In a previous posting, it was related how, in 1965, David Tyrrell, at the Common Cold Unit in Salisbury, England, grew a virus from a schoolboy with a cold (1). That virus, designated B814, would be the first known human coronavirus. With the aid of Swedish surgeon Bertil Hoorn, B814 was grown in human trachea organ cultures that were inoculated with throat swabs taken from the sick boy. The main point of the previous posting was that the detection of B814 depended on the exceptional electron microscopy skills of June Almeida. In addition, Almeida noted that the morphology of B814 resembled that of the previously isolated infectious bronchitis virus (IBV) of chickens. She then concluded that B814 and IBV are members of a previously unclassified family of viruses. Then, based on Almeida’s interpretation of B814’s morphology, she, Tyrrell, and Tony Waterson (a colleague of Tyrrell who recommended that Almeida be recruited to Tyrrell’s team), dubbed the new family “coronaviruses.”
Even though B814 is recognized as the first known human coronavirus, listings of coronaviruses that infect humans, as compiled in medical virology books and in the journal literature, generally include only seven coronaviruses, none of which has been shown to be identical to B814. These seven coronaviruses include four human coronaviruses that cause common colds (HCoVs 229E, NL63, OC43, and HKUI), and three animal coronaviruses that recently emerged in, and are highly virulent in humans (SARS, MERS, and SARS-CoV2). Where then does B814 fit in this compilation of coronaviruses? In search of an answer, here is a brief history of the discoveries of the human coronaviruses.
In 1966, at about the same time that Almeida was carrying out her electron microscopy analysis of B814, Dorothy Hamre and John Procknow, at the University of Chicago, recovered 5 viral isolates from medical students with colds (2). One of these isolates, 229E, was examined by Almeida, who found its morphology to be identical to that of B814. Then, in 1967, Robert Chanock and coworkers at the NIH used the organ culture technique to isolate yet other strains, including OC43, whose morphology likewise resembled that of B814 (3).
229E and OC43 were virtually the only HCoVs being studied during the next thirty years, mainly because they were the easiest of the HCoV strains to work with. As for B814, it could only be grown in organ culture (1). In contrast, clinical samples of 229E could be grown directly in cell culture. And while OC43 was originally grown in organ culture, it was subsequently adapted to growth in suckling mouse brain, and then to growth in cell culture.
Both 229E and OC43 are clinically significant, as each caused multiple epidemics in the United States, at intervals of 2 to 3 years. Moreover, reinfection with each of these strains was common (4); a point that may be relevant to the current COVID-19 pandemic. In any case, in 1970 it was reported that B814 is not serologically identical to either OC43 or 229E (5). Little else seems to be known about B814.
The winter of 2002-2003 saw the emergence of the SARS coronavirus; a virus far more lethal in humans than either 229E or OC43. By the end of the SARS outbreak, a total of 8,096 cases were reported, of which 744 were fatal, for a fatality rate of about 12%. Prior to the SARS outbreak, coronavirus infections were regarded as merely mild nuisances. But the SARS outbreak led to an awareness that animal coronaviruses could represent a significant health threat to humans.
But yet other relatively mild HCoVs remained to be discovered. In 2004, researchers at the Erasmus Medical Center in the Netherlands isolated NL63 from a 7-month-old girl with coryza, conjunctivitis, fever, and bronchiolitis (6). The same year, another group of investigators in the Netherlands isolated a coronavirus, designated NL, from an 8-month-old boy with pneumonia (7). And, in 2004, researchers in New Haven, Connecticut, used a PCR-based approach to isolate a virus, designated the New Haven coronavirus (HCoV-NH), from young children with respiratory disease, (8).
NL63, NL, and NH are so-called group I coronaviruses. They are closely related to each other and may very well be the same species. In any case, these viruses are believed to be a significant cause of respiratory tract disease in infants and children. And, like SARS-CoV2, HCoV-NH has been associated with Kowasaki disease in children (9).
