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Four of the seven human coronaviruses are endemic around the world but cause little more than the common cold. Currently, SARS-CoV-2 is a global epidemic, with the potential to be considered a pandemic. In one scenario, this outbreak may be contained, and the virus never seen again, like SARS-CoV. Alternatively, the virus may become an endemic virus with seasonality like influenza and the other human coronaviruses. However, it is too early to know whether SARS-CoV-2 spread will be affected by changing weather conditions. Nearly all cases of COVID-19 have been in China, where it is winter; whether cases will decrease as temperatures increase in the Northern Hemisphere, as is seen for influenza, remains to be seen.
As more cases of COVID-19 occur, it is becoming established that the most severe cases and mortality are associated with underlying health conditions. The most common associated comorbidities are pulmonary disease, diabetes, and old age (10). Interesting questions as to how these comorbidities impact viral pathogenesis are open for investigation. More severe SARS cases were also associated with age, and work in mice has demonstrated this (12–14). Severe MERS is associated with diabetes and other underlying health conditions (15, 16), and again, work in mice has shown that diabetes can impact the immune response to infection, leading to increased pathogenesis (17). It will be interesting to see whether SARS-CoV-2 infection is similarly impacted.
In 2003, the Chinese population was infected with a virus causing Severe Acute Respiratory Syndrome (SARS) in Guangdong province. The virus was confirmed as a member of the Beta-coronavirus subgroup and was named SARS-CoV,. The infected patients exhibited pneumonia symptoms with a diffused alveolar injury which lead to acute respiratory distress syndrome (ARDS). SARS initially emerged in Guangdong, China and then spread rapidly around the globe with more than 8000 infected persons and 776 deceases. A decade later in 2012, a couple of Saudi Arabian nationals were diagnosed to be infected with another coronavirus. The detected virus was confirmed as a member of coronaviruses and named as the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The World health organization reported that MERS-coronavirus infected more than 2428 individuals and 838 deaths. MERS-CoV is a member beta-coronavirus subgroup and phylogenetically diverse from other human-CoV. The infection of MERS-CoV initiates from a mild upper respiratory injury while progression leads to severe respiratory disease. Similar to SARS-coronavirus, patients infected with MERS-coronavirus suffer pneumonia, followed by ARDS and renal failure.
Recently, by the end of 2019, WHO was informed by the Chinese government about several cases of pneumonia with unfamiliar etiology. The outbreak was initiated from the Hunan seafood market in Wuhan city of China and rapidly infected more than 50 peoples. The live animals are frequently sold at the Hunan seafood market such as bats, frogs, snakes, birds, marmots and rabbits. On 12 January 2020, the National Health Commission of China released further details about the epidemic, suggested viral pneumonia. From the sequence-based analysis of isolates from the patients, the virus was identified as a novel coronavirus. Moreover, the genetic sequence was also provided for the diagnosis of viral infection. Initially, it was suggested that the patients infected with Wuhan coronavirus induced pneumonia in China may have visited the seafood market where live animals were sold or may have used infected animals or birds as a source of food. However, further investigations revealed that some individuals contracted the infection even with no record of visiting the seafood market. These observations indicated a human to the human spreading capability of this virus, which was subsequently reported in more than 100 countries in the world. The human to the human spreading of the virus occurs due to close contact with an infected person, exposed to coughing, sneezing, respiratory droplets or aerosols. These aerosols can penetrate the human body (lungs) via inhalation through the nose or mouth (Fig. 2),,,.
The clinical features of this cohort of 271 patients managed at a single institution were similar to those reported elsewhere, although diarrhea was present in only 11% of patients compared to rates of 20–73% reported in studies from Hong Kong and Canada. Like other SARS outbreaks, many cases (41.3%) were acquired after exposure in the hospital environment, with healthcare workers providing 34% of cases at this institution. Of note was a case of SARS possibly acquired in a diagnostic laboratory. There have since been a number of cases acquired in research laboratories.
Detection of SARS-CoV RNA by RT-PCR is only moderately useful in the early diagnosis of SARS, as the maximal viral load and RT-PCR sensitivity occurs in the second week of illness. In addition, the sensitivity of SARS-CoV RT-PCR on specimens collected from different sites and at different time points in the illness varies. Testing more than one clinical specimen increases the likelihood of obtaining a positive RT-PCR result. In one large study, 60% of patients with clinical SARS had a positive SARS-CoV RT-PCR in one or more clinical specimens, with the highest detection rates in sputum (55.6%), NPAs (29.6%) and nose/throat swabs (20%) collected within the first 5 days of illness. We found that the likelihood of a positive SARS-CoV RT-PCR was similar in serum (54.3%) and throat washes (56.6%) in the first 9 days of illness. We found the peak of SARS-CoV detection in throat washes to be between days 5 to 14, where 60.8% (42/69) of samples were positive, similar to reported rates in other respiratory specimens. The viral loads in throat washes decreased over time and were at levels between those in feces and sera at similar time points.
In other studies, throat swabs were RT-PCR positive in 37.5% of probable SARS cases, reaching 50–60% on days 7–10, and consistent with earlier studies showing peaks of virus shedding in the respiratory tract in the second week of illness. High viral loads were seen in NPAs in 14 patients with SARS, mainly in the second week of illness. The use of patient self-collected throat washings may reduce risks to healthcare workers, although lower respiratory tract samples such as sputum, NPAs or bronchoalveolar lavage fluid are likely to have higher viral loads and offer increased likelihood of SARS-CoV detection by RT-PCR. We were unable to correlate viral loads in the various clinical samples with ability to isolate virus or transmission to other people; whether viral load in the respiratory tract correlates with 'super-shedding' events is uncertain.
Although overall SARS-CoV detection rates and viral load in throat washes and stools were higher than in the serum, serum SARS-CoV RT-PCR is a useful investigation early in the illness as we found that 50% of sera had SARS-CoV detected in the first four days of illness. One study of sera from 8 probable SARS patients found a detectable SARS-CoV load ranging from 2 × 103 to 1 × 104 copies/ml serum in 50% of the samples, but not after 12 days after onset. Of interest was that occasional serum samples from individuals remained SARS-CoV RT-PCR positive (with moderate viral loads) over three weeks after onset of illness, a feature noted in another study.
High rates of SARS-CoV RT-PCR detection (as high as 86.3% between days 10–19) and high viral loads were found in fecal samples in the second to fourth weeks of disease. Rates of SARS-CoV detection in fecal samples began to decrease after one month, although many stools were still SARS-CoV RNA positive 40 days or more after the onset of the clinical illness. The SARS-CoV load in fecal samples collected after 40 days were higher than the peak load seen in sera collected early in disease, and comparable to the viral load in throat washes in the second week of illness. Both lower (27% in fecal samples collected 11–20 days after onset) and similar high detection rates (over 80% in stools collected 11–16 days after onset) have been reported elsewhere, as have fecal samples positive 40 days or more after onset. Despite the high SARS-CoV load in feces, diarrhea was not a prominent clinical feature in this cohort. Long-term fecal viral shedding may be an additional source of community spread of SARS, although the infectivity of feces may be better assessed with virus isolation.
Direct comparisons of the sensitivity and specificity of RT-PCR for the detection of SARS-CoV are hampered by the use of different types of clinical specimens, RNA extraction procedures and different RT-PCR techniques. The first published interlaboratory comparison showed sensitivities of 61% and 68% for 72 NPAs, 65% and 72% for 54 throat swabs, 50% and 54% for 78 urine samples and 58% and 63% for 19 stool specimens, with an overall specificity of 100%. To date, no significant differences in the sensitivity and specificity of various commercial and in-house RT-PCR or other molecular assays have been reported.
SARS-CoV infection results in a severe respiratory disease. It causes significant nosocomial infection and requires aggressive infection control practices rarely used for other causes of atypical pneumonia. Laboratory confirmation of SARS is crucial in the management of patients presenting with pneumonia, particularly as the clinical features of SARS make it difficult to distinguish from other causes of atypical pneumonia. Molecular methods for SARS diagnosis are useful, although their value is affected by the observation that maximal viral shedding occurs after the first week of illness rather than at the initial clinical presentation. The SARS outbreak was characterized by high infection rates in healthcare workers; patient self-collected specimens such as throat washes or feces, or serum may pose less risk to healthcare workers, particularly in the context of concerns about nosocomial acquisition. Although NPAs and other lower respiratory tract samples are the sample of choice for suspected respiratory viral infections, patient self-collected specimens are suitable for RT-PCR. Thus they offer diagnostic value, especially in SARS where the peak of viral shedding is after the first week of illness, and this sampling approach may reduce the safety issues of healthcare workers collecting NPAs. Patient self-collected specimens may be less appropriate for common seasonal respiratory virus infections such as influenza, where viral shedding is maximal at clinical presentation and virus is rarely detected outside the respiratory tract. Accurate and rapid laboratory diagnosis will become even more important as SARS becomes less common, or in the event of new outbreaks of SARS, especially if influenza or other seasonal respiratory viruses are co-circulating.
