (
14,
20,
35,
49) and the CoVDB CoV database (
51) for sequence retrieval. Additional primers were designed from the results of the first and subsequent rounds of sequencing. These primer sequences are available on request. The 5′ ends of the viral genomes were confirmed by rapid amplification of cDNA ends (RACE) using a 5′/3′ RACE kit (Roche Diagnostics GmbH, Mannheim, Germany). Sequences were assembled and manually edited to produce the final sequences of the viral genomes.
Genome analysis.The nucleotide sequences of the genomes and the deduced amino acid sequences of the open reading frames (ORFs) were compared to those of other CoVs for which complete genomes are available using the sequences in CoVDB (
51). Phylogenetic tree construction was performed using the maximum likelihood method and PhyML software, with bootstrap values being calculated from 100 trees. Protein family analysis was performed using the PFAM and InterProScan tools (
52,
53). Prediction of transmembrane domains was performed using the TMHMM server (
54). The structure of the ChRCoV HKU24 N-terminal domain (NTD) was predicted using a web-based homology-modeling server, SWISS-MODEL. A BLASTp search against the sequences in the Protein Data Bank (PDB) was performed with the default parameters to find suitable templates for homology modeling. On the basis of the higher sequence identity, the QMEAN Z-score, coverage, and lower E value, the crystal structure of the BCoV NTD (PDB accession number
4H14) was selected as the template. The predicted structure was visualized using the Jmol viewer.
Estimation of divergence dates.The divergence time was calculated on the basis of complete RdRp and HE gene sequence data using a Bayesian Markov chain Monte Carlo (MCMC) approach implemented in the BEAST (version 1.8.0) package, as described previously (
49,
55,
56). One parametric model (Constant Size) and one nonparametric model (Bayesian Skyline) tree priors were used for inference. Analyses were performed under the SRD06 model and by the use of both a strict and a relaxed molecular clock. The MCMC run was 2 × 108 steps long with sampling every 1,000 steps. Convergence was assessed on the basis of the effective sampling size after a 10% burn-in using Tracer (version 1.5) software (
55). The mean time of the most recent common ancestor (tMRCA) and the highest posterior density regions at 95% (HPDs) were calculated, and the best-fitting models were selected by use of a Bayes factor and marginal likelihoods implemented in Tracer (
56). Bayesian skyline under a relaxed-clock model with an uncorrelated exponential distribution was adopted for making inferences, as Bayes factor analysis for the RdRp and hemagglutinin-esterase (HE) genes indicated that this model fitted the data better than the other models tested. The tree was summarized in a target tree by the Tree Annotator program included in the BEAST package by choosing the tree with the maximum sum of posterior probabilities (maximum clade credibility) after a 10% burn-in.
Cloning and purification of His6-tagged recombinant ChRCoV HKU24 nucleocapsid protein and spike polypeptide.To produce fusion plasmids for protein purification, primers 5′-CTAGCTAGCATGTCTCATACGCCA-3′ and 5′-CTAGCTAGCTTATATTTCTGAGCTTCCC-3′ and primers 5′-CTAGCTAGCCAACCAATAGCAGATGTGTA-3′ and 5′-CTAGCTAGCTTATCTCTTGGCTCGCCATGT-3′ were used to amplify the nucleocapsid gene and a partial S1 fragment encoding amino acid residues 317 to 763 of the spike protein of ChRCoV HKU24, respectively, as described previously (
31,
49,
57,
58). The sequences, coding for a total of 443 amino acid (aa) and 447 aa residues, respectively, were amplified and cloned into the NheI site of expression vector pET-28b(+) (Merck, KGaA, Darmstadt, Germany) in frame and downstream of the series of six histidine residues. The His6-tagged recombinant nucleocapsid protein and the spike polypeptide were expressed and purified using Ni-nitrilotriacetic acid affinity chromatography (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Western blot analysis.To detect the presence of antibodies against the ChRCoV HKU24 N protein and spike polypeptide in rodent sera and to test for possible cross antigenicity between ChRCoV HKU24 and other βCoVs, 600 ng of purified His6-tagged recombinant N protein or spike polypeptide of ChRCoV HKU24 was loaded into the well of a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and subsequently electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The blot was cut into strips, and the strips were incubated separately with 1:2,000, 1:4,000, or 1:8,000 dilutions of sera collected from rodents for which serum samples were available, human sera from two patients with HCoV OC43 infection, sera from two rabbits with rabbit coronavirus (RbCoV) HKU14 infection, and sera from two patients with SARS-CoV infection. The antigen-antibody interaction was detected with 1:4,000 horseradish peroxidase-conjugated anti-rat IgG, anti-human IgG, or anti-rabbit IgG (Zymed) and an ECL fluorescence system (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) as described previously (
14,
58).
Nucleotide sequence accession numbers.The nucleotide sequences of the three genomes of ChRCoV HKU24 have been lodged within the GenBank sequence database under accession no.
KM349742 to
KM349744.
RESULTS
Identification of a novel CoV from Norway rats in China.Of 91 alimentary tract samples from rodents in China, RT-PCR for a 440-bp fragment in the RdRp gene of CoVs was positive for a potentially novel CoV in 3 samples from Norway rats (Rattus norvegicus) from a restaurant in Guangzhou (
Table 1). None of the 573 alimentary tract samples from rodents in Hong Kong, including those from Norway rats, was positive for CoVs. Sequencing results suggested that the potentially novel virus was most closely related to MHV with ≤85% nucleotide sequence identities and members of the species Betacoronavirus 1, including HCoV OC43, BCoV, equine coronavirus (ECoV) and porcine hemagglutinating encephalomyelitis virus, with ≤84% nucleotide sequence identities. Quantitative RT-PCR showed that the viral load in the positive samples ranged from 1.2 × 103 to 1.3 × 106 copies/g. Attempts to stably passage ChRCoV HKU24 in cell cultures were unsuccessful, with no cytopathic effect or viral replication being detected.
TABLE 1
Detection of ChRCoV HKU24 in rodents by RT-PCR and serological studies by Western blotting
Genome organization and coding potential of ChRCoV HKU24.Complete genome sequence data for the three strains of ChRCoV HKU24 were obtained by assembly of the sequences of the RT-PCR products from the RNA directly extracted from the corresponding individual specimens. The three genomes shared >99% nucleotide sequence similarity. Their genome size was 31,234 bases, with the G+C content (40%) being closer to that of murine coronavirus than to that of Betacoronavirus 1 (
Table 2). The genome organization was similar to that of other lineage A βCoVs and had the characteristic gene order 5′-replicase ORF1ab, hemagglutinin-esterase (HE), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′ (
Table 2 and
Fig. 1). Moreover, additional ORFs coding for nonstructural (NS) proteins NS2a, NS4, NS5, and N2 were found. A putative transcription regulatory sequence (TRS) motif, 5′-CUAAAC-3′, similar to that of αCoVs and the motif 5′-UCUAAAC-3′ in other lineage A βCoVs, was identified at the 3′ end of the leader sequence and preceded each ORF except the ORFs for the NS4, E, and N2 genes (
Table 3) (
26,
49,
59–61). However, there were base mismatches for HE and NS5, with alternative TRS motifs, 5′-CUGAAC-3′ and 5′-GUAAAC-3′, respectively, being detected.