In 2005, researchers at the University of Hong Kong used RT-PCR to recover a novel human coronavirus from a sample taken earlier from a 71-year old man who presented in Hong Kong with a fever and a cold (10). HKU1 is a so-called group II coronavirus. As such, it is distinct from OC43, the only other known group II human coronavirus. [229E is a group I coronavirus. Sequence analysis shows SARS to be sufficiently different from any of the known human and animal coronaviruses for it to occupy a separate group.] It is not known whether B814, or any of the other uncharacterized HCoV strains from the 1960s (i.e., those strains that were grown only in organ culture) are similar or identical to the more recently isolated HCoVs.
MERS-CoV is the second deadly coronavirus to emerge in humans. It was first isolated from a patient in Saudi Arabia in 2012. It caused fewer than 2,500 confirmed cases worldwide. Yet it killed an astounding 35% of people with confirmed diagnoses. At present, we are in what is still the early phase of the SARS-CoV-2 (COVID-19) pandemic. SARS-CoV-2 is clearly far more transmissible than either SARS or MERS. Its fatality rate is not known but estimates are at about 1%. In any case, the above compendium hopefully includes all the coronavirus strains known to infect humans.
References:
Norkin, L.C. June Almeida and the Discovery of the First Human Coronavirus, Posted on the blog June 2, 2020.
Hamre, D., and J.J. Procknow, 1966. A new virus isolated from the human respiratory tract. Soc. Exp. Biol. Med. 121:190–193.
McIntosh, K., J.H. Dees, W.B. Becker, A.Z. Kapikian, and R.M. Chanock, 1967. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Natl. Acad. Sci. USA. 57:933–940.
Callow, K.A., H.F. Parry, M. Sergeant, and D.A. Tyrrell, 1990. The time course of the immune response to experimental coronavirus infection of man. Infect. 105:435–446.
Bradburne, A.F., 1970. Antigenic relationships amongst coronaviruses. Gesamte Virusforsch. 31:352–364.
van der Hoek, L., K. Pyrc, M.F, Jebbink, et al., 2004, Identification of a new human coronavirus. Med. 10:368–373.
Fouchier, R.A., N.G. Hartwig, T.M. Bestebroer, et al., 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Natl. Acad. Sci. USA. 101:6212–6216.
Esper, F., R.A. Martinello, R.P. Boucher, et al., 2005. Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children. Infect. Dis. 191:492–498.
Esper, F., E.D. Shapiro, C. Weibel, D. Ferguson, M.L. Landry, and J.S. Kahn, 2005. Association between a novel human coronavirus and Kawasaki disease. Infect. Dis. 191:499–502.
Woo, P.C., S.K. Lau, C.M. Chu, et al., 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. Virol. 79:884–895.
At this still early phase of the COVID-19 crisis, it is not clear who, if any, individual might emerge as a hero comparable in stature to say Carlo Urbani during the SARS outbreak, or Peter Piot during the Ebola and HIV outbreaks. Nonetheless, June Almeida has been noted in the media recently for her critical role in the discovery of the first human coronavirus. Almeida made other significant contributions to virology. Yet, for a major part of her career, her only formal scientific training was as a laboratory technician.
David Tyrrell recounts the discovery of the first human coronavirus in his book “Cold Wars: The Fight Against the Common Cold” (Oxford University Press, 2002). In 1966, Tyrrell and co-researchers at the Common Cold Unit in Salisbury, England, were taking nasal swabs of people experiencing common colds. They then inoculated human volunteers with the swabs. Next, samples that caused colds in the volunteers were inoculated into cell cultures, so that possible viral agents might be grown for further analysis. But Tyrrell ran up against a serious impediment. Some samples, which caused colds in the volunteers, did not appear to grow in cell cultures. Consequently, those samples could not be studied further. Nothing more could be ascertained regarding their taxonomy, mode of replication, or ability to induce and respond to antibodies. Nor might they be grown to produce a possible vaccine. Consequently, Tyrrell and coworkers sought a more reliable means for growing their isolates in the lab.
“We decided to look at things from the virus point of view…we were interested in viruses that normally grow in the cells of the nose. Perhaps some of them were quite unable to grow anywhere else.” But while “the cells of the human nose are by far the best environment in which to grow the cold virus, …from the researcher’s point of view, they have the disadvantage of being attached to the rest of the human body.”