Coronaviruses belong to the Coronaviridae family in the Nidovirales order. Corona represents crown-like spikes on the outer surface of the virus; thus, it was named as a coronavirus. Coronaviruses are minute in size (65–125 nm in diameter) and contain a single-stranded RNA as a nucleic material, size ranging from 26 to 32kbs in length (Fig. 1). The subgroups of coronaviruses family are alpha (α), beta (β), gamma (γ) and delta (δ) coronavirus. The severe acute respiratory syndrome coronavirus (SARS-CoV), H5N1 influenza A, H1N1 2009 and Middle East respiratory syndrome coronavirus (MERS-CoV) cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) which leads to pulmonary failure and result in fatality. These viruses were thought to infect only animals until the world witnessed a severe acute respiratory syndrome (SARS) outbreak caused by SARS-CoV, 2002 in Guangdong, China. Only a decade later, another pathogenic coronavirus, known as Middle East respiratory syndrome coronavirus (MERS-CoV) caused an endemic in Middle Eastern countries.
Recently at the end of 2019, Wuhan an emerging business hub of China experienced an outbreak of a novel coronavirus that killed more than eighteen hundred and infected over seventy thousand individuals within the first fifty days of the epidemic. This virus was reported to be a member of the β group of coronaviruses. The novel virus was named as Wuhan coronavirus or 2019 novel coronavirus (2019-nCov) by the Chinese researchers. The International Committee on Taxonomy of Viruses (ICTV) named the virus as SARS-CoV-2 and the disease as COVID-19,,. In the history, SRAS-CoV (2003) infected 8098 individuals with mortality rate of 9%, across 26 contries in the world, on the other hand, novel corona virus (2019) infected 120,000 induviduals with mortality rate of 2.9%, across 109 countries, till date of this writing. It shows that the transmission rate of SARS-CoV-2 is higher than SRAS-CoV and the reason could be genetic recombination event at S protein in the RBD region of SARS-CoV-2 may have enhanced its transmission ability. In this review article, we discuss the origination of human coronaviruses briefly. We further discuss the associated infectiousness and biological features of SARS and MERS with a special focus on COVID-19.
Both SARS-CoV and MERS-CoV are emerging zoonotic pathogens that crossed the species barriers to infect humans [10, 53, 99]. Evidence showed that SARS-CoV and MERS-CoV originated from bats, the nature reservoirs, then transmitted to human via intermediate hosts civets and camels, respectively [10, 40, 53, 81, 100]. Human SARS-CoV infection originated from the direct contact between humans and civets in markets or restaurants. Closing wet markets and cleaning civet cut off the spread chain of SARS-CoV and effectively ended the SARS epidemic [40, 42, 101]. In contrast, MERS-CoV is believed to have existed in camels for a very long time and camels are widely distributed in Middle East and African countries, serving as important transport vectors and sources of meat and milk for the local population. Therefore, it is difficult to adopt the same strategy of SARS-CoV control in the prevention of future MERS-CoV outbreaks. Until a comprehensive approach is found, which most likely will involve the effective vaccination of camels against MERS-CoV among other measures, it is envisaged that sporadic human infection will persist for some time in the future [11, 70].
SARS, also known as “atypical pneumonia”, was the first well documented HCoV-caused pandemic in human history and the etiological agent is SARS-CoV, the third HCoV discovered 14,15. The first case of SARS can be traced back to late 2002 in Guangdong Province of China. The SARS epidemic resulted in 8,096 reported cases with 774 deaths, spreading across many countries and continents. Apart from the super-spreaders, it was estimated that each case could give rise to approximately two secondary cases, with an incubation period of 4 to 7 days and the peak of viral load appearing on the 10th day of illness 14,15.
Patients infected with SARS-CoV initially present with myalgia, headache, fever, malaise and chills, followed by dyspnea, cough and respiratory distress as late symptoms 14,15. Lymphopenia, deranged liver function tests, and elevated creatine kinase are common laboratory abnormalities of SARS 14,15. Diffuse alveolar damage, epithelial cell proliferation and an increase of macrophages are also observed in SARS patients 31. Approximately 20-30% of patients subsequently require intensive care and mechanical ventilation. In addition to lower respiratory tract, multiple organs including gastrointestinal tract, liver and kidney can also be infected in these severe cases, usually accompanied with a cytokine storm, which might be lethal particularly in immunocompromised patients. The virus was first isolated from the open lung biopsy of a relative of the index patient who travelled to Hong Kong from Guangzhou 14,15. Since then, tremendous efforts have been dedicated to HCoV research.
The first HCoV-229E strain was isolated from the respiratory tract of patients with upper respiratory tract infection in the year of 1966 27, and was subsequently adapted to grow in WI-38 lung cell lines 28. Patients infected with HCoV-229E presented with common cold symptoms, including headache, sneezing, malaise and sore-throat, with fever and cough seen in 10~20% cases 29. Later in 1967, HCoV-OC43 was isolated from organ culture and subsequent serial passage in brains of suckling mice 28. The clinical features of HCoV-OC43 infection appear to be similar to those caused by HCoV-229E, which are symptomatically indistinguishable from infection with other respiratory tract pathogens such as influenza A viruses and rhinoviruses 28.
Both HCoV-229E and HCoV-OC43 are distributed globally, and they tend to be predominantly transmitted during the season of winter in temperate climate 2. Generally, the incubation time of these two viruses is less than one week, followed by an approximately 2-week illness 28. According to a human volunteer study, healthy individuals infected with HCoV-229E developed mild common cold 30. Only a few immunocompromised patients exhibited severe lower respiratory tract infection.
Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, resulting in about 8,000 human infections with an 11% case fatality rate. SARS-CoV is a zoonotic pathogen that originated from bats and either entered the human population directly and/or cycled through palm civets and raccoon dogs as intermediate hosts,. Advanced age was significantly associated with increased SARS-related pathogenicity and deaths due to rapidly progressive respiratory compromise (acute respiratory distress syndrome [ARDS]),.
Based on epidemiological studies the SARS-CoV epidemic was characterized by different phases: zoonotic, early, middle and late phases. The zoonotic phase was characterized by strains isolated from palm civets and raccoon dogs in live animal markets. The early phase was characterized by several independent human cases, likely due to zoonotic transmission. The middle phase was characterized by initial human-to-human transmission, whereas the late phase was characterized by efficient human-to-human transmission and extensive global spread to over 30 countries.
The SARS-CoV spike (S) glycoprotein has been identified as the main mediator of virus entry and host range by binding to its receptor angiotensin 1-converting enzyme 2 (ACE-2),. A high mutation rate of the S glycoprotein was observed between the different isolates from both animals and humans and several amino acid changes have been identified as being critical for the transition from animal-to-human to human-to-human transmission,,.
The S glycoprotein has also been identified as a major target of protective immunity and as such has been the main focus of vaccine development,. While most vaccine candidates have been developed using nearly identical isolates from the late phase in the epidemic, it is not clear whether these late phase isolates will provide robust protection against infection with zoonotic and early human phase isolates, the most likely source of future outbreaks. Therefore a heterologous challenge model is needed to test cross-protection efficacy of vaccine candidates.
Currently, several animal models for SARS-CoV exist, including mice, hamsters, ferrets and non-human primates,,,,,,. While these models have been used for pathogenesis studies as well as vaccine development, the majority of these studies focused on isolates from the late phase of the outbreak. In fact, heterologous challenge of mice after vaccination against a late phase S glycoprotein offered only partial protection.
