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Human Coronaviruses associated with mild respiratory diseases
The first isolated human Coronaviruses were HCoV-229E and OC43 in 1967, from patients with upper respiratory tract infections (Almeida and Tyrrell, 1967). These two human Coronaviruses cause mild upper and lower respiratory tract infections, have a low mortality rate and are often associated with the common cold. Both Coronaviruses have seasonal infectious cycles, with a peak of infectivity usually situated during winter and early spring, in countries with a temperate climate. First infections usually occur during early childhood and re-infections are frequent throughout life and are symptomatic in 45% of the cases.
Shortly after the SARS epidemic, two more human Coronaviruses were identified: HCoV-NL63, first described in 2004 (van der Hoek et al., 2004) and HCoV-HKU1, which was discovered in 2006 in Hong-Kong (Woo et al., 2005). Both are also associated with mild respiratory diseases. HCoV-NL63 belongs to the Alpha-genus and diverged from HCoV-229E in the 1800s. It was first isolated from a 7 months old child in The Netherlands, suffering from bronchiolitis. This virus was independently reported by another Dutch group, from a respiratory sample, dating from 1988, of a young child suffering from pneumonopathy (Fouchier et al., 2004), therefore suggesting that this strain was already circulating among the human population without being identified before. In children infections, HCoV-NL63 has been associated with acute bronchiolitis (Ebihara et al., 2005). HCoV-HKU1 was first isolated from a 71 years old patient, suffering from pneumonia, but remains to this day an agent of mild and severe respiratory tract infection. The seriousness of the respiratory infection depends in majority of the underlying condition of the patient, whether he is immunocompromised or displays a chronic disease.
Severe acute respiratory syndrome virus-SARS-CoV
The SARS epidemic started in the Guangdong Province in Southern China in November 2002, with an outbreak of “acute respiratory syndrome” resulting in 300 human cases and 5 deaths. By the end of February 2003, it spread out of the Chinese frontiers to Hong Kong, where an infected physician contaminated 16 individuals, who travelled to at least 6 different countries and therefore triggered the second wave of the outbreak (Centers for Disease Control and Prevention (CDC), 2003). The epidemic resulted in more than 8270 cases and 775 deaths, making approximately a 10% death rate (Poon et al., 2004). The epidemic had a huge economic impact with a total loss of approximately 40 billion dollars and spread over to 30 different countries (Caulford, 2003). International cooperation of scientists and medical healthcare combined with drastic hygiene measures helped to control the outbreak. Quarantine was the only intervening strategy as no effective therapeutic exists.
After an incubation period of two to six days, patients develop general symptoms of flu, associated with fever, asthenia, shivering, anorexia and stiffness (Cheng et al., 2013). Severe respiratory symptoms occur several days after general symptoms. SARS-CoV has a strong tropism for the low respiratory tract and preferentially infects type 1 pneumocytes, where the infection is productive. It also infects macrophages and dendritic cells but infection is abortive and leads to the production of pro-inflammatory cytokines, such as CXCL10, CCL2… This immune dysregulation leads to pneumopathy. Patients can exhibit either localised or diffused alveolar damages. Pathologies of the lungs are associated with localised/diffused alveolar damages, increase of macrophages and epithelial cell proliferation. Over time, alveolar damages progress and lead to acute lung injury. In the most severe cases, damages develop into an acute respiratory distress syndrome, necessitating mechanical ventilation. For these patients, CD4 and CD8 lymphocytes drastically drop, whereas neutrophils and pro-inflammatory cytokines are elevated. SARS patients can also develop gastrointestinal symptoms (about 30%), as SARS-CoV has been shown to replicate in enterocytes (To et al., 2004). Enteric symptoms are associated with vomiting, diarrhoea and abdominal aches. For 10% of the patients, evolution of the disease lead to a fatal issue.