TABLE 2
Comparison of genomic features of ChRCoV HKU24 and other CoVs for which complete genome sequences are available and amino acid identities between the predicted proteins of ChRCoV HKU24 and the corresponding proteins of other CoVs
FIG 1
Comparison of genome organizations of ChRCoV HKU24, MHV, HCoV OC43, and HCoV HKU1. Papain-like proteases 1 and 2 (PL1pro and PL2pro, respectively) are represented by orange boxes. The residues at the cleavage site are indicated above or below the boundary of each nonstructural protein. The unique cleavage site in ChRCoV HKU24 is in bold.
TABLE 3
Coding potential and predicted domains in different proteins of ChRCoV HKU24
The coding potential and characteristics of putative nonstructural proteins (nsp's) of ORF1 of ChRCoV HKU24 are shown in
Tables 3 and
4. The ORF1 polyprotein possessed 68.6 to 75.0% amino acid sequence identities to the amino acid sequences of the polyproteins of other lineage A βCoVs. It possessed a unique putative cleavage site, G/L, between nsp1 and nsp2, in contrast to the G/V found in the other lineage A βCoVs except HCoV HKU1, which possessed a G/I cleavage site (
Table 4 and
Fig. 1). Other predicted cleavage sites were mostly conserved between ChRCoV HKU24 and other lineage A βCoVs. However, the lengths of nsp1, nsp2, nsp3, nsp13, nsp15, and nsp16 in ChRCoV HKU24 differed from those of the corresponding nsp's in members of Betacoronavirus 1 and murine coronavirus, as a result of deletions or insertions.
TABLE 4
Cleavage site used between nsp's in lineage A βCoVs
All lineage A βCoVs except HCoV HKU1 possess the NS2a gene between ORF1ab and the HE gene. Unlike RbCoV HKU14, in which the gene for NS2a is broken into several small ORFs (
49), ChRCoV HKU24 is predicted to possess a single NS2a protein, as in other lineage A βCoVs. This NS2a protein displayed from 43.7 to 62.0% amino acid sequence identities to the NS2a amino acid sequences of members of Betacoronavirus 1 and 45.7 to 47.3% amino acid sequence identities to the NS2a amino acid sequences of members of murine coronavirus. Although the βCoV-specific NS2 protein has been shown to be nonessential for
in vitro viral replication (
62), cyclic phosphodiesterase domains have been predicted in the NS2 proteins of some CoVs and toroviruses, and a possible role in viral pathogenicity has been suggested in MHV (
63,
64). In contrast to MHV and RCoV, such a domain was not found in ChRCoV HKU24.
Similar to other CoV S proteins, the S protein of ChRCoV HKU24 is predicted to be a type I membrane glycoprotein, with most of the protein (residues 16 to 1302) being exposed on the outside of the virus and with a transmembrane domain (residues 1303 to 1325) occurring at the C terminus (
Fig. 2). Two heptad repeats (HRs), important for membrane fusion and viral entry, were located at residues 1045 to 1079 (HR1) and 1253 to 1285 (HR2). The S protein of ChRCoV HKU24 possessed 66.7 to 69.6% amino acid sequence identities to the amino acid sequences of members of Betacoronavirus 1 and 62.4 to 64.3% amino acid sequence identities to the amino acid sequences of members of murine coronavirus. The amino acid sequence identities between the ChRCoV HKU24 NTD and the BCoV and MHV NTDs were 61 and 56%, respectively. BCoV and HCoV OC43 utilize
N-acetyl-9-
O-acetylneuramic acid as a receptor for the initiation of infection (
65,
66). In contrast, MHV utilizes carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as a receptor, and its receptor-binding domain does not bind sugars (
10,
67,
68). Recent structural studies showed that, among the four critical sugar-binding residues in BoV, a Glu → Gly substitution which may explain the reduction in sugar-binding affinity was found in one residue in MHV. In ChRCoV HKU24, a Glu → Ser substitution is found at this position (
Fig. 2). Comparison of the amino acid sequences between the S proteins of ChRCoV HKU24 and MHV showed that ChRCoV HKU24 possessed many amino acid substitutions in the region corresponding to the MHV NTD (
Fig. 2). In particular, 12 of the 14 important contact residues at the MHV NTD/murine CEACAM1a (mCEACAM1a) interface were not conserved between ChRCoV HKU24 and MHV. Similar to the MHV and BCoV NTDs, the ChRCoV HKU24 NTD is also predicted, using homology modeling, to contain a core structure with a β-sandwich fold like that in human galectins (galactose-binding lectins) (
10). Modeling showed that the β-sandwich core structure of ChRCoV HKU24 consists of one six-stranded β sheet and one seven-stranded β sheet that are stacked together through hydrophobic interactions (
Fig. 2). In addition, the S protein of ChRCoV HKU24 possessed a unique predicted cleavage site, RAKR, among lineage A βCoVs.
FIG 2
Predicted model of ChRCoV HKU24 spike protein and NTD using the SWISS-MODEL tool. (A) Predicted domain structure of ChRCoV HKU24 spike protein. NTD, N-terminal domain; RBD, receptor-binding domain; HR, heptad repeat; TM, transmembrane anchor. The signal peptide corresponds to residues 1 to 15 and is cleaved during molecular maturation. (B) Sequence alignment of the ChRCoV HKU24 NTD with the BCoV, HCoV OC43, and MHV NTDs, performed using the PROMALS3D program. The three strains of ChRCoV HKU24 characterized in this study are in bold. β strands are shown as yellow arrows, and the alpha helix is shown as a red wavy line. Loops 10 and 11 are boxed. The 14 contact residues at the MHV NTD/mCEACAM1a interface are highlighted in blue, the four BCoV critical sugar-binding residues are highlighted in brown, and the BCoV noncritical sugar-binding residues are highlighted in yellow. The location of the residue substitution that might decrease the sugar-binding affinity of BCoV NTD is marked by an inverted triangle. Asterisks, positions that have fully conserved residues; colons, positions that have strongly conserved residues; periods, positions that have weakly conserved residues. (C) Predicted structure of the ChRCoV HKU24 NTD constructed through homology modeling from BCoV NTD (4h14) and close-up view of the pocket above the β-sandwich core. The global model quality estimation score of 0.83 and QMEAN4 Z-score of −1.82 indicate reliable overall model quality.