We previously characterized the in vitro and in vivo virulence of an isogenic panel of recombinant SARS-CoV isolates bearing the S glycoprotein from zoonotic, early, middle and late phase isolates. The late and early human phase viruses replicated to high titers in human ciliated airway epithelial cultures whereas the zoonotic isolates did not. Interestingly, the early human (GZ02) and zoonotic phase (HC/SZ/61/03) isolates were lethal in an aged mouse model resulting in severe lung infection progressing to an early organizing phase of ARDS and death,.
In this study we compare the pathogenesis of three recombinant SARS-CoV isolates bearing the S glycoprotein of zoonotic, early and late phase isolates in cynomolgus macaques. The Urbani strain and recombinant Urbani SARS-CoV from our molecular clone have previously been tested in a non-human primate model and served as a control in the current study. Clinical, virological and histological parameters as well as the host responses were compared to more fully understand the effect of S glycoprotein variation on virus replication and the host response to SARS-CoV infection with the ultimate goal of characterizing heterologous challenge models for SARS-CoV vaccine development. Importantly, all viruses replicated to near similar titers and induced similar pathologic changes of mild disease, demonstrating the availability of heterologous challenge viruses for future vaccine studies.
Avian infectious bronchitis virus was the first CoV isolated in 1930. Different CoVs were subsequently isolated in infected rodents and domestic animals, including mouse, pig, cow, turkey, cats, and dogs. CoVs were once believed not to cause human disease, but this was changed after the successful isolation of HCoV strain B814 from the clinical specimen of patients with common cold by serial passage of inoculum in tracheal organ culture in 1962. In the 1960s, several novel HCoVs were described but no further characterization was performed in most cases. 229E and OC43 were known as causative agents of the common cold and upper respiratory tract infection, accounting for up to 30% of cases with common cold. 229E is a prototype strain isolated using tracheal organ culture. OC43 was isolated from organ culture and subsequent serial passage in the brain of suckling mice. The clinical features of 229E and OC43 infection were characterized in human volunteer study. In most cases, natural infection with HCoVs results in mild common cold-like symptoms. Severe lower respiratory tract infection develops only in immunocompromised patients. Apart from a respiratory infection, 229E and OC43 were suspected to infect the central nervous system (CNS) as mRNA and CoV-like particles were detected in CNS samples of patients with multiple sclerosis. This claim was further supported by the susceptibility of human neural primary culture to 229E and OC43. However, the influence of 229E and OC43 on the development and progression of multiple sclerosis awaits further investigations.
In the pre-SARS era, it was generally accepted that HCoVs cause mild respiratory disease only. This concept was changed after the emergence of SARS-CoV. The first reported case of SARS-CoV infection was retrospectively dated back to November 2002 in Guangdong Province of China. In the subsequent seven months, the SARS epidemic resulted in over 8000 reported cases in 37 countries with a case fatality of 9.6%. The superspreading events in SARS-CoV transmission caused fears in the society. Although the exact cause of superspreading remains to be understood, the host but not the virus only is thought to play a key role in the release of large amounts of virions in superspreading. In this regard, the use of immunosuppressive agents such as high-dose steroid in an early phase of viral infection as a treatment modality might boost viral replication leading to the shedding of large amounts of virus. Likewise, the immunocompromised status of the superspreader could have the same effect. In addition, mutation of virus susceptibility genes encoding restriction factors implicated in host antiviral defence would also result in the shedding of extraordinarily large quantities of the virus. In other words, compromising host antiviral defence or decoupling host antiviral immune response from viral replication might allow or facilitate superspreading.
SARS-CoV was isolated and identified as the causative agent of SARS. Unlike 229E and OC43, SARS-CoV infection causes lower respiratory tract infection, accompanied by a cytokine storm in patients with poor outcome. Life-threatening ARDS was developed in some critically ill patients. Apart from a respiratory infection, gastrointestinal and CNS infection was also found in some patients with SARS. FRHK4 and Vero-E6 cells played an important role in the discovery of SARS-CoV. The same as chicken embryos in which other CoVs including avian infectious bronchitis virus were isolated and cultured, these cells are highly susceptible to SARS-CoV infection because they are type I interferon-defective. Consistent with this, infection of STAT1−/− mice with SARS-CoV resulted in a lethal outcome with a profibrotic phenotype in the lung. These mice cannot clear the virus. All these highlight the importance of host antiviral response in the control of SARS-CoV infection at the cellular and organismal level.
In the post-SARS era, CoV research was brought back to the limelight and more effort was put into the search for novel HCoVs. The search was fruitful and two new HCoVs were identified in human samples positive for HCoV but not for SARS-CoV. NL63 and HKU1 were first isolated from a child suffering from bronchiolitis and a patient with pneumonia, respectively. HKU1 is difficult to culture and can only be propagated in primary human airway epithelial cells cultured at the air–liquid interface. Similar to 229E and OC43, NL63 and HKU1 were found worldwide, causing mild respiratory diseases. Particularly, NL63 infection was associated with virus-induced croup in children. All these four viruses are community-acquired HCoVs that are well adapted to humans. Only in rare cases, they might be accidentally mutated to cause more severe lower respiratory tract disease. For example, a subtype of NL63 was recently found to be associated with severe lower respiratory tract infection in China.
Another highly pathogenic HCoV outbreak emerged in 2012 from Saudi Arabia. A new HCoV subsequently named MERS-CoV was isolated from patients who developed acute pneumonia and renal failure. Exported MERS cases were also reported outside Arabian Peninsula occasionally. One relatively big secondary outbreak with 186 confirmed cases occurred in South Korea in 2015. Up to January 2020, >2500 laboratory-confirmed case were reported with a case fatality of 34.4%. Clinical symptoms were diverse in MERS patients, ranging from asymptomatic to ARDS. Acute renal injury was unique in MERS patients, but it is more commonly observed in the Middle East than in South Korea. MERS-CoV replicates well in many different types of cells and extrapulmonary tissues including the kidney and intestinal tract. MERS-CoV is endemic in Arabian Peninsula with sporadic, but recurrent outbreaks occurring continuously since 2012.
It is clear now that SARS-CoV-2 can be transmitted by human-to-human despite the majority of the early cases had contact history with the Huanan Seafood market 11,18,28. Analysis of 425 patients with confirmed COVID-19 showed that the incubation period is 3 to 7 days. The mean was 5.2 days (95% CI: 4.1 to 7.0), and the 95th percentile of the distribution is 12.5 days (95% CI: 9.2 to 18) 11. Notably, it was reported that the incubation period could be as long as 24 days in a rare case 25. The basic reproductive number (R0) up to the period of 4 Jan 2020 was estimated based on the study of 425 patients to be 2.2 (meaning that one patient has been spreading infection to 2.2 other people) 11, slightly smaller than the value of 2.68 by a modelling in another 29. The R0 of SARS-CoV-2 from both of these two studies is smaller than that of SRAS, which are 3 before public health measures were implemented 30. However, subsequent investigation based on the analysis of high-resolution real-time human travel and infection data estimated that the R0 is much larger, ranging from 4.7 to 6.6 before the control measures 31, implying that SARS-CoV-2 is highly contagious and more infectious than initially estimated. This conclusion is consistent with the wide spread of SARS-CoV-2 within a short period time and was also echoed by the finding that SARS-CoV-2 Spike (S) protein had 10- to 20-fold higher affinity to human angiotensin-converting enzyme 2 (ACE2) receptor than that of SARS-CoV based on the Cryo-EM structure analysis of S proteins 32. Similar to SARS-CoV, the entry of SARS-CoV-2 into host cells depends on the recognition and binding of S protein to ACE2 receptor of the host cells 14,33. The high affinity of S protein to ACE2 receptor likely contributes to the quick spreading of virus. The finding of ACE2 as the receptor of SARS-CoV-2 also indicates that human organs with high ACE2 expression level, such as lung alveolar epithelial cells and enterocytes of the small intestine, are potentially the target of SARS-CoV-2 34.
As a new coronavirus, it is not known yet about how SARS-CoV-2 spreads. Current knowledge for SARS-CoV-2 transmission is largely based on what is known from the similar coronaviruses, particularly SARS-CoV and MERS-CoV, in which human-to-human transmission occurs through droplets, contact and fomites. SARS-CoV is predominantly transmitted through indirect or direct contact with mucous membranes in the mouth, eyes, or nose 35. It has been shown that unprotected eyes and exposed mucous membranes are vulnerable to SARS-CoV transmission 36. A member of the national expert panel on pneumonia was infected by SARS-CoV-2 after the inspection in Wuhan 37. As he wore a N95 mask but not any eye protector, and experienced eye redness before the onset of pneumonia, it was thus suspected that unprotected exposure of the eyes to SARS-CoV-2 might be another transmission pathway 37. However, SARS-CoV-2 was not detected from the conjunctival swab sample in a confirmed COVID-19 patent with conjunctivitis 38, suggesting that more evidences are needed before concluding the conjunctival route as the transmission pathway of SARS-CoV-2. The mode of transmission by MERS-CoV is not well understood but is believed to spread largely via the respiratory close contact route 39,40.