Middle East respiratory syndrome virus-MERS-CoV
MERS-CoV was first identified in Saudi Arabia in 2012 and has since been circulating widely in the Arabian Peninsula (Zaki et al., 2012). As a new emerging pathogen with a symptomatology close to the SARS-CoV infection, it is under scrutiny of the WHO. Its infection differs among patients and while it can be asymptomatic for some, it can also cause fever, cough, respiratory distress, pneumonia, leading to death in 30% of the cases. Most severe and fatal cases have been found in immunocompromised patients, people with chronic diseases, such as diabetes or cancer and elderly patients. Some gastrointestinal symptoms have been reported as well as kidney failure. To this date, MERS-CoV displays a limited human-to-human transmission. All cases are linked to the Middle East region (Arabi et al., 2014).
Feline enteric Coronavirus and feline infectious peritonitis virus
Feline Coronavirus infections are widely spread among this population. Ten to 50% of household cats are infected and this number can reach 80% of seropositive animals in cat-breeding facilities. Avirulent FCoV, which cause usually mild enteric symptoms are referred as FECV strains, for feline enteric Coronavirus. Virulent strains, named FIPV, induce the feline infectious peritonitis, a lethal disease. In 2% to 5% of the cases, FECV infected cats develop FIP, after mutation of FECV into FIPV (Pedersen et al., 1981)(See chapter IV.3.1).
Unlike FECV, which replicates in enterocytes, FIPV has a major tropism for monocytes/macrophages (Kipar et al., 2005). Infection of these cells leads to the secretion of pro-inflammatory cytokines like TNFα, Il-1β and GM-CSF. These cytokines attract neutrophil cells at the site of infection and in turn, provoke pyogranulomatous lesions, which are pathognomonic of FIP infections (Berg et al., 2005). Secretion of TNFα triggers the apoptotic death of T cells and CD8+ in particular, leading to lymphopenia, frequently observed during the course of infection (de Groot-Mijnes et al., 2005). Evolution of the FIPV infection can have two different symptomatic forms: an effusive or a non-effusive one (wet or dry forms). The wet form is characterised by accumulation of protein-rich liquid in the abdomen or pleural cavity of the infected animal. For the dry form, symptoms vary according to the affected organs. In almost 10% of the cases, neurological symptoms are linked with ataxia, vestibular disorders or seizures. Uveitis is also frequently associated with neurological disorders. Abdominal organs may in turn be affected, leading to renal or hepatic failure.
Different canine CoVs (CCoVs) belong to the Alpha and Betacoronaviruses. CCoVs from the Alpha-genus were described since the 1970s and are important entero-pathogens of canine population. They are widely spread, especially in kennels and animal shelters. Canine CoV infections present a high morbidity and a low mortality, except for younglings. Infection is mainly restricted to the intestinal tract. Symptoms include loss of appetite, diarrhoea, vomiting and eventually death. Death of the animal is often a consequence of co-infection with canine parvovirus, canine distemper virus or other pathogens.
In 2007, an Italian group reported fatal cases of infected pups by a new virulent strain of Alphacoronavirus, named canine pantropic CoV. The infection caused a systemic disease and death within two days after pups developed symptoms. Those symptoms include fever, haemorrhagic diarrhoea, vomiting, leukopenia, ataxia, anorexia, depression and seizure. Viral RNA was detected in intestinal contents of pups, as well as lungs, spleen, liver, kidney and brain. A difference in mortality rate exists between young pups and older pups, as the latter of 6 months old recover from the disease (Decaro et al., 2007). Epidemiological studies conducted in different European countries indicate that this new strain is widely distributed in dog populations (Decaro et al., 2013).
In 2003, a new canine Coronavirus with pulmonary tropism was detected by RT-PCR, in a kennel in United Kingdom. This new virus, CRCoV (Canine Respiratory Coronavirus), presents a close relationship with BCoV for both the spike and the replicase genes and belongs to the beta-genus (Erles et al., 2003). It is responsible for mild respiratory infections, and its implication in the kennel cough syndrome is under investigations.
Interaction with the host innate immune response
Type I Interferon (IFN) has been discovered in 1987 by Isaacs and Lindemaan (Isaacs and Lindenmann, 1987) and constitutes the first defence line by a host against a viral pathogen. It influences protein synthesis, growth and processes of cell survival, besides regulating innate and adaptive immune system. Still, viruses have developed many different strategies to circumvent host innate antiviral response. Viral proteins may interfere with multiple steps of the innate response, to establish a sustainable infection. Coronaviruses encode several proteins affecting type I IFN pathway and have been extensively studied for the SARS-CoV.