Other predicted domains in the HE, S, NS4, NS5, E, M, and N proteins of ChRCoV HKU24 are summarized in
Table 3 and
Fig. 1. The NS4 of ChRCoV HKU24 shared 37 to 42% amino acid sequence identity to the NS4 proteins of members of murine coronavirus. In most members of Betacoronavirus 1, NS4 is split into smaller proteins. NS5 of ChRCoV HKU24 is homologous to NS5/NS5a of members of Betacoronavirus 1, having 47.7% to 51.4% amino acid sequence identities, but it has only 39.5% amino acid sequence identity to NS5 of members of MHV. Interestingly, NS5 is not found in the genome of RCoV. The absence of a preceding TRS upstream of the E of ChRCoV HKU24 suggests that the translation of this E protein may be cap independent via an internal ribosomal entry site (IRES), as demonstrated in MHV (
69). Similarly, the E proteins of RCoV and HCoV HKU1 were also not preceded by a TRS. This is in contrast to the findings for members of Betacoronavirus 1, which possess a preceding TRS upstream of their E proteins (
49,
61). Downstream of the N gene, the 3′ untranslated region contains a predicted bulged stem-loop structure of 69 nucleotides (nt; nucleotide positions 30944 to 31012) that is conserved in βCoVs (
70). Overlapping with the bulged stem-loop structure by 5 nt, a conserved pseudoknot structure (nucleotide positions 31008 to 31059) that is important for CoV replication is found. Since nonstructural proteins in CoVs may possess unique functions for replication and virulence (
71,
72), further studies are warranted to understand the potential function of the nsp's and NS proteins in ChRCoV HKU24.
Phylogenetic analyses.Phylogenetic trees constructed using the amino acid sequences of the RdRp, S, and N proteins of ChRCoV HKU24 and other CoVs are shown in
Fig. 3, and the corresponding pairwise amino acid sequence identities are shown in
Table 2. For all three genes, the three ChRCoV HKU24 strains formed a distinct cluster among lineage A βCoVs, occupying a deep branch at the root of members of the species Betacoronavirus 1 and being the most closely related to members of the species Betacoronavirus 1. Comparison of the amino acid sequences of the seven conserved replicase domains for CoV species demarcation (
3), ADP-ribose 1″-phosphatase (ADRP), nsp5 (3C-like protease [3CLpro]), nsp12 (RdRp), nsp13 (Hel), nsp14 (exonuclease [ExoN]), nsp15 (nidoviral uridylate-specific endoribonuclease [NendoU]), and nsp16 (2′-
O-ribose methyltransferase [O-MT]), showed that these domains of ChRCoV HKU24 possessed 69.5 to 81.7%, 82.2 to 86.8%, 88.1 to 92.6%, 88.9 to 94.8%, 80.2 to 88.7%, 70.1 to 79.5%, and 83.8 to 89.7% amino acid identities to those of other lineage A βCoVs, respectively (
Table 5). Based on the present results, we propose a novel species, ChRCoV HKU24, to describe this virus under Betacoronavirus lineage A and distinguish it from RCoV.
FIG 3
Phylogenetic analyses of the RdRp, S, N, and HE proteins of ChRCoV HKU24. The trees were constructed by the maximum likelihood method using the WAG+I+G substitution model and bootstrap values calculated from 100 trees. Bootstrap values below 70% are not shown. Nine hundred twenty-eight, 1,358, 443, and 425 aa positions in RdRp, S, N, and HE, respectively, were included in the analyses. The scale bar represents 0.3 substitution per site. The three strains of ChRCoV HKU24 characterized in this study are in bold. Definitions of the abbreviations are provided in footnote
a of
Table 2.
TABLE 5
Pairwise comparisons of Coronaviridae-wide conserved domains in replicase polyprotein 1ab between ChRCoV HKU24 and other lineage A betacoronaviruses
HE proteins are glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting as both lectins and receptor-destroying enzymes which aid viral detachment from sugars on infected cells (
68,
73). Related HEs have been found in influenza C viruses, toroviruses, and lineage A βCoVs, but not other CoVs. It has been suggested that HEs of lineage A βCoVs have arisen from an influenza C virus-like HE fusion protein, likely as a result of relatively recent lateral gene transfer events (
73). Phylogenetic analysis of the HE proteins of lineage A βCoVs, toroviruses, and influenza C viruses showed that they fell into three separate clusters (
Fig. 3). The HE of ChRCoV HKU24 also forms a deep branch at the root of all members of the species Betacoronavirus 1 except ECoV and is distinct from members of murine coronavirus. Previous studies have demonstrated the heterogeneity of gene expression of HE proteins among different MHV strains (
74). Since the HE of ChRCoV HKU24 is not preceded by a perfectly matched TRS, further studies are required to determine if it is expressed and functional.
Estimation of divergence dates.Using the uncorrelated relaxed-clock model on complete RdRp gene sequences, the date of tMRCA of ChRCoV HKU24, members of Betacoronavirus 1, and RbCoV HKU14 was estimated to be 1402 (HPDs, 918.05 to 1749.91) (
Fig. 4). The date of divergence between HCoV OC43 and BCoV was estimated to be 1897 (HPDs, 1826.15 to 1950.05), consistent with results from previous molecular clock studies (
27). Using the uncorrelated relaxed-clock model on complete HE gene sequences, the date of tMRCA of ChRCoV HKU24, members of Betacoronavirus 1, and RbCoV HKU14 was estimated to be 1337 (HPDs, 724.59 to 1776.78) (
Fig. 4). The date of divergence between HCoV OC43 and BCoV was estimated to be 1871 (HPDs, 1764.55 to 1944.37). The estimated mean substitution rates of the RdRp and HE data sets were 1.877 × 10−4 and 4.016 × 10−4 substitution per site per year, respectively, which are comparable to previous estimates for other lineage A βCoVs (
26,
27,
39).
FIG 4
Estimation of tMRCA of the ChRCoV HKU24 strains, BCoV/HCoV OC43, and ChRCoV HKU24/members of Betacoronavirus 1/RbCoV HKU14 on the basis of the complete sequences of the RdRp and HE genes. The mean estimated dates (above the branch) and Bayesian posterior probabilities (below the branch) are labeled and are represented by gray squares. The taxa are labeled with their sampling dates.
