Although human coronaviruses (CoV) have been recognized as human pathogens since the 1960s, their virus family gained notoriety in 2002 and 2003 with the first outbreak of the SARS coronavirus epidemic and the recent emergence of the MERS coronavirus in 2012.
Coronaviruses are enveloped single stranded RNA viruses with positive RNA genomes that belong to the Coronaviridae family. Their genome is approximately 26–32 kilobases long, making it the longest known viral RNA genome. Coronaviruses got their name from electron microscopy photographs that sparked the imaginations of early electron microscopy analysts who thought the viruses had a crown-like surface. As a result, these researchers named the viruses corona, after the Latin word for crown.
Until now, all known coronaviruses have a similar genome organization and expression profile: The structural proteins (spike/S, envelope/E, membrane/M, nucleocapsid/N) are encoded by ORFs located 3′ of the viral genome, followed by the nonstructural proteins (nsp1–16).
Within the coronavirus family, four genera are known as
- Alpha CoV (or group 1)
- Beta-CoV (group 2)
- Gamma-CoV (group 3)
- Delta-CoV (group 4)
With group 2 coronaviruses consisting of four lineages known as A, B, C, and D, respectively. It is worth noting in this context that the lineage A viruses of group 2 CoVs encode a smaller protein called hemagglutinin esterase (HE), which appears to be functionally similar to the S protein.
Organization of the Human coronaviruses Genome (HCoV)
Human coronaviruses, as previously stated, have a non-segmented positive stranded RNA genome. Approximately 60–70% of this genome is made up of two large, overlapping open reading frames (ORF1a and ORF1b) that encode for the polyproteins pp1a and pp1ab, which are then processed into the 16 nonstructural proteins 1–16.
The structural proteins E, M, N, and S share the rest of the viral genome’s ORFs and are accompanied by a variable number of accessory proteins. The long genomes are thought to be the result of a unique replication fidelity, which in turn is the result of a set of viral enzymes with RNA-processing functions.
Clinical Symptoms Human coronaviruses Genome (HCoV)
In general, HCoV infections in humans cause self-limiting disease courses involving the upper respiratory and gastrointestinal tracts. In permissive patients, symptoms range from a common cold to bronchitis and pneumonia, with renal involvement occurring on rare occasions.
In this context, it is important to note that the clinical manifestations of the two most serious (but also least common) HCoVs, SARS coronavirus and MERS coronavirus, are more serious and frequently life-threatening. Despite the ongoing endemic MERS outbreak in the Arabian region and single outbreaks in South Korea, these two pathogens remain limited to single outbreaks (in the case of SARS-CoV) and endemic zoonotic transmissions in the Middle East region.
In any case, none of the remaining human coronaviruses can be identified based solely on clinical symptoms, and co-infections with other respiratory viruses are as common as with other respiratory pathogens, making it difficult to determine which pathogen is the “leading” pathogen in multiple infections.
Epidemiology of Human coronaviruses Genome (HCoV)
Six human coronaviruses have been discovered to date, including the human coronaviruses OC43 and 229E, NL63 and HKU1, as well as the SARS and MERS coronaviruses. Except for the latter two, all human coronaviruses have been observed worldwide and are mostly associated with a seasonality that coincides with the typical flu-like symptom season. Because coronavirus nomenclature is far from logical, these viruses are described in greater detail in the following section in their systematic order.
1. Human Coronavirus 229E (Group 1/Alpha-Coronavirus)
The human coronavirus type 229E, which is found worldwide, was discovered in 1966 during a trial to identify several newly recognized pathogens associated with the common cold. Malaise, headache, sneezing, sore throat, fever, and cough are some of the clinical symptoms associated with 229E. The time between infection and clinical symptoms has been reported to be 2 to 5 days, with clinical symptoms lasting 2 to 18 days. As previously stated, there is no clinical difference between 229E infections and other viral pathogen-caused respiratory infections such as rhinovirus or influenza A.
It was recently proposed that 229E arose from a recombination event between the alpaca alpha-coronavirus. This recombination took place within the S gene and was followed by a deletion within the same gene.
2. Human Coronavirus NL63 (Group 1/Alpha-Coronavirus)
Since its discovery in 2004, the human coronavirus NL63 has been linked to respiratory infections in children, the elderly, and immune-compromised patients. The virus was discovered in two separate laboratories in the Netherlands, one in Amsterdam and one in Rotterdam, in quick succession. In general, NL63 infections cause mild respiratory symptoms such as cough, rhinorrhea, tachypnea, fever, and hypoxia and are self-limiting. Croup is a common “complication” that occurs in approximately 5% of NL63 infections.
3. Human Coronavirus HKU1 (Group 2/Betacoronavirus, Lineage A)
With the description of the human metapneumovirus in 2001, a new era in virology began, focusing on viral discovery methods that combined traditional virological techniques with modern molecular methods. The subsequent wave of virus discoveries spawned a new trend in molecular diagnostics, with singleplex step-by-step methods being replaced by multiplexing technologies capable of screening for multiple pathogens at the same time. HKU1 was discovered in 2005 at Hong Kong University during this time period (which is also the institution from which the name HKU1 was derived). HKU1 was isolated from an elderly patient suffering from bronchiolitis and pneumonia. Fatal infections are uncommon, and the infections are difficult to distinguish from other viral respiratory infections. HKU, like the other “common cold” coronaviruses, is found all over the world.