Some structural proteins function as interferon antagonists. M and N proteins of the SARS-CoV inhibit the synthesis of type I IFN (Siu et al., 2009)(Kopecky-Bromberg et al., 2007). Non-structural proteins also modulate the innate immune response through diverse mechanisms. Nsp1 antagonises type I IFN by three different mechanisms: inactivation of host protein translation, degradation of host mRNA whereas viral RNA is not affected by this process and third, inhibition of STAT1 phosphorylation, a critical transcription factor of IFN signalling (Kamitani et al., 2006). Nsp3 inhibits the IFN synthesis by antagonising IRF3 (IFN-regulatory factor 3), a key element in the IFN inducing pathway (Devaraj et al., 2007). Finally, the IFN antagonist effect of nsp16 is due to its 2’ O-methylation activity of viral RNA that protects the genome from recognition by MDA5, an inducer of the IFN pathway (Daffis et al., 2010). Some accessory proteins may also counteract the IFN pathway (see below).
Roles of the accessory proteins in pathogenesis
The accessory proteins are virus-specific and are dispensable for the viral replication when studied in vitro (Haijema et al., 2004)(Yount et al., 2005)(Shen et al., 2003)(Hodgson et al., 2006)(Casais et al., 2005). Still, as they are conserved under selective pressure, they somehow confer a selective advantage to the virus, in in vivo infections. During the intense studies that followed the SARS outbreak, one noticeable aspect of the SARS-CoV was the uncommonly high number of these accessory proteins it displayed. SARS-CoV contains eight ORFs, namely ORF3a, 3b, 6, 7a, 7b, 8a, 8b and 9b (McBride and Fielding, 2012). They, as well as accessory proteins of some other Coronaviruses, have been extensively studied and many modulate the viral replication or virulence of the virus or even help evade the innate immune system. They are therefore involved in viral pathogenesis. Properties, localisation and functions of studied accessory proteins of Coronaviruses widely differ from each other and are summarised in table 5.
Despite their high diversity in sequences and subcellular localisation, they share some common functions and properties.
Three of them have been identified as viroporins: ORF4a of the HCoV-229E, ORF3 of PEDV and ORF3a of the SARS-CoV. They share common membrane topology with three transmembrane domains and all seem to localise in the assembling budding site of CoVs. They regulate membrane permeability and regulate viral production, probably by influencing the release step of viral particles.
HCoV-OC43 strain: a strictly human respiratory virus with bovine origins
Until 2005, only partial sequences of HCoV-OC43 strain were available, thus challenging studies of this human virus. Publication of the full genomic characterization of HCoV-OC43 by Vijgen et al. lifted the veil on the animal origins of this virus (Vijgen et al., 2006). Acquisition of the full sequence of the lab strain (named VR759) showed the close relationship between the human strain HCoV-OC43 and the porcine strain PHEV with the E sequence, sharing 99.6% of sequence identity. Concerning other sequences such as HE, S, M and N, a strong relationship was also established with the bovine Coronavirus, BCoV. Molecular clock calculations permitted the authors to date the zoonotic transmission event back to 1890, where a common ancestor for both bovine and human virus is most plausible. The most likely circumstance for the emergence of this human strain would be a host-jump phenomenon and an adaptation of the bovine strain to the human species. The authors observed the presence in the human strain of a 290 nucleotides deletion at the N-terminus of the N protein sequence, leading to two small ORFs. In the bovine strain, this ORF located inside the N sequence is intact and encodes a 207 a.a length protein, of unknown function (Vijgen et al., 2006). Significance of this deletion is unknown and unfortunately, no further studies on this observation were carried out. However, it is plausible that this deletion occurred during the host switch and may have played a role in the adaptation of the virus to the human strain.