Based on the transmission mode of SARS-CoV and MERS-CoV, a serial of preventive measures have been recommended, including avoiding close contact with people suffering from acute respiratory infections and frequent hand-washing 41. The viruses of SARS-CoV-2 were also detected in the stool samples in some patients but not all 18,22, suggesting that a possible fecal-oral transmission occurs 42. A systematic study showed that viruses could be detected in oral swabs, anal swabs and blood samples of the patients, and the anal swabs and blood could test positive when oral swab tested negative 43. Furthermore, a trend of shift from more oral positive in the collected samples during the early period of patient infection to more anal positive during later period of infection was also found 43. Therefore, a multiple shedding routes of SARS-CoV-2 might exist.
One of the challenges for preventive control of SARS-CoV-2 spreading is that the viruses are likely transmitted by asymptomatic contact. A German businessman was found infected by SARS-CoV-2 after attending a conference together with a colleague, who had no signs or symptoms of infection but had become ill due to the SARS-CoV-2 infection later 44. This observation suggests that infected patients likely start to shed viruses before the onset of any symptom, which undoubtedly will bring great challenge to the current practice of preventive control by measuring body temperature. Despite the claim of the transmission by asymptomatic contact has been challenged 45, other asymptomatic carriers were also observed to transmit the viruses of SARS-CoV-2 46,47. Consistently, a study found that an asymptomatic patient had a similar vial loads in the samples of nasal and throat swabs to that of the symptomatic patients 48.
Prior to the outbreaks of SARS and MERS, the clinical importance and epidemic possibility of CoVs had been recognized by researchers, (Table 1). In 2002, a SARS epidemic that originated in Guangdong Province in China resulted in 916 deaths among more than 8098 patients in 29 countries, identifying SARS as the first new infectious disease of the 21st century. Ten years later, the World Health Organization (WHO) published 2254 laboratory-confirmed cases of MERS-CoV that occurred from 2012 to 16 September 2018, with at least 800 deaths in 27 countries. Remarkably, more than 80% of recent research into the virology and genetics of this infection indicated that bats could be the possible natural reservoirs of both SARS and MERS-CoV. Palm civets and dromedary camels are also possible intermediary hosts of SARS and MERS, respectively, before dissemination to humans.
The transmission mechanism of SARS-CoV and MERS-CoV has yet to be fully understood. For transmission from animals to humans, direct contact with the intermediary host might be one route. Recent reports demonstrated that camel workers in Saudi Arabia with high prevalence of MERS-CoV infection may contribute to the transmission of MERS. Some customs and habits may also be conducive to transmission, such as the consumption of milk, urine, or uncooked meat. In this way, MERS-CoV was transmitted from dromedary camels directly to humans, principally in the Arabian Peninsula, and this is considered to be the main route of transmission from animals to humans, causing significant morbidity and mortality. Human-to-human spread has also been detected, especially through nosocomial transmission. Delays in diagnosis in hospitals might lead to secondary cases among healthcare workers, family members, or other patients sharing rooms. Among the reported cases of SARS, 22% were healthcare workers in China and more than 40% were healthcare workers in Canada. Nosocomial transmission for MERS has similarly been seen in the Middle East and in the Republic of Korea. Outbreaks in other countries all resulted from the reported cases in the Middle East or North Africa, and transmission was the result of international travel. Both SARS and MERS caused large outbreaks with significant public health and economic consequences.
Virus replication of all three SARS-CoV strains was mainly restricted to the respiratory tract (trachea, bronchi and lung lobes) and low levels of replication in spleen, cervical and bronchial lymph nodes (Fig. 2A and B). Viral replication peaked in the respiratory tract on day 1 p.i. (Fig. 2A) with a reduction in virus titers and numbers of virus positive tissues on day 4 p.i. (Fig. 2B), similar to our findings in mice. No infectious virus could be isolated on 14 days p.i. in any of the tissues (Data not shown). Virus titers in Urbani infected animals were generally 1 to 2 logs higher compared to GZ02 and HC/SZ/61/03 infected animals. Viral RNA could be detected in the lungs of infected animals up to 14 days p.i. (data not shown). The levels of viral RNA were similar for each SARS-CoV strain at each time point but decreased over time as observed with the viral titers.
As a measure of virus shedding, nasal, oral and rectal swabs were assayed for infectious virus. In nasal swabs, virus titers peaked by day 2 but could be detected up to day 7 and 11 p.i. (Fig. 3A) Virus titers in oral swabs peaked on day 1 and were on or below detectable titers by day 4 p.i. (Fig. 3B). Low levels of virus replication could be observed in rectal swabs but only in a few animals (Fig. 3C).
To date, MERS‐CoV only has two mature models. This section will deal with additional non‐human prime models for SARS‐CoV. Rhesus, cynomolgus (Macaca fasicularis), and African green (Chlorocebus aethiops sabaeus or Cercopithecu aethiops sabaeus) monkeys have been used to investigate vaccine immunogenicity or efficacy against SARS‐CoV. Squirrel monkeys (Saimiri sciureus) and mustached tamarins (Saguinus mystax) have been shown to be incapable of being infected.
In the case of cynomolgus monkeys, clinical evidence, such as lethargy, temporary skin rash or respiratory distress, has not been reported. However, 4 to 6 days post‐inoculation (dpi), there was diffuse alveolar damage and extensive loss of epithelium from alveolar and bronchiolar walls.28, 34
Regarding African green monkeys, clearance of the virus takes approximately 4 dpi, and the infection niduses are patchy. Respiratory secretions cannot accurately reflect the viral titers. However, there is a report that showed that the titer is higher and the residence time is longer in African green monkeys than in two other kinds of old world monkeys (cynomolgus and rhesus).34
As a novel disease, COVID-19 has just started to manifest its full clinical course throughout thousands of patients. In most cases, patients can recover gradually without sequelae. However, similar to SARS and MERS, COVID-19 is also associated with high morbidity and mortality in patients with severe cases. Therefore, building a prognosis model for the disease is essential for health-care agencies to prioritize their services, especially in resource-constrained areas. Based on clinical studies reported thus far, the following factors may affect or be associated with the prognosis of COVID-19 patients (Table 3):Age: Age was the most important factor for the prognosis of SARS 99, which is also true for COVID-19. COVID-19 mainly happened at the age of 30-65 with 47.7% of those patients being over 50 in a study of 8,866 cases as described above 37. Patients who required intensive care were more likely to have underlying comorbidities and complications and were significantly older than those who did not (at the median age of 66 versus 51) 34, suggesting age as a prognostic factor for the outcome of COVID-19 patients.Sex: SARS-CoV-2 has infected more men than women (0.31/100,000 versus 0.27/100,000), as described above 37.Comorbidities and complications: Patients with COVID-19 who require intensive care are more likely to suffer from acute cardiac injury and arrhythmia 34. Cardiac events were also the main reason for death in SARS patients 55,65,99. It has been reported that SARS-CoV-2 can also bind to ACE2-positive cholangiocytes, which might lead to liver dysfunctions in COVID-19 patients 100. It is worth noting that age and underlying disease are strongly correlated and might interfere with each other 55.Abnormal laboratory findings: The C-reactive protein (CRP) level in blood reflects the severity of inflammation or tissue injury and has been proposed to be a potential prognostic factor for disease, response to therapy, and ultimate recovery 101. The correlation of CRP level to the severity and prognosis of COVID-19 has also been proposed 101. In addition, elevated lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) may also help predict the outcome. These enzymes are expressed extensively in multiple organs, especially in the heart and liver, and are released during tissue damage 102,103. Thus, they are traditional markers for heart or liver dysfunctions.Major clinical symptoms: Chest radiography and temporal progression of clinical symptoms should be considered together with the other issues for the prediction of outcomes and complications of COVID-19.Use of steroids: As described above, steroids are immunosuppressant commonly used as an adjunctive therapy for infectious diseases to reduce the severity of inflammatory damage 104. Since a high dosage of corticosteroids was widely used in severe SARS patients, many survivors suffered from avascular osteonecrosis with life-long disability and poor life quality 105. Thus, if needed, steroids should be used at low dosage and for a short time in COVID-19 patients.Mental stress: As described above, during the COVID-19 outbreak many patients have suffered from extraordinary stress as they often endured long periods of quarantine and extreme uncertainty and witnessed the death of close family members and fellow patients. It is imperative to provide psychological counseling and long-term support to help these patients recover from the stress and return to normal life 66.