Serological studies.Western blot analysis using recombinant ChRCoV HKU24 N protein was performed using sera from 144 rodents for which serum samples were available, sera from two patients with HCoV OC43 infection, sera from two rabbits with RbCoV HKU14 infection, and sera from two patients with SARS-CoV infection. Among the sera tested from 74 Norway rats from Guangzhou for which serum samples were available, 60 (81.1%) were positive for antibody against recombinant ChRCoV HKU24 N protein with prominent immunoreactive bands of about 50 kDa (
Table 1 and
Fig. 5). These 60 positive samples included the 3 serum samples collected from the three Norway rats positive for ChRCoV HKU24 in their alimentary tract samples. In addition, 15 (48.4%) of 31 Norway rats from Hong Kong were also positive for antibody against recombinant ChRCoV HKU24 N protein, although the virus was not detected in alimentary tract samples from these rats. Moreover, 7 (77.8%) of 9 oriental house rats but only 4 (0.13%) of 30 black rats were positive for antibody against recombinant ChRCoV HKU24 N protein. Possible cross antigenicity between ChRCoV HKU24 and other βCoVs, including lineage A and B βCoVs, was found. Sera from two patients with HCoV OC43 infection, sera from two rabbits with RbCoV HKU14 infection, and sera from two patients with SARS-CoV infection were also positive for antibody against recombinant ChRCoV HKU24 N protein by Western blot assay (
Fig. 5).
FIG 5
Western blot analysis for antibodies against purified His6-tagged recombinant ChRCoV HKU24 N protein (∼50-kDa) (A) and spike polypeptide (∼50-kDa) (B) in rodent serum samples and serum samples from other animals or humans infected by different βCoVs, including HCoV OC43 (Betacoronavirus lineage A), RbCoV HKU14 (Betacoronavirus lineage A), and SARS-CoV (Betacoronavirus lineage B). Lanes: 1, negative control; 2, oriental house rat serum sample negative for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 3, Norway rat serum sample negative for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 4, oriental house rat serum sample positive for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 5, Norway rat serum sample positive for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 6 and 7, serum samples from rabbits infected by RbCoV HKU14; 8 and 9, serum samples from patients with HCoV OC43 infection; 10 and 11, serum samples from patients with SARS-CoV infection; 12, positive control (anti-His antibody).
Western blot analysis using recombinant ChRCoV HKU24 spike polypeptide was performed to verify the specificity of antibodies against ChRCoV HKU24 N protein using positive rodent sera and sera from two patients with HCoV OC43 infection, sera from two rabbits with RbCoV HKU14 infection, and sera from two patients with SARS-CoV infection. Among the sera from the 60 Norway rats positive for antibodies against the ChRCoV HKU24 N protein, 21 were positive for antibodies against the ChRCoV HKU24 spike polypeptide with prominent immunoreactive bands of about 50 kDa (
Table 1 and
Fig. 5). However, serum samples from the three Norway rats positive for ChRCoV HKU24 in their alimentary tract samples were negative for anti-ChRCoV HKU24 spike polypeptide antibody. Of the seven oriental house rats positive for antibodies against the ChRCoV HKU24 N protein, two were positive for antibodies against the ChRCoV HKU24 spike polypeptide. However, serum samples from the 4 black rats and 15 Norway rats from Hong Kong positive for antibodies against the ChRCoV HKU24 N protein were negative for antibodies against the ChRCoV HKU24 spike polypeptide. In contrast to N protein, no cross antigenicity between the ChRCoV HKU24 spike polypeptide and sera positive for antibodies against other βCoVs, including lineage A and B βCoVs, was detected. Sera from two patients with HCoV OC43 infection, sera from two rabbits with RbCoV HKU14 infection, and sera from two patients with SARS-CoV infection were all negative for antibody against recombinant ChRCoV HKU24 spike polypeptide by Western blot assay (
Fig. 5).
DISCUSSION
We discovered a novel lineage A βCoV, ChRCoV HKU24, from Norway rats in southern China. Betacoronavirus lineage A comprises the traditional group 2 CoVs, including members of murine coronavirus and Betacoronavirus 1, HCoV HKU1, and RbCoV HKU14. ChRCoV HKU24 possessed <90% amino acid sequence identities to the amino acid sequences of all other lineage A βCoVs in five of the seven conserved replicase domains used for CoV species demarcation by ICTV (
3), supporting the suggestion that ChRCoV HKU24 belongs to a separate species. The genome of ChRCoV HKU24 also possesses features distinct from those of other lineage A βCoVs, including a unique putative nsp1/nsp2 cleavage site and a unique putative cleavage site in S protein. Phylogenetically, its position at the root of Betacoronavirus 1, being distinct from the positions of murine coronavirus and HCoV HKU1, suggests that ChRCoV HKU24 may represent the murine ancestor for Betacoronavirus 1, after branching off from the common ancestor of murine coronavirus and HCoV HKU1. Interestingly, the genome of ChRCoV HKU24 possessed features that resemble those of the genomes of both Betacoronavirus 1 and murine coronavirus. It is more similar to Betacoronavirus 1 than murine coronavirus by the higher sequence identities in most predicted proteins, including NS2a, NS5, and S. On the other hand, it is more similar to murine coronavirus than to Betacoronavirus 1 in terms of its G+C content, the presence of a single NS4, and the absence of a TRS upstream of the E gene. Therefore, it is most likely that ChRCoV has evolved from the ancestor of murine coronavirus to infect other mammals, resulting in the generation of Betacoronavirus 1 with the acquisition of a TRS for the E gene. The tMRCAs of ChRCoV HKU24, members of Betacoronavirus 1, and RbCoV HKU14 were estimated to be 1402 (HPDs, 918.05 to 1749.91) and 1337 (HPDs, 724.59 to 1776.78) using complete RdRp and HE gene analysis, respectively, suggesting that interspecies transmission from rodents to other mammals occurred at least several centuries ago before the emergence of HCoV OC43 in humans in about the 1890s.
Western blot assays based on recombinant ChRCoV HKU24 N protein and spike polypeptide showed a high seroprevalence of ChRCoV HKU24 infection among Norway rats from Guangzhou. We evaluated the cross-reactivities of both N protein and spike polypeptide assays using sera from infections caused by other lineage A βCoVs, HCoV OC43 in humans and RbCoV HKU14 in rabbits, as well as SARS-CoV, a lineage B βCoV. Cross-reacting antibodies against N proteins were observed, a finding which is in line with previous findings on cross-reactivity between N proteins of different βCoVs (
49,
57). In contrast, no cross-reactivities between spike polypeptides were detected, supporting the specificity of CoV spike polypeptide-based assays and their ability to rectify cross-reactivities (
57,
58). Using the present assays, 60 of 74 Norway rats from Guangzhou were positive for antibodies against the ChRCoV HKU24 N protein, and among these rats, 21 were positive for antibodies against the ChRCoV HKU24 spike polypeptide, indicating that these 21 rats had previously been infected with ChRCoV HKU24. Interestingly, the three Norway rats positive for ChRCoV HKU24 in their alimentary tract samples were positive for antibodies against ChRCoV HKU24 N protein but negative for antibodies against ChRCoV HKU24 spike polypeptide. This is likely due to a delay in mounting neutralizing antibodies against spike protein during acute infection in these three rats, where antibodies against N protein may arise earlier as a result of the high abundance and antigenicity of CoV N proteins, or the response to the N protein may be a result of cross-reactions to N proteins from other βCoVs. The finding is also in keeping with previous findings on SARS-related Rhinolophus bat CoV, in which a negative correlation between the viral load and neutralizing antibody titer was observed (
14). Besides Norway rats, antibodies against ChRCoV HKU24 N protein and spike polypeptide were also detected in two oriental house rats from Guangzhou, although antibodies against spike polypeptide were relatively weak. This suggests possible cross-species infection with ChRCoV HKU24 or cross-reactivity from a very close lineage A βCoV. Four black rats and 15 Norway rats in Hong Kong were also positive for antibodies against the ChRCoV HKU24 N protein but not the spike polypeptide. This suggests a possible past infection by another βCoV(s) and cross-reactivity between the N protein(s) of that βCoV(s) and the N protein of ChRCoV HKU24. More studies with diverse rodent species from China and other countries are required to determine the natural reservoir and host range of ChRCoV HKU24 and other murine lineage A βCoVs.