Emergence of the MERS-CoV: role of the dromedary camel
Soon after the discovery of MERS-COV, it was hypothesized that the virus emerged from an animal reservoir. Bat CoV-HKU4 and HKU5 are phylogenetically close to the MERS-CoV, suggesting a common ancestor in bats. This hypothesis was reinforced by the detection of small genomic fragments of MERS-like-CoV in bats from various countries (Lelli et al., 2013). However, to date, no complete sequence of MERS-like-CoV was recovered from bats. It has been recently demonstrated that MERS-CoV arises from an ancestor bat CoV named Neoromicia capensis bat CoV (NeoCoV) that infects this particular bat species in Africa (Corman et al., 2014).
Furthermore, bats do not have direct contact with human or at least rare and for a very short time and potential for an intermediate host intervention was sought. As such, samples were collected in different domesticated and wild animals from the Middle East region and neutralizing antibodies to the MERS-CoV were found in camels, leading to the speculation that they represent the intermediate host (Meyer et al., 2014). Camels and dromedaries are companion animals that live close to their owners in that region, as they are raised in farms for both races and leisure. Further investigations revealed the presence of MERS-CoV RNA in camels in close contact with infected humans (Nowotny and Kolodziejek, 2014). Recently, Azhar et al isolated and sequenced MERS-CoV from a patient and one of his dromedary camels. Isolates from both the patient and the camel were found identical and serological investigations conducted in parallel indicated that MERS-CoV infected the camel in a first place and later, the patient (Azhar et al., 2014). These findings strongly suggest that camels can transmit MERS-CoV to humans in close contact. In contrast to the SARS-CoV, person-to-person transmission of MERS-CoV occurs in peculiar situations of a prolonged contact or the presence of risk factors such as immunodeficiency.
Table of contents :
CHAPTER ONE: CORONAVIRUSES
I. History and classification
II. Biology of Coronaviruses
II.1. Morphology of the virion
II.2. Genome organisation
II.3. Structural proteins
II.3.1. The spike S glycoprotein
II.3.2. The envelope E protein
II.3.3. The M protein
II.3.4. The N protein
II.3.5. The hemagglutinin-esterase (HE) protein
II.4. Non-structural proteins
II.5. Accessory proteins
III. Virus life cycle
III.1. Virus attachment and entry
III.2. Viral RNA synthesis
III.3. Assembly and release of the virions
IV. Human and veterinary diseases
IV.1. Human diseases
IV.1.1. Human Coronaviruses associated with mild respiratory diseases
IV.1.2. Severe acute respiratory syndrome virus-SARS-CoV
IV.1.3. Middle East respiratory syndrome virus-MERS-CoV
IV.2. Domesticated animal diseases
IV.2.1. Infectious bronchitis virus
IV.2.2. Porcine Coronaviruses
IV.2.3. Feline enteric Coronavirus and feline infectious peritonitis virus
IV.2.4. Canine Coronaviruses
IV.3. Molecular determinants of pathogenesis
IV.3.1. Tropism switch and pathogenesis
IV.3.2. Interaction with the host innate immune response
IV.3.3. Roles of the accessory proteins in pathogenesis
CHAPTER TWO: CORONAVIRUS INTERSPECIES TRANSMISSIONS AND ADAPTATION TO A NEW HOST
I. Understanding the interspecies transmission
I.3. Host adaptation
II. From animal Coronaviruses to zoonotic diseases
II.1. HCoV-OC43 strain: a strictly human respiratory virus with bovine origins
II.2. From bat SARS-CoV to SARS-CoV
II.3. Emergence of the MERS-CoV: role of the dromedary camel
III. Interspecies transmissions among animal Coronavirus
III.1. Avian Coronaviruses
III.2. Feline, canine and porcine Coronaviruses
III.3. Available models for the study of Coronavirus inter-species transmissions
OBJECTIVES OF THESIS PROJECT
INFECTIONS OF CATS WITH ATYPICAL FELINE CORONAVIRUSES HARBOURING A TRUNCATED FORM OF THE CANINE TYPE I NON-STRUCTURAL ORF3 GENE .
CHARACTERIZATION OF DIFFERENT FORMS OF THE ACCESSORY GP3 CANINE CORONAVIRUS TYPE I PROTEIN IDENTIFIED IN CATS
I. Infections of cats by strains harbouring the CCoV-I ORF3 gene
II. Characterisation of the deleted forms of the accessory glycoprotein 3 (gp3)