According to demographic studies so far, COVID-19 seems to have different epidemiological features from SARS. In addition to replicating in the lower respiratory tract, SARS-CoV-2 can efficiently replicate in the upper respiratory tract and causes mild or no symptoms in the early phase of infection, similar to other CoVs that cause common colds 106. Therefore, infected patients at the early phase or incubation period can produce a large amount of virus during daily activities, causing great difficulty for the control of the epidemic. However, the transmission of SARS-CoV was considered to occur when the patients are severely ill, while most transmission did not happen at the early phase 107. Thus, the current outbreak of COVID-19 is much more severe and difficult to control than the outbreak of SARS.
Great efforts are currently underway in China including the lockdown of Wuhan and surrounding cities and continuous quarantine of almost the entire population in hopes of interrupting the transmission of SARS-CoV-2. Although these actions have been dramatically damaging the economy and other sectors of the country, the number of new patients is declining, indicating the slowdown of the epidemic. The most optimistic estimate is that the outbreak will end by March and the downswing phase will last for 3-4 months 108. However, some other experts are not that optimistic. Paul Hunter, et al., estimated that COVID-19, which seems substantially more infectious than SARS, will not end in 2020 109. Ira Longini, et al., established a model to predict the outcome of the epidemic and suggested that SARS-CoV-2 could infect two-thirds of the global population 110.
A Canadian group reported that SARS-CoV-2 was detected in both mid-turbinate and throat swabs of patients who recovered and left the hospital 2 weeks earlier 111, which indicates that the newly identified virus could become a cyclical episode similar to influenza. However, promising signs have occurred in China based on the declining number of new cases, indicating the current strategies might have been working. Ebola was originally predicted to cause up to a million cases with half a million deaths. However, via strict quarantine and isolation, the disease has eventually been put under control 112,113. It is possible, similar to SARS-CoV, that SARS-CoV-2 might become weaker in infectivity and eventually die down or become a less pathogenic virus co-existent with humans. A comparison of the epidemic of COVID-19 with that of SARS and MERS is provided below (Fig. 5).
We searched MEDLINE, ScienceDirect, Embase, the Cochrane Library, WanFang Database, VIP Database, SinoMed, China National Knowledge Infrastructure (CNKI), the CDC for COVID-19 website (https://www.cdc.gov/coronavirus/2019-ncov/publications.htm), Chinese Scientific Research Academic Exchange Platform for COVID-19 (http://medjournals.cn/2019NCP/index.do), and relevant references for papers related to "ophthalmology and SARS-CoV-2/COVID-19"; published till 12th March 2020. The search strategy was as follows: (SARS-CoV-2 or 2019-nCov or COVID-19 or NCP or coronavirus or "severe acute respiratory syndrome coronavirus 2" [Supplementary Concept] or "COVID-19" [Supplementary Concept]) and (ocular or eye or ophthalm* or ophthalmologist or tear or conjunctiv* or "Conjunctivitis"[Mesh] or "Conjunctivitis, Viral"[Mesh]).
We identified 33 articles in total published by Chinese scholars directly relevant to ophthalmology and SARS-CoV-2/COVID-19. Twenty-seven articles are published in Chinese journals, most articles are reviews, almost all regarding ophthalmic precautions and ocular surface transmission of SARS-CoV-2 infection (Table 1).
Following reports of the last case of the severe acute respiratory syndrome (SARS) epidemic in July 2003, there has been remarkable progress in several areas of research on the molecular identification of the pathogen and its pathogenesis, replication, genetics, and host immunogenicity, as well as elegant epidemiological studies. The sequence of epidemiological events that unfolded early in the outbreak gave researchers a glimpse into the first new pathogen of the era of globalization. As the year 2002 drew to a close, multiple reports of an "infectious atypical pneumonia" caught public health officials across the globe by surprise and suggested that a new human pathogen had emerged in the Guangdong Province in China. By the end of February 2003, this outbreak of SARS had infected almost 800 patients and caused 31 deaths in the Province. One month later, the disease had spread throughout Asia and into Europe and North America. This epidemic eventually affected more than 8000 people and resulted in approximately 800 deaths worldwide, with mortality rates reaching over 40% in certain populations.
Electron microscope analysis quickly identified the putative SARS agent as having features associated with coronaviruses. The SARS agent was later unambiguously identified as a new coronavirus member and named SARS-coronavirus (SARS-CoV). Coronaviruses are enveloped, plus-stranded RNA viruses with the largest RNA genomes known (on the order of 30 kb). Coronaviruses have long been important in the world of veterinary viral diseases. However, previously known human coronaviruses such as HCoV-229E and HCoV-OC43 cause only minor health problems such as the common cold and gastrointestinal diseases. In contrast, the SARS-CoV pathogen causes fever, pulmonary edema, and diffuse alveolar damage in severely affected individuals (collectively termed severe acute respiratory syndrome). SARS-CoV is also a unique coronavirus in that, to date, it is the only member known to cause severe morbidity and mortality in humans. Demonstration that SARS-CoV can cause serious public health problems has focused attention on the need to understand the viral replicative strategy and devise prophylactic measures.
The clinical symptoms of SARS are those of a lower respiratory tract infection and are accompanied by damage to the lungs. Gastrointestinal involvement is also common, with more than 20% of patients presenting with watery diarrhea. Fecal samples from SARS patients taken up to 25 days after onset of disease contain viral RNA, which suggests viral shedding through the bowels. Liver dysfunction has also been reported based on observed necrosis in hepatocytes. Post-mortem tissue examination of SARS patients has found the virus presence in lung, bowel, lymph node, liver, heart, kidney, and skeletal muscle samples. The primary mode of SARS-CoV transmission is airborne via droplets. However, there are also reports of the presence of replicating virus in blood cells (peripheral blood mononuclear cells) and in the small and large intestine. Alternative modes of transmission, such as blood-borne or fecal-oral are therefore possible.
The virus has been isolated from wild animals (Himalayan palm civets and raccoon dogs) found in the animal markets of Guangdong, China. The actual natural reservoir for SARS-CoV is still unknown. Once transmitted to humans, SARS-CoV appears to evolve to facilitate to human-human transmission. Sequence analysis of different SARS-CoV isolates from early in the epidemic show deletion events occurring in open reading frame 8 (Orf 8). Identical deletions in Orf 8 have also been seen in animal coronaviruses supporting the idea that SARS-CoV was introduced to humans via an animal intermediate. In addition to deletion events occurring early and late in the epidemic, a slowing of missense mutations is seen over time, with the most extensive changes occurring in the S protein during the early stages of the outbreak. This suggests the virus has undergone some level of adaptation but has ultimately stabilized at a time in the epidemic where SARS-CoV has become more virulent. Deciphering the evolutionary passage of this virus will undoubtedly provide valuable information on preventing future outbreaks.
In the wake of the SARS epidemic, a number of excellent review articles on the clinical and molecular aspects of SARS epidemiology have been published. These reviews have focused primarily on rapid advances made in the identification and characterization of SARS-CoV genomes as well as describing the etiology of the virus and clinical features of the disease. Now the SARS-CoV story has entered a new phase, a search for preventative strategies and a cure. In this review, we highlight the progress made in revealing the molecular aspects of SARS-CoV biology and how such information may lead to strategies for disease prevention.
Coronaviruses can be found in many kinds of birds.23 Within Hong Kong alone, CoV‐HKU11 has been found in nightingales, CoV‐HKU12 in thrushes, CoV‐HKU13 in munias, CoV‐HKU16 in white‐eyes, CoV‐HKU17 in sparrows, and CoV‐HKU18 in magpies. Fortunately, avian coronaviruses are not that closely related to SARS‐CoV. Additionally, these are not migratory birds and therefore do not expand the range of the pathogen. Moreover, unlike bats, these birds were accessible enough to sterilize. However, in light of the findings above, randomly hunting them for food or for pets is unwise.