The present results extend our knowledge on the evolutionary origin of CoVs. While birds are important sources for γCoVs and δCoVs, bats host diverse αCoVs and βCoVs that may be the ancestral origins of various mammalian CoVs, including human CoVs. For human αCoVs, both HCoV NL63 and HCoV 229E likely originated from bat CoVs. HCoV NL63 has been shown to share a common ancestry with αCoVs from the North American tricolored bat, with the most recent common ancestor between these viruses occurring from approximately 563 to 822 years ago (
75). Moreover, immortalized lung cell lines derived from this bat species allowed replication of HCoV NL63, supporting potential zoonotic-reverse zoonotic transmission cycles between bats and humans. HCoV 229E also shared a common ancestor with diverse αCoVs from leaf-nosed bats in Ghana, with the most recent common ancestor dating to 1686 to 1800 (
76). However, no complete genomes are available for the putative bat ancestors of HCoV NL63 and HCoV 229E. For human βCoVs, SARS-CoV and MERS-CoV are also known to share common ancestors with bat CoVs. Soon after the SARS epidemic, horseshoe bats in China were found to be the reservoir for SARS-CoV-like viruses, which were postulated to have jumped from bats to civets and, later, humans (
8,
14,
15). A recent study also reported the isolation of a SARS-like bat CoV in Vero E6 cells and the ability of this bat virus to use the angiotensin-converting enzyme 2 (ACE2) from humans, civets, and Chinese horseshoe bats for cell entry (
77). MERS-CoV belongs to Betacoronavirus lineage C, which was known to consist of only two bat viruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5, before the MERS epidemic (
35–37). This has led to the speculation that bats may be the zoonotic origin of MERS-CoV. However, recent evidence supports dromedary camels as the immediate source of human MERS-CoV (
78–80). Nevertheless, a conspecific virus from a South African Neoromicia capensis bat has been found to share 85% nucleotide sequence identity to the nucleotide sequence of the MERS-CoV genome, suggesting the acquisition of MERS-CoV by camels from bats in sub-Saharan Africa, from where camels on the Arabian peninsula are imported (
81). In contrast, there has been no evidence that bats are the origin of human lineage A βCoVs, such as HCoV OC43 and HCoV HKU1. HCoV OC43, being closely related to BCoV, is believed to have emerged relatively recently from bovine-to-human transmission in about 1890 (
27,
30,
39). Both viruses belonged to the promiscuous CoV species Betacoronavirus 1, which consists of many closely related mammalian CoVs, implying a low threshold for cross-mammalian species transmission and a complex evolutionary history among these viruses (
40–47,
49). However, the ancestral origin of members of Betacoronavirus 1 remains elusive. As for HCoV HKU1, no recent zoonotic ancestor has yet been identified, although the virus is most closely related to members of murine coronaviruses (
20,
42). Although rodents constitute approximately 40% of all mammalian species, murine coronavirus has been the only CoV species known to exist in rodents. This is in contrast to the large diversity of CoVs found in bats, which make up another 20% of all species of mammals (
6,
33,
36). The present results suggest that rodents may be an important reservoir for lineage A βCoVs and may harbor other ancestral viruses of Betacoronavirus 1 and HCoV HKU1 (
Fig. 6). Nevertheless, many mysteries about the evolution of lineage A βCoVs remain unresolved, such as the origin of their HE proteins. For example, both toroviruses and influenza C viruses can be found in bovine and porcine samples. Further studies are required to determine if the HE proteins of potential rodent CoV ancestors of Betacoronavirus lineage A may have been acquired from cattle or pigs.
FIG 6
Evolution of CoVs from their ancestors in bat, bird, and rodent hosts to virus species that infect other animals. Dashed arrows, possible routes of transmission from bats or birds to rodents before establishment of Betacoronavirus lineage A.
The potential pathogenicity and tissue tropism of ChRCoV HKU24 remain to be determined. While CoVs are associated with a wide spectrum of diseases in animals, some CoVs, especially those from bats, were detected in apparently healthy individuals without obvious signs of disease (
8,
14,
15,
31,
33). The detection of ChRCoV HKU24 in the alimentary tract samples of Norway rats suggested a possible enteric tropism. However, the three positive rats did not show obvious signs of disease. MHV, the prototype CoV most extensively studied before the SARS epidemic, can cause a variety of neurological, hepatic, gastrointestinal, and respiratory diseases in mice, depending on the strain tropism and route of inoculation. The virus, originally isolated from a mouse with spontaneous encephalomyelitis, causes disseminated encephalomyelitis with extensive destruction of myelin and focal necrosis of the liver in experimentally infected mice (
82–84). Strain MHV A59 is primarily hepatotropic, while strain MHV JHM is neurotropic. Enterotropic strains can spread quickly as a result of the high level of excretion in feces and cause significant environmental contamination in animal houses. Respiratory tract-tropic or polytropic strains, although uncommon, are the strains that commonly contaminate cell lines. As for RCoV, it causes diseases primarily in the respiratory tract, with strain sialodacryoadenitis virus (SDAV) being more associated with upper respiratory tract, salivary and lacrimal gland, and eye infections and strain RCoV Parker causing pneumonia in experimentally infected rats (
85,
86). Further investigations are required to study the tissue tropism and pathogenicity of ChRCoV HKU24 in Norway rats and other potential rodent reservoirs.