Tyrrell learned of a possible solution to his dilemma from Sir Christopher Andrewes, a colleague who had been to Sweden to receive an honorary doctorate. While in Sweden, Andrewes met Bertil Hoorn, a surgeon at the University of Lund, who discovered that he could make organ cultures from human trachea tissue, that would support the growth of influenza virus. Hoorn established these cultures from small squares of airway-lining tissue that attached to specially treated surfaces of plastic petri dishes. The cultures were then maintained under liquid medium. They retained characteristics of the actual airway tissue in situ, such as beating cilia. If virus grew in these cultures, it would cause the cilia to cease beating. Moreover, progeny viruses could be recovered from the media.
Influenza viruses are a family of viruses that are distinct from the rhinoviruses already known to cause common colds. Nonetheless, Tyrrell recognized the potential of Hoorn’s discovery for his own work. Consequently, he invited Hoorn to come to Salisbury, to test whether his respiratory tract organ cultures might support the growth of cold viruses. Hoorn accepted Tyrrell’s invitation. He came by ferry from Sweden and arrived at the driveway of the CCU in his two-horsepower Citroën, crammed in the back with his glassware, fluids, and surgical instruments. At the CCU, Hoorn duplicated his influenza findings. Then, without exception, he was able to grow all of the cold virus samples that Tyrrell had at hand.
But, while Hoorn’s work was underway in Salisbury, Tyrrell’s team acquired new virus samples from a group of boarding-school boys with colds. Tyrrell was taken aback when one of these samples, dubbed B814, caused no apparent change in Hoorn’s organ cultures. Yet this was the case even though B814 appeared to replicate in the organ cultures, as shown by the fact that fluids from the inoculated cultures caused colds in human volunteers.
Tyrrell grew his first rhinovirus in cell culture in 1946. Yet B814 was, thus far, his only sample that did not cause cell damage (cytopathic effects) in Hoorn’s organ cultures. In the undifferentiated cell cultures that Tyrrell used earlier, rhinoviruses would cause the cells to darken, round up, and detach from the surface of the culture dish. In Hoorn’s organ cultures, rhinoviruses generally caused the cilia to stop beating. So, might B814 not be a rhinovirus?
There was another clue that B814 might not be a rhinovirus. Since rhinoviruses are not enclosed within lipid envelopes, they are not inactivated by fat solvents. But B814 was inactivated by fat solvents, suggesting that it has a lipid envelope and, consequently, is not a rhinovirus. [If B814 were in fact enveloped, that might explain why it caused no apparent damage in the Hoorn’s organ cultures. Enveloped viruses generally acquire their envelopes during their release from the cell. This occurs via a process called “budding,” which usually leaves behind an intact cell. In contrast, release of non-enveloped viruses is generally facilitated by cell damage.] But, without an image of B814, Tyrrell had no means to verify whether B814 is enveloped, and whether it is something other than a rhinovirus. Moreover, Tyrrell had no way of detecting other B814-like isolates, except by infecting volunteers.
Again, a colleague suggested a solution to a Tyrrell dilemna. Tony Waterson, a virologist at St Thomas’ Hospital in London (the same hospital that treated British Prime Minister Boris Johnson when he was suffering from COVID-19) had just hired an electron microscopist, June Almeida, who he claimed was extending the range of electron microscopy to new limits. In Tyrrell’s words: “It was generally believed at the time that you had to have concentrated and purified viruses to detect them with such an instrument. But she claimed that she would be able to find the virus particles in our organ cultures with her new, improved techniques. We were not too hopeful but felt it was worth a try.”
“Samples were prepared, each one infected with a well-known virus such as influenza or herpes, and included amongst them was the B814 virus. The cultures were put into bottles with coded labels and sent by train to London. June Almeida duly dabbed the surface of the tissues onto her microscope grids and examined each one. The results exceeded all our hopes. She recognized all our viruses, and her pictures revealed their structure beautifully. But, more importantly, she also saw virus particles in the B814 specimens!”