The 2019-nCoV causes an ongoing outbreak of lower respiratory tract disease called novel coronavirus pneumonia (NCP) by the Chinese government initially. The disease name was subsequently recommended as COVID-19 by the World Health Organization. Meanwhile, 2019-nCoV was renamed SARS-CoV-2 by the International Committee on Taxonomy of Viruses. As of February 24, 2020, more than 80,000 confirmed cases including more than 2,700 deaths have been reported worldwide, affecting at least 37 countries. The WHO has declared this a global health emergency at the end of January 2020. The epicenter of this ongoing outbreak is in the city of Wuhan in Hubei Province of central China and the Huanan seafood wholesale market was thought to be at least one of the places where SARS-CoV-2 from an unknown animal source might have crossed the species barrier to infect humans.
A pioneering study conducted in the city of Shenzhen near Hong Kong by a group of clinicians and scientists from the University of Hong Kong has provided the first concrete evidence for human-to-human transmission of SARS-CoV-2. This is an excellent example of how a high-quality clinical study can make a major difference in policy setting. Several important clinical features of COVID-19 have also been documented in this study. First, an attack rate of 83% within the family context is alarmingly high, indicating the high transmissibility of SARS-CoV-2. Second, the clinical manifestations of COVID-19 in this family range from mild to moderate, with more systematic symptoms and more severe radiological abnormalities seen in older patients. Generally, COVID-19 appears to be less severe than SARS. Third, an asymptomatic child was found to have ground-glass opacities in his lung and SARS-CoV-2 RNA in his sputum sample. This finding of asymptomatic virus shedding raises the possibility for transmission of SARS-CoV-2 from asymptomatic carriers to others, which is later confirmed by others. Finally, the presentation of diarrhea in two young adults from the same family also suggests the possibility for gastrointestinal involvement in SARS-CoV-2 infection and fecal–oral transmission. The study has set the stage for the control and management of COVID-19. The work was completed timely and the investigators showed great courage and leadership in a very difficult time when the Chinese authority failed to recognize widespread person-to-person transmission of SARS-CoV-2 before January 20, 2020.
Several interesting papers on SARS-CoV-2 and COVID-19 have been published in the past few weeks to report on the evolutionary reservoir, possible intermediate host and genomic sequence of SARS-CoV-2 as well as clinical characteristics of COVID-19 [6, 7]. In view of these findings and the urgent needs in the prevention and control of SARS-CoV-2 and COVID-19, in this commentary we highlight the most important research questions in the field from our personal perspectives.
The first question concerns how SARS-CoV-2 is transmitted currently in the epicenter of Wuhan. In order to minimize the spreading of SARS-CoV-2, China has locked down Wuhan and nearby cities since January 23, 2020. The unprecedented control measures including suspension of all urban transportation have apparently been successful in preventing further spreading of SARS-CoV-2 to other cities. However, the number of confirmed cases in Wuhan continued to rise. It is therefore crucial to determine whether the rise is due to a large number of infected individuals before the lock down and/or failure in the prevention of widespread intra-familial, nosocomial or community transmission. Based on the number of exported cases from Wuhan to cities outside of mainland China, it was predicted that there might be more than 70,000 individuals infected with SARS-CoV-2 on January 25, 2020 in Wuhan. This should be determined experimentally in Wuhan as discussed below and it will reveal whether the real numbers of infected people and asymptomatic carriers are indeed underestimated severely. In addition to viral RNA detection, measurement of IgM and IgG antibodies as well as antigens would be very helpful. Several representative residential areas should be selected for detailed analysis so that a big picture can be deduced. The analysis should include all healthy and diseased individuals within the area with the aim of identifying people who have recovered from an infection or are having an active infection. The ratio of asymptomatic carriers should also be determined. The analysis should also be extended to detect RNA and antigen of influenza viruses. The activity of seasonal flu in Wuhan also reached a peak at the beginning of 2020. It will be of interest to see whether the flu season had ended and how many people having a fever now are actually infected with influenza virus. Precision control measures for SARS-CoV-2 should be tailor-designed for high-risk groups based on the results of this analysis. Differentiating people having a flu and preventing them from infecting with SARS-CoV-2 in a hospital setting might also be critical.
The second question is how transmissible and pathogenic is SARS-CoV-2 in tertiary and quaternary spreading within humans. Continued transmission of SARS-CoV-2 in Wuhan suggests that tertiary and quaternary spreading has occurred. Compared to the primary and secondary spreading during which SARS-CoV-2 was transmitted from animal to human and from human to human, has the transmission rate increased and has the pathogenicity decreased? Alternatively, is the virus less transmissible after several passages in humans? Retrospective analysis of all confirmed cases in Wuhan should be very informative. The answers to the above questions hold the key to the outcome of the outbreak. If the transmission is weakened, the outbreak may ultimately come to an end at which SARS-CoV-2 is eradicated from humans. On the contrary, if effective transmission can be sustained, the chance is increased that SARS-CoV-2 will become another community-acquired human coronavirus just like the other four human coronaviruses (229E, OC43, HKU1 and NL63) causing common cold only. The basic reproductive number (R0) of SARS-CoV-2 has been estimated to be 2.68, resulting in an epidemic doubling time of about 6.4 days. Other estimates of R0 could go up to 4, higher than that of SARS-CoV, which is lower than 2. Determining the real R0 will shed light on whether and to what extent infection control measures are effective.
The third question relates to the importance of asymptomatic and presymptomatic virus shedding in SARS-CoV-2 transmission. Asymptomatic and presymptomatic virus shedding posts a big challenge to infection control [1, 2]. In addition, patients with mild and unspecific symptoms are also difficult to identify and quarantine. Notably, the absence of fever in SARS-CoV-2 infection (12.1%) is more frequent than in SARS-CoV (1%) and Middle East respiratory syndrome coronavirus (MERS-CoV; 2%) infection. In light of this, the effectiveness of using fever detection as the surveillance method should be reviewed. However, based on previous studies of influenza viruses and community-acquired human coronaviruses, the viral loads in asymptomatic carriers are relatively low. If this is also the case for SARS-CoV-2, the risk should remain low. Studies on the natural history of SARS-CoV-2 infection in humans are urgently needed. Identifying a cohort of asymptomatic carriers in Wuhan and following their viral loads, clinical presentations and antibody titers over a time course will provide clues as to how many of the subjects have symptoms in a later phase, whether virus shedding from the subjects is indeed less robust, and how often they might transmit SARS-CoV-2 to others.
The fourth question relates to the importance of fecal–oral route in SARS-CoV-2 transmission. In addition to transmission via droplets and close contact, fecal–oral transmission of SARS-CoV has been shown to be important in certain circumstances. Gastrointestinal involvement of SARS-CoV-2 infection and isolation of SARS-CoV-2 from fecal samples of patients are in support of the importance of fecal–oral route in SARS-CoV-2 transmission. Although diarrhea was rarely seen in studies with large cohorts [6, 7], the possibility of SARS-CoV-2 transmission via sewage, waste, contaminated water, air condition system and aerosols cannot be underestimated, particularly in cases such as the Diamond Princess cruise ship with 3,700 people, among whom at least 742 have been confirmed to be infected with SARS-CoV-2 plausibly as the result of a superspreading event. Further investigations are required to determine the role of fecal–oral transmission in these cases and within the representative residential areas selected for detailed epidemiological studies in Wuhan as discussed earlier.
The fifth question concerns how COVID-19 should be diagnosed and what diagnostic reagents should be made available. RT-PCR-based SARS-CoV-2 RNA detection in respiratory samples provides the only specific diagnostic test at the initial phase of the outbreak. It has played a very critical role in early detection of patients infected with SARS-CoV-2 outside of Wuhan, implicating that widespread infection of the virus had occurred in Wuhan at least as early as the beginning of 2020. This has also pushed the Chinese authority to acknowledge the severity of the situation. Due to difficulties in sampling and other technical issues in this test, at one point in early February clinically diagnosed patients with typical ground glass lung opacities in chest CT were also counted as confirmed cases in order to have the patients identified and quarantined as early as possible. ELISA kits for detection of IgM and IgG antibodies against N and other SARS-CoV-2 proteins have also been available more recently. This has made specific diagnosis of ongoing and past infection possible. Particularly, seroconversion for IgM antibodies normally occurs a few days earlier than that of IgG. ELISA reagents for detection of SARS-CoV-2 antigens such as S and N are still in urgent need, and would provide another test highly complementary to viral RNA detection.