Elucidating the receptor of ChRCoV HKU24 will be important to understand the mechanism of host adaptation and interspecies transmission from rodents to other mammals. The higher sequence identity to Betacoronavirus 1 than to murine coronavirus of the S protein and NTD of ChRCoV HKU24 is in line with the findings for other regions of the genome. Homology modeling showed that the conformation of the sugar-binding loop in the BCoV NTD is conserved in the ChRCoV HKU24 NTD. Moreover, 3 of the 4 critical sugar-binding residues in BCoV but only 2 of the 14 contact residues at the MHV NTD/mCEACAM1a interface are conserved in ChRCoV HKU24. While it remains to be ascertained if ChRCoV HKU24 may utilize sugar or CEACAM1 as a receptor, its predicted NTD appears to resemble that of BCoV more than that of MHV. On the basis of the presence of a β-sandwich fold in the NTDs of MHV and BCoV, it has been proposed that CoV NTDs may have originated from a host galectin with sugar-binding functions but evolved new structural features in MHV for binding to CEACMA1 (
10,
87). If rodents are indeed the host origin for Betacoronavirus lineage A, including Betacoronavirus 1, it would be interesting to study the sugar-binding activity of NTDs of different rodent βCoVs to understand their evolutionary history. Although some lineage A βCoVs, such as Betacoronavirus 1 and MHV, can replicate in cell lines such as BSC-1 and HRT-18, attempts to isolate ChRCoV HKU24 from the three positive samples were unsuccessful. Future studies to isolate the virus from more rodent samples will allow characterization of its receptor usage and pathogenicity.
ACKNOWLEDGMENTS
We thank the following for facilitation of and assistance with sample collection: Wing-Man Ko, Secretary for the Food and Health Bureau; Vivian Lau, Kwok-Hau Sin, and M. C. Yuen of FEHD; Alan C. K. Wong, Siu-Fai Leung, Thomas Hon-Chung Sit, Howard Kai-Hay Wong, Chung-Tong Shek, and Joseph W. K. So of AFCD. We are grateful for the generous support of Carol Yu, Richard Yu, Hui Hoy, and Hui Ming in the genomic sequencing platform.
The views expressed in this paper are those of the authors only and do not represent the opinion of FEHD, AFCD, or the government of the HKSAR.
This work is partly supported by a Research Grant Council grant, University Grant Council; the Committee for Research and Conference Grants, the Strategic Research Theme Fund, and the University Development Fund, The University of Hong Kong; the Health and Medical Research Fund of the Food and Health Bureau of HKSAR; and the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health.
FOOTNOTES
Received 22 August 2014.
Accepted 23 December 2014.
Accepted manuscript posted online 31 December 2014.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Bottom line - why has Dr Shi's work been censored? Why did the glorious CCP party taken offline close to 1000 academic papers in coronavirus research.
https://www.washingtonpost.com/opinions/2021/02/05/coronavirus-origins-mystery-china/
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A critical database went offline
At the core of Dr. Shi’s work is a database at the institute. According to research by DRASTIC, a network of researchers and scientists, this is the most important bat coronavirus database in China. Overall, it holds records of some 22,000 samples and some of their genetic sequences, including for WIV virus sampling trips going back many years. The institute collected more than 15,000 samples from bats, covering over 1,400 bat viruses. The database holds more than 100 unpublished sequences of bat coronaviruses that could significantly help the probe into the origins of the pandemic.
Of particular interest are the full sequences of eight viruses sampled in 2015 in an unidentified location in Yunnan province, which was only recently disclosed. In 2012, six people who were clearing bat feces from an abandoned mine in Yunnan developed an illness with symptoms very similar to covid-19. Three eventually died. The results of the investigation into the cause of their illness have not been fully disclosed. A bat-virus sampling trip by WIV-EcoHealth was underway in nearby locations while these six people were infected. A virus designated RaTG13 was sampled from the mine in 2013 and has been described as the closest known relative of SARS-CoV-2. Based on limited information about their sequences, the other eight viruses are very similar to RaTG13 and may hold evolutionary clues.
"
China clamps down in hidden hunt for coronavirus origins
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...
apnews.com
China clamps down in hidden hunt for coronavirus origins
By DAKE KANG, MARIA CHENG and SAM MCNEILDecember 30, 2020
1 of 13
FILE - In this Tuesday, March 10, 2020 file photo, people walk by a giant TV screen at a quiet shopping mall in Beijing broadcasting news of Chinese President Xi Jinping talking to medical workers at the Huoshenshan Hospital in Wuhan in central China's Hubei Province, as he visited the center of the global virus outbreak. The government is handing out hundreds of thousands of dollars in grants to scientists researching the virus’ origins in southern China and with the military, The Associated Press has found. But it is monitoring their findings and mandating that the publication of any data or research must be approved by a new task force managed by China’s cabinet, under direct orders from President Xi Jinping, according to internal documents obtained by the AP. (AP Photo/Andy Wong)
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 ON THE PANDEMIC
The government is handing out hundreds of thousands of dollars in grants to scientists researching the virus’ origins in southern China and affiliated with the military, the AP has found. But it is monitoring their findings and mandating that the publication of any data or research must be approved by a new task force managed by China’s cabinet, under direct orders from President Xi Jinping,
according to internal documents obtained by the AP. A rare leak from within the government, the dozens of pages of unpublished documents confirm what many have long suspected: The clampdown comes from the top.
As a result, very little has been made public. Authorities are severely limiting information and impeding cooperation with international scientists.
“What did they find?” asked Gregory Gray, a Duke University epidemiologist who oversees a lab in China studying the transmission of infectious diseases from animals to people. “Maybe their data were not conclusive, or maybe they suppressed the data for some political reason. I don’t know … I wish I did.”
The AP investigation was based on dozens of interviews with Chinese and foreign scientists and officials, along with public notices, leaked emails, internal data and the documents from China’s cabinet and the Chinese Center for Disease Control and Prevention. It reveals a pattern of government secrecy and top-down control that has been evident throughout the pandemic.
As the AP previously documented, this culture has
delayed warnings about the pandemic,
blocked the sharing of information with the World Health Organization and
hampered early testing. Scientists familiar with China’s public health system say the same practices apply to sensitive research.
“They only select people they can trust, those that they can control,” said a public health expert who works regularly with the China CDC, declining to be identified out of fear of retribution. “Military teams and others are working hard on this, but whether it gets published all depends on the outcome.”
The pandemic has crippled Beijing’s reputation on the global stage, and China’s leaders are wary of any findings that could suggest they were negligent in its spread. The Chinese Ministry of Science and Technology and the National Health Commission, which are managing research into the coronavirus’ origins, did not respond to requests for comment.
“The novel coronavirus has been discovered in many parts of the world,” China’s foreign ministry said in a fax. “Scientists should carry out international scientific research and cooperation on a global scale.”
Notices obtained by The AP link new research restrictions to Xi Jinping, China's most authoritarian leader in decades.
Some Chinese scientists say little has been shared simply because nothing of significance has been discovered.
“We’ve been looking, but we haven’t found it,” said Zhang Yongzhen, a renowned Chinese virologist.
