Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses

Human Coronavirus 229E (alphacoronavirus)

Strain 229E was discovered during 1966, when researchers were characterizing five novel agents that were isolated from the respiratory tract of humans who had contracted the common cold [23]. 229E was adapted to grow in WI-38 lung fibroblast cell lines, and was later shown to be morphologically identical to IBV and MHV [24]. Symptoms of 229E infection include general malaise, headache, nasal discharge, sneezing, and a sore throat [25]. A small portion of patients (10–20%) will also exhibit fever and cough. The incubation time is approximately 2–5 days, followed by illness lasting between 2 and 18 days, and clinically indistinguishable from respiratory tract infections caused by other pathogens, such as rhinovirus and influenza A [18]. 229E is distributed globally (Figure 1A).

Human Coronavirus OC43 (betacoronavirus, lineage A)

Strain OC43 was discovered, during 1967, in the nasopharyngeal wash of a patient with the common cold, and is adapted to grow in organ cultures containing suckling mouse brains. Similar to 229E, OC43 is morphologically indistinguishable from IBV and MHV [24], and patients infected with OC43 present the same clinical symptoms as that of 229E [24]. However, there is no serological cross-reactivity between 229E and OC43 [24]. OC43 is also distributed globally (Figure 1A).

SARS-CoV (betacoronavirus, lineage B)

Patients infected with SARS-CoV initially present with fever, myalgia, headache, malaise, and chills, followed by a nonproductive cough, dyspnea, and respiratory distress generally 5 to 7 days later, which may result in death (http://www.who.int/csr/sars/en/WHOconsensus.pdf?ua=1). Other notable features in some cases include infection of the gastrointestinal tract, liver, kidney, and brain. Diffuse alveolar damage, epithelial cell proliferation, and an increase in macrophages is seen in SARS-CoV infection of the lung. Lymphopenia, hemophagocytosis in the lung, in addition to white-pulp atrophy of the spleen observed in SARS patients, are similar to fatal H5N1 influenza virus infections [1]. Diarrhea is observed in approximately 30–40% of SARS infections (http://www.cdc.gov/sars/about/faq.html).

An outbreak of disease caused by SARS-CoV, originating from Guangdong Province in southern China during November 2002, eventually spread to other countries in Asia, in addition to North America and Europe (37 countries/regions in total) over 9 months (http://www.who.int/ith/diseases/sars/en/) (Figure 1B). An eventual 8273 cases were reported, with 775 deaths for a case fatality rate (CFR) of 9%, and the majority of cases and deaths occurred in mainland China and in Hong Kong [19]. The elderly were more susceptible to SARS disease, with a mortality rate of over 50% (http://www.cdc.gov/sars/surveillance/absence.html).

Human Coronavirus NL63 (alphacoronavirus)

NL63 is primarily associated with young children, the elderly, and immunocompromised patients with respiratory illnesses [26]. NL63 was a novel HCoV isolated from a 7-month-old child with coryza, conjunctivitis, fever, and bronchiolitis in the Netherlands during late 2004 [26], and an independent investigation described the isolation of virtually the same virus from a nasal sample collected from an 8-month-old boy suffering from pneumonia in the Netherlands [27]. NL63 was also detected in New Haven, USA, during 2005 amongst 79 of 895 children, and was initially called HCoV-NH (NH), but genetic and phylogenetic analyses showed that NH and HKU1 likely are the same species 26, 28. Infections with NL63 typically result in mild respiratory disease similar to the common cold, characterized by cough, rhinorrhea, tachypnea, fever, and hypoxia, and resolve on their own [29]. Obstructive laryngitis, also known as croup, is frequently observed with NL63 infections. A study estimated that NL63 accounts for an estimated 4.7% of common respiratory diseases [26]. NL63 is distributed globally (Figure 1A).

Human Coronavirus HKU1 (betacoronavirus, lineage A)

HKU1 was first discovered during January 2005 in a 71-year-old patient from Hong Kong who had been hospitalized with pneumonia and bronchiolitis [30]. The symptoms of HKU1 respiratory tract infections are not able to be separated from those caused by other respiratory viruses. Most patients present with fever, running nose, and cough for infections in the upper respiratory tract, whereas a fever, productive cough, and dyspnea are the common symptoms presenting for infections in the lower respiratory tract [31]. Most HKU1 infections are self-limiting, with only two deaths reported in patients with pneumonia due to HKU1 [32]. Although HKU1 is clinically relatively mild in children, HKU1 infection is associated with a high incidence of seizures, and was found in a patient with meningitis 33, 34. HKU1 cases outside Asia were detected in New Haven, USA, in 2 out of 851 children [35], and also in Australia [36], France [37], and Brazil [38], indicating a global distribution for the virus (Figure 1A).