The sixth question concerns how COVID-19 should be treated and what treatment options should be made available. COVID-19 is a self-limiting disease in more than 80% of patients. Severe pneumonia occurred in about 15% of cases as revealed in studies with large cohorts of patients. The gross case fatality is 3.4% worldwide as of February 25, 2020. This rate is 4.4% for patients in Wuhan, 4.0% for patients in Hubei and 0.92% for patients outside of Hubei. The exceedingly high fatality in Wuhan might be explained by the collapse of hospitals, a large number of undiagnosed patients, suboptimal treatment or a combination of these. Up to date, we still do not have any specific anti-SARS-CoV-2 agents but an anti-Ebola drug, remdesivir, may hold the promise. As a nucleotide analog, remdesivir was shown to be effective in preventing MERS-CoV replication in monkeys. Severity of disease, viral replication, and lung damage were reduced when the drug was administered either before or after infection with MERS-CoV. These results provide the basis for a rapid test of the beneficial effects of remdesivir in COVID-19. Other antiviral agents worthy of further clinical investigations include ribavirin, protease inhibitors lopinavir and ritonavir, interferon α2b, interferon β, chloroquine phosphate, and Arbidol. However, we should also bear in mind the side effects of these antiviral agents. For example, type I interferons including interferon α2b and interferon β are well known for their antiviral activity. Their beneficial effects at an early phase of infection are well expected. However, administration at a later stage carries the risk that they might worsen the cytokine storm and exacerbate inflammation. Notably, steroids have been experimentally used widely in the treatment of SARS and are still preferred by some Chinese physicians in the treatment of COVID-19. It is said to be capable of stopping the cytokine storm and preventing lung fibrosis. However, the window in which steroids might be beneficial to patients with COVID-19 is very narrow. In other words, steroids can only be used when SARS-CoV-2 has already been eliminated by human immune response. Otherwise, SARS-CoV-2 replication will be boosted leading to exacerbation of symptoms, substantial virus shedding, as well as increased risk for nosocomial transmission and secondary infection. In this regard, it will be of interest to determine whether the report of fungal infection in the lungs of some patients in Wuhan might be linked to misuse of steroids. Nevertheless, the screening of new pharmaceuticals, small-molecule compounds and other agents that have potent anti-SARS-CoV-2 effects will successfully derive new and better lead compounds and agents that might prove useful in the treatment of COVID-19.
The seventh question is whether inactivated vaccines are a viable option for SARS-CoV-2. The chance that SARS-CoV-2 will become endemic in some areas or even pandemic has increased in view of its high transmissibility, asymptomatic and presymptomatic virus shedding, high number of patients with mild symptoms, as well as the evidence for superspreading events. Thus, vaccine development becomes necessary for prevention and ultimate eradication of SARS-CoV-2. Inactivated vaccines are one major type of conventional vaccines that could be easily produced and quickly developed. In this approach, SARS-CoV-2 virions can be chemically and/or physically inactivated to elicit neutralizing antibodies. In the case of SARS-CoV and MERS-CoV, neutralizing antibodies were successfully and robustly induced by an inactivated vaccine in all types of animal experiments, but there are concerns about antibody-dependent enhancement of viral infection and other safety issues. While inactivated vaccines should still be tested, alternative approaches include live attenuated vaccines, subunit vaccines and vectored vaccines. All of these merit further investigations and tests in animals.
The eighth question relates to the origins of SARS-CoV-2 and COVID-19. To make a long story short, two parental viruses of SARS-CoV-2 have now been identified. The first one is bat coronavirus RaTG13 found in Rhinolophus affinis from Yunnan Province and it shares 96.2% overall genome sequence identity with SARS-CoV-2. However, RaTG13 might not be the immediate ancestor of SARS-CoV-2 because it is not predicted to use the same ACE2 receptor used by SARS-CoV-2 due to sequence divergence in the receptor-binding domain sharing 89% identity in amino acid sequence with that of SARS-CoV-2. The second one is a group of betacoronaviruses found in the endangered species of small mammals known as pangolins, which are often consumed as a source of meat in southern China. They share about 90% overall nucleotide sequence identity with SARS-CoV-2 but carries a receptor-binding domain predicted to interact with ACE2 and sharing 97.4% identity in amino acid sequence with that of SARS-CoV-2. They are closely related to both SARS-CoV-2 and RaTG13, but apparently they are unlikely the immediate ancestor of SARS-CoV-2 in view of the sequence divergence over the whole genome. Many hypotheses involving recombination, convergence and adaptation have been put forward to suggest a probable evolutionary pathway for SARS-CoV-2, but none is supported by direct evidence. The jury is still out as to what animals might serve as reservoir and intermediate hosts of SARS-CoV-2. Although Huanan seafood wholesale market was suggested as the original source of SARS-CoV-2 and COVID-19, there is evidence for the involvement of other wild animal markets in Wuhan. In addition, the possibility for a human superspreader in the Huanan market has not been excluded. Further investigations are required to shed light on the origins of SARS-CoV-2 and COVID-19.
The ninth question concerns why SARS-CoV-2 is less pathogenic. If the reduced pathogenicity of SARS-CoV-2 is the result of adaptation to humans, it will be of great importance to identify the molecular basis of this adaptation. The induction of a cytokine storm is the root cause of pathogenic inflammation both in SARS and COVID-19. SARS-CoV is known to be exceedingly potent in the suppression of antiviral immunity and the activation of proinflammatory response. It is therefore intriguing to see how SARS-CoV-2 might be different from SARS-CoV in interferon-antagonizing and inflammasome-activating properties. It is noteworthy that some interferon antagonists and inflammasome activators encoded by SARS-CoV are not conserved in SARS-CoV-2. Particularly, ORF3 and ORF8 in SARS-CoV-2 are highly divergent from ORF3a and ORF8b in SARS-CoV that are known to induce NLRP3 inflammasome activation. ORF3 of SARS-CoV-2 is also significantly different from the interferon antagonist ORF3b of SARS-CoV. Thus, these viral proteins of SARS-CoV and SARS-CoV-2 should be compared for their abilities to modulate antiviral and proinflammatory responses. The hypothesis that SARS-CoV-2 might be less efficient in the suppression of antiviral response and the activation of NLRP3 inflammasome should be tested experimentally.
Much progress has been made in the surveillance and control of infectious diseases in China after the outbreak of SARS-CoV in 2003. Meanwhile, virological research in the country has also been strengthened. The new disease report and surveillance system did function relatively well during the 2009 pandemic of swine flu. New viral pathogens such as avian influenza virus H7N9 and severe-fever-with-thrombocytopenia syndrome bunyavirus have also been discovered in recent years [11, 12], indicating the strength and vigor of Chinese infectious disease surveillance and virological research. However, the ongoing outbreak of SARS-CoV-2 has not only caused significant morbidity and mortality in China, but also revealed major systematic problems in control and prevention of infectious diseases there. Unfortunately, many of the lessons from the 2003 outbreak have not been learned. Importantly, disease control professionals, practicing physicians and scientists are disconnected in the fight against SARS-CoV-2 and COVID-19. In addition, important decisions were not made by experts in the field. Hopefully, these issues will be dealt with swiftly and decisively during and after the outbreak.
Above we have discussed the two possibilities that this outbreak will unfold. If SARS-CoV-2 is not eliminated from humans through quarantine and other measures, it can still be eradicated by vaccination. If it attenuates to become another community-acquired human coronavirus causing mild respiratory tract disease resembling the other four human coronaviruses associated with common cold, it will not be a disaster either. Before SARS-CoV-2 attenuates further to a much less virulent form, early diagnosis and improved treatment of severe cases hold the key to reduce mortality. We should remain vigilant, but there are grounds for guarded optimism. Redoubling our research efforts on SARS-CoV-2 and COVID-19 will solidify the scientific basis on which important decisions are made.