China’s leaders are far from alone in politicizing research into the origins of the virus. In April, President Donald Trump shelved a U.S.-funded project to identify dangerous animal diseases in China and Southeast Asia, effectively severing ties between Chinese and American scientists and complicating the search for virus origins. Trump also has accused China of setting off the pandemic through an accident at a Wuhan lab — a theory that some experts say cannot be ruled out but as yet has no evidence behind it.
Research into COVID-19’s origins is critical to the prevention of future pandemics. Although a World Health Organization international team plans to visit China in early January to investigate what started the pandemic, its members and agenda had to be approved by China.
Some public health experts warn that China’s refusal to grant further access to international scientists has jeopardized the global collaboration that pinpointed the source of the SARS outbreak nearly two decades ago. Jonna Mazet, a founding executive director of the UC Davis One Health Institute, said the lack of collaboration between Chinese and U.S. scientists was “a disappointment” and the inability of American scientists to work in China “devastating.”
“There’s so much speculation around the origins of this virus,” Mazet said. “We need to step back...and let scientists get the real answer without the finger-pointing.”
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The hidden hunt for the origins of COVID-19 shows how the Chinese government has tried to steer the narrative.
The search started in the Huanan Seafood market in Wuhan, a sprawling, low-slung complex where many of the first human coronavirus cases were detected. Scientists initially suspected the virus came from wild animals sold in the market, such as civet cats implicated in the spread of SARS.
In mid-December last year, Huanan vendor Jiang Dafa started noticing people were falling ill. Among the first was a part-time worker in his 60s who helped clean carcasses at a stall; soon, a friend he played chess with also fell ill. A third, a seafood monger in his 40s, was infected and later died.
Patients began trickling into nearby hospitals, triggering alarms by late December that alerted the China CDC. CDC chief Gao Fu immediately sent a team to investigate.
At first, research appeared to be moving swiftly.
Overnight on Jan. 1, the market suddenly was ordered shut, barring vendors from fetching their belongings, Jiang said. China CDC researchers collected 585 environmental samples from door handles, sewage and the floor of the market, and authorities sprayed the complex down with sanitizer. Later, they would cart out everything inside and incinerate it.
Internal China CDC data obtained by the AP shows that by Jan. 10 and 11, researchers were sequencing dozens of environmental samples from Wuhan. Gary Kobinger, a Canadian microbiologist advising WHO, emailed his colleagues to share his concerns that the virus originated at the market.
“This corona(virus) is very close to SARS,” he wrote on Jan. 13. “If we put aside an accident ... then I would look at the bats in these markets (sold and ‘wild’).”
By late January, Chinese state media announced that 33 of the environmental samples had tested positive. In a report to WHO, officials said 11 specimens were more than 99% similar to the new coronavirus. They also told the U.N. health agency that rats and mice were common in the market, and that most of the positive samples were clustered in an area where vendors traded in wildlife.
In the meantime, Jiang avoided telling people he worked at Huanan because of the stigma. He criticized the political tussle between China and the U.S.
“It’s pointless to blame anyone for this disease,” Jiang said.
As the virus continued spreading rapidly into February, Chinese scientists published a burst of research papers on COVID-19. Then a paper by two Chinese scientists proposed without concrete evidence that the virus could have leaked from a Wuhan laboratory near the market. It was later taken down, but it raised the need for image control.
Internal documents show that the state soon began requiring all coronavirus studies in China to be approved by high-level government officials — a policy that critics say paralyzed research efforts.
A China CDC lab notice on Feb. 24 put in new approval processes for publication under “important instructions” from Chinese President Xi Jinping.
Other notices ordered CDC staff not to share any data, specimens or other information related to the coronavirus with outside institutions or individuals.
Then on March 2, Xi emphasized “coordination” on coronavirus research,
state media reported.
The next day, China’s cabinet, the State Council, centralized all COVID-19 publication under a special task force.
The notice, obtained by the AP and marked “not to be made public,” was far more sweeping in scope than the earlier CDC notices, applying to all universities, companies and medical and research institutions.
The order said communication and publication of research had to be orchestrated like “a game of chess” under instructions from Xi, and
propaganda and public opinion teams were to “guide publication.” It went on to warn that those who publish without permission, “causing serious adverse social impact, shall be held accountable.”
“The regulations are very strict, and they don’t make any sense,” said a former China CDC deputy director, who declined to be named because they were told not to speak to the media. “I think it’s political, because people overseas could find things being said there that might contradict what China says, so it’s all being controlled.”
After the secret orders, the tide of research papers slowed to a trickle. Although China CDC researcher Liu Jun returned to the market
nearly 20 times to collect some 2,000 samples over the following months, nothing was released about what they revealed.
On May 25, CDC chief Gao finally broke the silence around the market in
an interview with China’s Phoenix TV. He said that, unlike the environmental samples, no animal samples from the market had tested positive.
The announcement surprised scientists who didn’t even know Chinese officials had taken samples from animals. It also ruled out the market as the likely source of the virus, along with further research that showed many of the first cases had no ties to it.
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With the market proving a dead end, scientists turned more attention to hunting for the virus at its likely source: bats.
Nearly a thousand miles away from the wet market in Wuhan, bats inhabit the maze of underground limestone caves in Yunnan province. With its rich, loamy soil, fog banks and dense plant growth, this area in southern China bordering Laos, Vietnam and Myanmar is one of the most biologically diverse on earth.
Scientists worry about contact between bats and humans in caves like this one, in southern China's Yunnan province.
At one Yunnan cave visited by the AP, with thick roots hanging over the entrance, bats fluttered out at dusk and flew over the roofs of a nearby small village. White droppings splattered the ground near an altar in the rear of the cave, and Buddhist prayer strings of red and yellow twine hung from the stalactites. Villagers said the cave had been used as a sacred place presided over by a Buddhist monk from Thailand.
Contact like this between bats and people praying, hunting or mining in caves alarms scientists. The coronavirus’ genetic code is strikingly similar to that of bat coronaviruses, and most scientists suspect COVID-19 jumped into humans either directly from a bat or via an intermediary animal.
Since bats harboring coronaviruses are found in China and throughout Southeast Asia, the wild animal host of COVID-19 could be anywhere in the region, said Linfa Wang at Duke-NUS Medical School in Singapore.
“There is a bat somewhere with a 99.9% similar virus to the coronavirus,” Wang said. “Bats don’t respect these borders.
COVID-19 research is proceeding in countries such as Thailand, where Dr. Supaporn Wacharapluesadee, a coronavirus expert, is leading teams of scientists deep into the countryside to collect samples from bats. During one expedition in August, Supaporn told the AP the virus could be found “anywhere” there were bats.
Chinese scientists quickly started testing potential animal hosts. Records show that Xia Xueshan, an infectious diseases expert, received
a 1.4 million RMB ($214,000) grant to screen animals in Yunnan for COVID-19. State media reported in February that his team collected hundreds of samples from bats, snakes, bamboo rats and other animals, and
ran a picture of masked scientists in white lab coats huddled around a large, caged porcupine.