MERS-CoV (betacoronavirus, lineage C)

MERS-CoV was first isolated from the lungs of a 60-year-old patient who had died from a severe respiratory illness in Jeddah, Saudi Arabia, 2012 [39]. Clinical manifestations of MERS-CoV infection range from asymptomatic to severe pneumonia with acute respiratory distress, septic shock, and renal failure resulting in death [40]. A typical disease course begins with fever, cough, chills, sore throat, myalgia, and arthralgia, followed by dyspnea and rapid progression to pneumonia 40, 41, 42, 43. Approximately one-third of patients present with gastrointestinal symptoms, such as diarrhea and vomiting. Acute renal impairment was the most striking feature of disease caused by MERS-CoV, which is thus far unique for human CoV infections 40, 44. Seventy-five percent of patients with MERS disease also had at least one other comorbidity, and patients who died were more likely to have a pre-existing/underlying condition [40]. Countries around the Arabian Peninsula are known to be endemic for MERS-CoV, and Saudi Arabia has reported the most cases, but since its discovery in 2012, cases have been occasionally exported to other countries through travel, sometimes causing clusters of secondary outbreaks (Figure 1B) [45]. As of December 31, 2015, a total of 1621 laboratory-confirmed infections have been reported, with 584 deaths (CFR = 36.0%) over 26 countries (http://www.who.int/csr/disease/coronavirus_infections/en), making MERS-CoV one of the most dangerous viruses known to humans.

Phylogenetic Analysis

The phylogenetic tree for the CoVs of zoonotic and human origin was constructed based on the full genome. It can be observed that 229E and NL63 belong to the alpha-CoV genus and are grouped together with porcine epidemic diarrhea virus (PEDV), TGEV, porcine respiratory coronavirus (PRCV), feline infectious peritonitis virus (FIPV), and some bat-derived coronaviruses (Figure 3 ). The topology of the phylogeny is similar to the tree constructed based on the RNA-dependent RNA polymerase gene [55].

OC43, HKU1, SARS-CoV, and MERS-CoV belong to the beta-CoV genus. Lineage A includes OC43 and HKU1, MHV, porcine hemagglutinating encephalomyelitis virus (PHEV), in addition to equine, rabbit, camel, bovine, antelope-derived animal coronaviruses. Lineage B includes SARS-CoV, SARS-like viruses of bat and palm civet origin, and some bat-derived CoVs. Lineage C includes MERS-CoV and some bat-derived viruses. Lineage D contains only bat-derived coronavirus (Figure 3). In contrast to alpha- and beta-CoVs, gamma-CoVs consist mainly of avian coronaviruses (such as IBV), as well as CoVs isolated from aquatic animals, whales, and dolphins. CoVs of wild-bird origin are clustered into the delta-CoV, which also contains some swine-derived CoVs (Figure 3).

In general, alpha- and beta-CoVs primarily include CoVs from mammals, whereas gamma- and delta-CoVs mostly include CoVs of avian origin. Notably, all currently known HCoVs belong to either the alpha- or betacoronavirus genera, which also contain CoVs of mainly bat origin.

Mutation and Genetic Recombination in Coronaviruses

The estimated mutation rates in CoV are moderate to high compared to other single-stranded RNA (ssRNA) viruses, and the average substitution rate for CoVs was ∼10⁻⁴ substitutions per year per site [63]. The nucleotide mutation rate of IBV for the hypervariable region in the S gene was estimated to be 0.3–0.6 × 10^–2 per site per year [64]. The S gene of OC43 possessed an average rate of 6.410.58 × 10^–4 substitution rates per site per year [65]. Additionally, the S gene of 229E possessed a rate of ∼3 × 10^{− 4} substitutions per site per year [63]. For SARS-CoV, the mutation rate in the whole genome was estimated to be 0.80–2.38 × 1^–3 nucleotide substitutions per site per year, and the nonsynonymous and synonymous substitution rates were estimated to be 1.16–3.30 × 10^–3 and 1.67–4.67 × 10^–3 per site per year, respectively, which is similar to other RNA viruses [66]. The evolutionary rate for MERS-CoV genomes was estimated as 1.12 × 10⁻³ substitutions per site per year (95% credible interval [95% CI], 8.76 × 10⁻⁴; 1.37 × 10⁻³) [67]. Furthermore, the large RNA genome in CoV allows for extra plasticity in genome modification by mutations and recombinations, thereby increasing the probability for intraspecies variability, interspecies ‘host jump’, and novel CoVs to emerge under the right conditions 68, 69, 70.