But what exactly was B814? As expected, it was not a rhinovirus. Rather, with its envelope and spikes, it looked more like influenza virus. Indeed, that was what most researchers believed it to be. But, based on the unique short spikey projections that Almeida detected on B814, she was quite certain that it represented a previously unknown family of viruses.
As Almeida was certain that she had identified a previously unknown type of virus, she, Tyrrell, and Waterson met to decide the new family’s name. In Tyrrell’s words: “So, what should we call them? ‘Influenza-like’ seemed a bit feeble, somewhat vague, and probably misleading. We looked more closely at the appearance of the new viruses and noticed that they had a kind of halo surrounding them. Recourse to a dictionary produced the Latin equivalent, corona, and so the name coronavirus was born.” Thus, Almeida revealed the first known human coronavirus, and was part of the trio that gave the virus family its name.
In 1965, Tyrell published a report on B814 in the British Medical Journal, referring to it as “a novel cold virus.” But, when Almeida submitted her paper on the structure of B814 to a peer-reviewed journal, it was rejected because the referees believed that her images of the new virus were just bad pictures of influenza virus. Almeida was finally able to publish her photographs of B814 in 1967, in the Journal of General Virology.
Before the discoveries of SARS and MERS, and now of COVID-19, virologists associated coronaviruses with clinically mild common colds. Consequently, coronaviruses were viewed as not particularly significant. In Tyrrell’s words: “Perhaps one of the common cold’s main problems is that it is not sufficiently virulent. ‘Familiarity breeds contempt’ is a proverb which could have been devised expressly to describe our attitude towards it. If it was more frequently life-threatening, like smallpox, polio, yellow fever, and AIDS, then perhaps research into its effects and a possible cure might have begun much earlier, been more widespread, and been maintained over a longer period.”
In any case, such sentiments help explain why Almeida moved on to investigating other viruses, rather than continuing with B814. In 1967, she produced the first electron micrographs of the rubella virus. And in 1970, she generated the first electron micrographs of hepatitis B virus (HBV), which she detected in the blood of a hemophiliac patient. That was an achievement of singular medical importance, since it was not yet clear whether the “Australia antigen” originally discovered in the blood of leukemia patients by Baruch Blumberg, and later in the blood of hepatitis B patients, might be an actual virus particle or a non-viral particle related to it, or, indeed the actual agent of hepatitis B. [See Note 1.] In fact, many scientists were skeptical of any relationship between the antigen and hepatitis B disease. But Almeida’s electron micrographs of a blood sample of a hemophiliac patient (Note 1), who tested positive for the Australia antigen, were instrumental in convincing the scientific community that the antigen is a component of the virus. Moreover, she convinced skeptics that HBV is the agent of hepatitis B. Furthermore, Almeida’s studies of individual differences in the immune response to HBV provided key insights into the variety of patient outcomes to the infection. And, Almeida provided the first high quality micrographs of HIV, the agent responsible for AIDS. These, and yet other important contributions, are testimony to Almeida’s outstanding scientific career. Yet, if not for the current COVID-19 crisis, she would not have come to the attention of the general public.
[Note 1: Leukemia and hemophiliac patients were more likely than the general population to have received multiple blood transfusions. Moreover, prior to 1972, a large percentage of the blood supply came from paid donors, at least some of whom were syringe-sharing, intravenous drug abusers and, consequently, more likely than most to be HBV carriers. Thus, leukemia and hemophiliac patients were more likely than most to have been recipients of donated blood, some of which was contaminated with HBV. By 1972, all donated blood in the United States had to be screened for HBV. See: Baruch Blumberg: The Hepatitis B Virus and Vaccine, posted on the blog June 2, 2016.]
June Almeida (nee June Hart) was born in 1930, in Glasgow, Scotland. Although she excelled in science in secondary school, her family lacked the means to send her to university. Consequently, June trained to be a laboratory technician in the department of histopathology at the Glasgow Royal infirmary. She then had a similar position at St. Bartholomew’s Hospital in London.
In 1954 June married Venezuelan artist, Henry Almeida. Two years later, June and Henry immigrated to Canada. The move was a watershed event in June’s life, since it gave her the opportunity to take a technician position at the newly opened Ontario Cancer Institute (OCI), under immunologist and expert electron microscopist, Allan F. Howatson, who set June was on the path to becoming an expert and innovative electron microscopist in her own right.