Severe Acute Respiratory Syndrome (SARS) disease hit the world in late 2002 and in 4 months swiftly spread to 29 countries infecting over 8,000 people and killing over 700. The etiological agent of SARS disease was determined to be of the coronavirus (CoV) family; the largest family of single-stranded, positive-sense RNA genomes known. The overall mortality rate of SARS corona virus (SARS-CoV) infection was ∼10% but this rate was 50% in patients over 65. Prior to the emergence of the SARS virus, coronaviruses were known to cause mild upper-respiratory tract diseases in humans. In contrast, SARS-CoV infection caused severe disease in the lower respiratory tract disease with symptoms ranging from flu-like and viral pneumonia to acute respiratory distress syndrome (ARDS) and fatal outcome–. The virus emerged from the Guangdong Province in China where it crossed to humans from a zoonotic reservoir. The most established theory puts horseshoe bats as the ultimate reservoir for the SARS-CoV and implicates palm civets as the intermediate species that passed the virus to humans. Aggressive public health intervention strategies are credited with successfully minimizing the SARS-CoV infection range, although it is uncertain if these same public health strategies would sufficiently contain a future SARS-CoV or SARS-like-CoV outbreak due to virus evolution.
Importantly, coronaviruses have a propensity toward frequent host-shifting events and over the past 30 years there have been many CoV cross-species transmission incidents giving rise to new animal and human CoV -based diseases. Coronaviruses infect a broad range of species lending further chance for recombination events and the advent of new CoV species. Moreover, coronaviruses can change cell type, tissue and host species barriers with ease,. Typically, the spike (S) protein of coronaviruses determines the host infectivity and the organization of the SARS-CoV S protein shows significant similarity with other aggressive class I viral fusion proteins: influenza virus HA, HIV-1 Env, Simian virus 5, and Ebola virus Gp2. The promiscuity of coronaviruses coupled with the tendency for mutations to occur gives reason for concern that another CoV outbreak is likely and highlights the need for continuous viral surveillance and forward development of CoV vaccination strategies and therapeutics.
Although entry of SARS-CoV into mammalian cells has been determined to be facilitated by the angiotensin-1 converting enzyme 2 (ACE2) molecule, the mechanisms by which the virus evades host immune responses causing generalized inflammation, increasing viral burden, and severe lung pathology still remain a significant scientific problem. Previous studies have shown substantial problems with potential CoV vaccines where the vaccines cause disease exacerbation opposed to initiating immunological protection,. Recently, several groups have described the immunologic response during SARS-CoV infection and some have investigated the use of a mouse adapted SARS-CoV in the mouse model–. The mouse-adapted SARS-CoV (MA15) is a valuable animal model for investigating the immune response and possible therapeutic and prophylactic strategies for SARS-CoV disease. Although the model helped to elucidate immune-pathological events during SARS-CoV infection and protection–, the caveat of this model is that it is based on an adapted virus and not a wild-type SARS-CoV that has naturally occurred in nature and cause disease in humans and animals. Although death is not observed in our wt TOR2 SARS-CoV ferret model, there are still several advantages where the use of both models is perhaps of equal importance as results from the mouse model compliment findings from the ferret model and vice versa. Specifically, use of the ferret model provides several benefits. As mentioned above, ferrets are susceptible to wild-type SARS-CoV infection from strains isolated from humans,. Furthermore, when infected with respiratory viruses including the SARS-CoV ferrets display many of the symptoms and pathological features as seen in infected humans as ferrets and humans have similar lung physiology–. Quantitative clinical signs displayed by ferrets include a rise in core body temperature (fever), nasal discharge (sneezing and runny nose) and weight loss.
Here we investigated the immune response transcriptome of SARS-CoV pathogenesis in a ferret model infected with an unadapted SARS-CoV and subsequently evaluated gene expression signatures induced with SARS-CoV reinfection. Furthermore, ferrets were immunized with a SARS-CoV vaccine and then challenged to compare immunological profiles with the SARS-CoV reinfected animals. The objective of this study was to identify immune correlates of protection upon reinfection with SARS-CoV in ferrets and provide a comprehensive profile of an effective and nonpathological immune response to SARS-CoV challenge following immunization. This information will not only provide a foundation for direct comparison with future SARS vaccine studies, but will also allow us to determine what immune mediators are responsible for the successful antiviral response.
The epidemic of unknown acute respiratory tract infection broke out first in Wuhan, China, since 12 December 2019, possibly related to a seafood market. Several studies suggested that bat may be the potential reservoir of SARS-CoV-2 [9, 10]. However, there is no evidence so far that the origin of SARS-CoV-2 was from the seafood market. Rather, bats are the natural reservoir of a wide variety of CoVs, including SARS-CoV-like and MERS-CoV-like viruses [11–13]. Upon virus genome sequencing, the COVID-19 was analyzed throughout the genome to Bat CoV RaTG13 and showed 96.2% overall genome sequence identity, suggesting that bat CoV and human SARS-CoV-2 might share the same ancestor, although bats are not available for sale in this seafood market. Besides, protein sequences alignment and phylogenetic analysis showed that similar residues of receptor were observed in many species, which provided more possibility of alternative intermediate hosts, such as turtles, pangolin and snacks.
Human-to-human transmission of SARS-CoV-2 occurs mainly between family members, including relatives and friends who intimately contacted with patients or incubation carriers. It is reported that 31.3% of patients recent travelled to Wuhan and 72.3% of patients contacting with people from Wuhan among the patients of non-residents of Wuhan. Transmission between healthcare workers occurred in 3.8% of COVID-19 patients, issued by the National Health Commission of China on 14 February 2020. By contrast, the transmission of SARS-CoV and MERS-CoV is reported to occur mainly through nosocomial transmission. Infections of healthcare workers in 33–42% of SARS cases and transmission between patients (62–79%) was the most common route of infection in MERS-CoV cases [17, 18]. Direct contact with intermediate host animals or consumption of wild animals was suspected to be the main route of SARS-CoV-2 transmission. However, the source(s) and transmission routine(s) of SARS-CoV-2 remain elusive.
SARS first emerged in late 2002 in Guangdong Province, southern China, as a novel clinical severe disease (termed “atypical pneumonia”) marked by fever, headache and subsequent onset of respiratory symptoms including cough, dyspnea and pneumonia. Being highly transmissible among humans, SARS rapidly spread to Hong Kong and other provinces across China and then to other 28 countries [6, 7]. By July 2003, it had caused 8096 confirmed cases of infection in 29 countries, 774 (9.6 %) of which were fatal (http://www.who.int/csr/sars/country/table2004_04_21/en/). The second outbreak in 2004 only caused 4 infections with no mortality nor further transmission.
The MERS epidemic emerged in the Kingdom of Saudi Arabia (KSA) since June 2012, with a similar clinical syndrome to SARS but seemingly less transmissible. In addition to respiratory illness, renal failure was identified in some severe cases [9–11]. Unlike SARS which had numerous super-spreader events, most MERS cases were independent clusters and limited to countries in the Middle East, particularly in KSA. Limited MERS cases have been reported in African and European countries and the United States of America, but exclusively in individuals travelling back from the Middle East. Some patients were reported to have a history of contact with camels while many other cases lacked this epidemiological link [9–11]. The MERS pandemic in the Republic of Korea in 2015 was caused by a single person who returned from travel in the Middle East. This made the Republic of Korea to be home to the second largest MERS epidemic with a total of 185 confirmed cases and 36 deaths [11, 12]. By 18 August 2015 a total of 1413 laboratory-confirmed cases of MERS have been reported worldwide with a median age of 50 years, including 502 related deaths. The mortality of MERS (approximately 35 %) is much higher than that of SARS (around 10 %).
Based on the current information, most patients had a good prognosis, while a few patients were in critical condition, especially the elderly and those with chronic underlying diseases. As of 1 March 2020, a total of 79,968 confirmed cases, including 14,475 (18.1%) with severe illness, and 2873 deaths (3.5%) in mainland China had been reported by WHO. Complications included acute respiratory distress syndrome (ARDS), arrhythmia, shock, acute kidney injury, acute cardiac injury, liver dysfunction and secondary infection. The poor clinical outcome was related to disease severity. The disease tends to progress faster in elderly people, with the median number of days from the occurrence of the first symptoms to death shorter among people aged 65 years or more [56, 57]. Similar to H7N9 patients, the elderly male with comorbidities and ARDS showed a higher death risk. Additionally, more than 100 children were infected, with the youngest being 30 h after birth. Neonates and the elderly need more attention and care due to their immature or weak immune system.