4. Human Coronavirus OC43 (Group 2/Betacoronavirus of Lineage A)
The strain OC43 is one of the oldest known human coronaviruses, having been discovered in 1967. OC43 and 229 can be distinguished solely by molecular methods or by serological methods, and both viruses have the same morphology and clinical spectrum.
5. SARS Coronavirus (Group 2 Covid /Beta coronavirus of Lineage B)
Since the SARS coronavirus was first detected in 2002/2003 during a Chinese outbreak, much has been speculated; even more has been confirmed. The ensuing pandemic was halted due to strict hygienic procedures and intervention measures implemented before a worldwide disaster could occur. Indeed, the discovery of this virus was made possible solely by the first alarming observations made by Dr. Carlo Urbani, a physician who was confronted with patients suffering from fever, myalgia, headache, malaise, and chills, followed by a dry cough, dyspnea, and respiratory distress; infections of the liver, kidney, gastrointestinal tract, and brain occurred in some cases. The overall mortality rate is 9%, but it rises as one gets older. To date, the SARS coronavirus has only caused one outbreak, which has since spread to other locations as a result of travel. This initial SARS coronavirus outbreak has now been identified as an archetypic zoonosis outbreak caused by this virus or other SARS-like coronaviruses. Such coronaviruses circulating in their natural reservoirs should not be excluded during an outbreak and necessitate a fine-grained surveillance network.
6. MERS Coronavirus (Group 2/Betacoronavirus, Lineage C)
When the MERS coronavirus was isolated for the first time in Saudi Arabia in 2012, it piqued the scientific community’s interest. It is the leading cause of severe pneumonia with acute respiratory distress (ARDS) and is frequently associated with gastrointestinal symptoms. It is worth noting that renal impairment is frequently observed. Patients with an underlying comorbidity, in particular, are vulnerable to MERS-CoV infections and have a high mortality rate. Although the virus appears to be endemic, spontaneous outbreaks due to imported cases are possible, as most recently reported from South Korea, where an index patient’s roommate left the hospital on his own account, causing a local outbreak. It is worth noting that in the case of MERS-CoV, it is assumed that the viral spike protein allows the virus to evade the immune system by preventing neutralizing antibodies from binding.
Virus Ecology of Human Coronaviruses
To date, it appears that the coronaviruses NL63, HKU1, 229E, and OC43 are well adapted human viruses that are still present in the human reservoir; these coronaviruses originated from zoonotic transmission a long time ago. MERSCoV and SARS-CoV, on the other hand, are less adapted to the human host and are most likely zoonosis originating in their natural reservoirs, camels and bats, respectively.
Diagnostics of of Human Coronaviruses
The diagnosis of a human coronavirus infection does not always result in a specific treatment plan. While coronaviruses NL63, HKU1, OC43, and 229E do not necessitate “special” care, isolation of patients is strictly required in the case of SARS-CoV and should be considered in the case of MERS-CoV.
Neither cell culture-based nor electron microscopy-based diagnostic methods are the first choices. Rather, molecular techniques such as RT-qPCR, LAMP, or multiplexing should be used. Several groups have described RT-qPCR protocols, which are the method of choice for the new coronaviruses. Corman and colleagues recommend using the upE region and Orf1a as PCR targets for MERS coronavirus, while Orf1b has a lower sensitivity. It is also recommended that parts of the RdRp- and/or N-genes be sequenced to confirm the results. Internal and external controls, which are available, for example, from Public Health England, should be included in every PCR run.
Several validated and approved multiplex assays for other coronaviruses are available, including the RespiFinder assay (Pathofinder, Maastricht, Netherlands), the film array (former IDAHO film assay, now produced and distributed by bioMerieux, Lyon, France), and the Luminex RVP (Luminex, Austin, Texas, USA). All of these assays have the advantage of having a high sensitivity as well as the ability to detect multiple pathogens at the same time. Furthermore, Roche/novel TIBMOLBIOL’s Light Mix Modular Assays could be used as an alternative for coronavirus diagnostics.
Advanced Molecular Techniques Relevant to Human Coronaviruses
The discovery of novel coronaviruses in the last 15 years is an excellent example of the importance of advanced molecular techniques that must be combined with traditional virological methods. For example, the SARS coronavirus was discovered solely through a sophisticated combination of detailed and timely clinical observation, followed by attempts to isolate the virus in cell culture (classical method), and subsequent characterization by modern molecular techniques. The latter method, known as random reverse transcriptase PCR, was used to identify the novel genome of the SARS coronavirus and resulted in the amplification and subsequent sequencing of the first known SARS genomes.
Another example is van der Hoek and colleagues’ discovery of the human coronavirus NL63. VIDISCA is a novel method developed by these researchers (virus discovery cDNA-AFLP). For this method, the viral DNA or cDNA is digested with enzymes that target short recognition sequences found in almost all viruses. After that, the fragments are ligated to adaptors and amplified using an adaptor-specific PCR. Meanwhile, the VIDISCA method (Fig. 1) has been refined and is now applicable as a sensitive assay for virus detection in clinical samples.
Concluding Remarks
Coronaviruses have been identified as a major cause of severe airway infections. Recent experiences with the MERS coronavirus and the SARS coronavirus outbreak have demonstrated that these zoonotic viruses can cross the species barrier and, along with influenza viruses, are the most likely candidates for future outbreaks. In conjunction with newer studies on virus ecology, it has become clear that coronaviruses are ubiquitous pathogens infecting a wide range of mammals that frequently come into contact with humans, laying the groundwork for future zoonotic outbreaks.
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