The move to Canada was fortunate for June for yet another reason. Not having a university degree was less of an impediment to her in Canada than it would have been had she remained in the United Kingdom, where there was more emphasis on formal academic degrees. So, in Canada, with no university degree, June Almeida rose to the position of junior scientist at the OCI. Moreover, she was encouraged to pursue her own independent research. What’s more, her expertise at electron microscopy is credited with helping to establish the reputation of the OCI in the new fields of tumor viruses and structural virology. Indeed, Almeida was a key player in the OCI group that characterized a new tumor virus, recently discovered by Ludwig Gross, as the prototype virus of the polyomavirus family of small DNA tumor viruses.
Almeida moved back to London in 1964. It happened after she met Tony Waterson when he happened to be visiting Toronto. Waterson had just been appointed chair of microbiology at London’s St Thomas‘s Hospital Medical School, one of the most prestigious medical schools in the United Kingdom. In any case, Almeida’s micrographs so impressed Waterson, that he used the occasion of being in Toronto to recruit her to his research team in London. That is how Almeida had been with Waterson when he recommended her to Tyrrell, so that she might help him sort out B814.
Henry Almeida soon regretted the couple’s mutual decision to leave Canada. He wanted to return to Canada, but June was unwilling to move back. So, the couple divorced in 1967. June then raised their daughter Joyce, born in 1960, as a single parent, while continuing her demanding work with Waterson.
Photograph of June Almeida in the 1960s using a Philips EM300 electron microscope. Credit: Joyce Almeida, in L. Marks, 2020.
After spending three years at St Thomas’ Hospital, Almeida moved, with Waterson, to the Hammersmith Postgraduate Medical School, where she was appointed a research fellow in the Department of Virology. At Hammersmith, she carried out her important investigations of HBV. In 1970, she was promoted at Hammersmith to the position of Senior Lecturer. Then, in 1972, she was recruited to the Wellcome Research Laboratories in Beckenham, Kent, where she worked on vaccines and diagnostics for different viruses, including HBV. Moreover, her pioneering contributions to the development of immune-electron microcopy significantly advanced diagnostic virology in general. And, her techniques for resolving viral morphologies were especially important for classifying newly detected viruses. Almeida remained at the Wellcome Laboratories until her retirement in 1984.
Lara Marks (in What is Biotechnology?) states that Almeida authored or co-authored 103 peer-reviewed publications during her career—a notable record by any standards. Nonetheless, Almeida had always been modest about her accomplishments, claiming that she was just fortunate to be in the right place at the right time. Actually, it was her nearly unique proficiency at electron microscopy that drew others to her with their projects.
Also, bear in mind that for a major part of Almeida’s career, her only academic qualification was her training, while a teenager, to be a laboratory technician. In 1971 she was awarded a D.Sc. from The University of London, based on the submission of her highly regarded scientific publications.
In 1982 Almeida was remarried to Phillip Gardner, a clinical virologist who had a hand in developing immunofluorescence-electron microscopy—an important technique for basic research, as well as for the rapid diagnosis of many viral infections, especially of the respiratory tract. The couple met at a conference organized by the European Group for Rapid Virus Diagnosis, which Gardner had founded. Gardner had earlier suffered a serious heart attack that would force him to retire in 1984. June joined him in retirement the same year, and the couple moved to the seaside town of Bexhill. But even in retirement, June helped colleagues at St Thomas’ Hospital to generate some of the first high-quality micrographs of HIV. June Almeida died in Bexhill, England, on Dec. 1, 2007. She was 77.
References:
David Tyrrell and Michael Fielder, Cold Wars: The Fight Against the Common Cold, Oxford University Press, 2002.
Tyrrell D. A. J., and M. L. Bynoe. 1965. Cultivation of a novel type of common-cold virus in organ cultures. British Medical Journal1:1467-1470.
Almeida, J. D., and D. A. J. Tyrrell. 1967. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture. Journal of General Virology1:175-178.
Leonard Norkin Virology Site 