The major reason for recombination may be at the replication step in the virus life cycle. During replication, a set of subgenomic RNAs is generated, increasing the homologous recombination rate among closely related genes from different lineages of CoVs or other viruses by template switching [33]. Concurrently, circulating CoVs in multiple host species likely contribute to increases in the rate of recombination events. However, the exact mechanism of genetic recombination in CoVs remains unclear: recombination site ‘breakpoints’ in the viral genome – the crossing point of the recombinant genes between two distinct viral strains or genotypes – appear to be random, as different recombinant strains have different breakpoints.

Genetic recombination has been previously documented for animal CoVs, for instance: MHV [71], TGEV [72], as well as feline and canine coronaviruses 73, 74. Genetic recombination for other HCoVs including OC43, NL63, HKU1, SARS-CoV [51], and MERS-CoV have also been documented and are discussed in detail below.

OC43 has diverged into five distinct genotypes (A to E). Genotype A is typified by the prototype VR759 strain that was isolated in 1967 [75]. Genotypes B and C are two naturally circulating viruses. Genotype D emerged via recombination between genotypes B and C viruses at the breakpoint of 2500–5000 bp in the NSP2–NSP3 gene and 15500 bp-3′end in NSP12-N genes [75] (Figure 4 , Key Figure). Genotype E, identified in 2014, emerged by recombination amongst the genotype B, C, and D viruses with different breakpoints in the genes compared to the generation of genotype D [76] (Figure 4). A study in France, between 2001 and 2013, demonstrated that OC43 strains have more recombination patterns in Lower Normandy, and circulating OC43 viruses have a high genetic diversity [17].

NL63 also has the potential for genetic recombination, and one such example is between the Amsterdam 1 and 496 strains. Recombination site breakpoints were identified in two parts of the S gene [63] (Figure 4).

Recombination events have been observed for different genotypes of HKU1, in which recombination breakpoints were found on genotypes B and C in the nsp6-nsp7 and nsp16-HE genes, respectively [70] (Figure 4). HKU1 was also shown to be able to recombine with other animal beta-CoVs, such as MHV, at the nsp3, nsp4, nsp6, nsp7, nps9, and nsp10 genes [77] (Figure 4).

SARS-CoV has a possible recombinant history with lineages of alpha- and gamma-CoV [78]. Many specific breakpoints and a large number of smaller recombinant regions had been identified in the RNA-dependent RNA polymerase (RDRP, nsp12 gene) at the following locations: nucleotides 13 392–13 610, 15 259–15 342, and 15 974–16 108 based on the sequence of the SARS-CoV TOR2 strain [78]. Recombination locations were also identified in the nsp9 and most of nsp10 gene located at 12 613–13 344, and parts of nsp14 (18 117–18 980) [78]. Another study analyzing the genome of the SARS-CoV CV7 strain had shown that seven putative recombination locations were found between SARS-CoV and six other coronaviruses: PEDV, TGEV, BCV, 229E, MHV, and IBV (Figure 4). This further suggests that SARS-CoV emerged via serial horizontal transmission and by genetic recombination, enhancing the adaptation process to its new host [79].

Recombination was also detected between the SARS-related bat-associated CoV (SARSr-Bat CoVs), in which recombination between the Rp3 strain from Guangxi Province and Rf1 strain from Hubei Province, China, generated a new strain (SARSr-Civet CoV SZ3) in civets, with a breakpoint at the nsp16/spike and S2 region [51] (Figure 4). Furthermore, the SARS-CoV Rf1/2004 strain is itself a recombinant from the bat CoV 279/2004, bat CoV Rm1/2004 and civet CoV SZ3/2003 [51] (Figure 4).

A recent study showed that the epidemic MERS-CoV experienced recombination events between the different lineages, which occurred in dromedary camels in Saudi Arabia [62]. Lineages 3 and 5 viruses were predominant during July and December 2014; however, lineage 5 became dominant within the camel population by 2015. Lineage 5, which is closely associated with the MERS-CoV causing the outbreak in South Korea in 2015, as well as recent human infections in Riyadh, Saudi Arabia, is a recombinant virus between lineages 3 and 4, or groups 3 and 5 of clade B (Figure 4) 62, 80.