Then the government restrictions kicked in. Data on the samples still has not been made public, and Xia did not respond to requests for an interview. Although Xia has co-authored more than a dozen papers this year, an AP review shows,
only two were on COVID-19, and neither focused on its origins.
Today, the caves that scientists once surveyed are under close watch by the authorities. Security agents tailed the AP team in three locations across Yunnan, and stopped journalists from visiting the cave where researchers in 2017 identified the species of bats responsible for SARS. At an entrance to a second location, a massive cave teeming with tourists taking selfies, authorities shut the gate on the AP.
“We just got a call from the county,” said a park official, before an armed policeman showed up.
Vehicles blocking AP journalists from visiting a mine shaft where the closest known relative of the COVID-19 virus was found.
Particularly sensitive is the mine shaft where the closest relative of the COVID-19 virus — called “RaTG13” — was found.
RaTG13 was discovered after an outbreak in 2012, when six men cleaning the bat-filled shaft fell ill with mysterious bouts of pneumonia, killing three. The Wuhan Institute of Virology and the China CDC both studied bat coronaviruses from this shaft. And although most scientists believe the COVID-19 virus had its origins in nature, some say it or a close relative could have been transported to Wuhan and leaked by mistake.
Wuhan Institute of Virology bat expert Shi Zhengli has repeatedly denied this theory, but Chinese authorities haven’t yet allowed foreign scientists in to investigate.
Some state-backed scientists say research is proceeding as usual. Famed virologist Zhang, who received
a 1.5 million RMB ($230,000) grant to search for the virus’ origins, said partnering scientists are sending him samples from all over, including from bats in Guizhou in southern China and rats in Henan hundreds of miles north.
“Bats, mice, are there any new coronaviruses in them? Do they have this particular coronavirus?” Zhang said. “We’ve been doing this work for over a decade. It’s not like we just started today.”
Zhang declined to confirm or comment on reports that his lab was briefly closed after publishing the virus’ genetic sequence ahead of authorities. He said he hasn’t heard of any special restrictions on publishing papers, and the only review his papers go through is a routine scientific one by his institution.
But scientists without state backing complain that getting approval to sample animals in southern China is now extremely difficult, and that little is known about the findings of government-sponsored teams.
___
Even as they controlled research within China, Chinese authorities promoted theories that suggested the virus came from elsewhere.
The government gave Bi Yuhai, the Chinese Academy of Sciences scientist
tapped to spearhead origins research, a 1.5 million RMB grant ($230,000),
records show.
A paper co-authored by Bi suggested an outbreak in a Beijing market in June could have been caused by packages of contaminated frozen fish from Europe.
China’s government-controlled media
used the theory to suggest the original outbreak in Wuhan could have started with seafood imported from abroad — a notion international scientists reject. WHO has said it is very unlikely that people can be infected with COVID-19 via packaged food, and that it is “highly speculative” to suggest COVID-19 did not start in China. Bi did not respond to requests for an interview, and China has not provided enough virus samples for a definitive analysis.
Posters in southern China's Yunnan province advising people not to eat wild animals.
The Chinese state press also has widely covered initial studies from Europe suggesting COVID-19 was found in wastewater samples in Italy and
Spain last year. But scientists have largely dismissed these studies, and the researchers themselves acknowledged they did not find enough virus fragments to determine conclusively if it was the coronavirus.
And in the last few weeks, Chinese state media has
taken out of context research from a German scientist, interpreting it to suggest that the pandemic began in Italy. The scientist, Alexander Kekule, director of the Institute for Biosecurity Research, has said repeatedly that he believes the virus first emerged in China.
Internal documents show that various government bodies also sponsored studies on the possible role of the Southeast Asian pangolin, a scaly anteater once prized in traditional Chinese medicine, as an intermediary animal host. Within the span of three days in February, Chinese scientists put out
four separate papers on coronaviruses related to COVID-19 in trafficked Malayan pangolins from Southeast Asia seized by customs officials in Guangdong.
But many experts now say the theory is unlikely. Wang of the Duke-NUS Medical School in Singapore said the search for the coronavirus in pangolins did not appear to be “scientifically driven.” He said blood samples would be the most conclusive evidence of COVID-19’s presence in the rare mammals, and so far, no incriminating matches have been found.
WHO has said more than 500 species of other animals, including cats, ferrets and hamsters, are being studied as possible intermediary hosts for COVID-19.
The Chinese government is also limiting and controlling the search for patient zero through the re-testing of old flu samples.
Chinese hospitals collect thousands of samples from patients with flu-like symptoms every week and store them in freezers. They could easily be tested again for COVID-19, although politics could then determine whether the results are made public, said Ray Yip, the founding director of the U.S. CDC office in China.
“They’d be crazy not to do it,” Yip said. “The political leadership will wait for that information to see, does this information make China look stupid or not? ... If it makes China look stupid, they won’t.”
In the U.S., CDC officials long ago
tested roughly 11,000 early samples collected under the flu surveillance program since Jan. 1. And in Italy,
researchers recently found a boy who had fallen ill in November 2019 and later tested positive for the coronavirus.
But in China, scientists have only published retrospective testing data
from two Wuhan flu surveillance hospitals — out of
at least 18 in Hubei province alone and
well over 500 across the country. The data includes just 520 samples out of the 330,000 collected in China last year.
These enormous gaps in the research aren’t due just to a lack of testing but also to a lack of transparency. Internal data obtained by the AP shows that by Feb. 6, the Hubei CDC had tested over 100 samples in Huanggang, a city southeast of Wuhan. But the results have not been made public.
The little information that has dribbled out suggests the virus was circulating well outside Wuhan in 2019 — a finding that could raise awkward questions for Chinese officials about their early handling of the outbreak. Chinese researchers found that
a child over a hundred kilometers from Wuhan had fallen ill with the virus by Jan. 2, suggesting it was spreading widely in December. But earlier samples weren’t tested, according to a scientist with direct knowledge of the study.
“There was a very deliberate choice of the time period to study, because going too early could have been too sensitive,” said the scientist, who declined to be named out of fear of retribution.
A WHO report written in July but published in November said Chinese authorities had identified 124 cases in December 2019, including five cases outside Wuhan. Among WHO’s aims for its upcoming visit to China are reviews of hospital records before December.
Coronavirus expert Peter Daszak, a member of the WHO team, said identifying the pandemic’s source should not be used to assign guilt.
“We’re all part of this together,” he said. “And until we realize that, we’re never going to get rid of this problem.”
___
Kang reported from Beijing and Cheng reported from London. Associated Press journalists Han Guan Ng and Emily Wang in Wuhan, China, Haven Daley in Stinson Beach, California, and Tassanee Vejpongsa in Kanchanaburi, Thailand, contributed to this report.
