Detailed account on Coronaviruses

The History of Discovery

First human coronavirus was isolated by D. Tyrrell and M.L. Bynoe from patient with acute rhinitis in 1965.

It was generally assumed earlier that coronaviruses cause only mild short-lasting respiratory or enteric diseases with complete patient’s recovery. But in 2002 the epidemics of new severe disease emerged in Southeast Asia, especially in China and Vietnam. The disorder primarily affected respiratory tract, resulting in fatal outcome near 10% of total disease cases. The disease was termed as “severe acute respiratory syndrome”, or SARS. In 2003 it was firmly established that the causative agent of SARS is the previously unknown new coronavirus (C. Urbani and coworkers, 2003). The virus was named as SARS coronavirus (SARS CoV) and placed into separate group of coronaviruses. Investigating SARS, Italian doctor Carlo Urbani contracted the infection and died.

Finally, in 2012 the severe disorder similar to SARS emerged in Saudi Arabia. Its agent, a novel coronavirus, was isolated by Egyptian virologist doctor Ali Mohamed Zaki.

By analogy with SARS, the illness was termed as MERS (Middle East respiratory syndrome), and its causative agent was entitled as MERS coronavirus (MERS CoV).

Classification of Coronaviruses

The family Coronaviridae pertain to the order Nidovirales.

The family comprises two subfamilies – Coronavirinae and Torovirinae.

Coronavirinae subfamily contains 4 genera each of several species. To date 6 species of coronaviruses are registered, which are definitely pathogenic for humans.

SARS and MERS coronavirus species pertain to genus Betacoronavirus.

According to serological and genetic properties, 3 groups of coronaviruses are known; groups 1 and 2 harbor mammal pathogens; group 3 includes avian viruses.

Human pathogens are present in both 1st and 2nd group; SARS and MERS coronaviruses pertain to group 2.

Structure and Properties of Coronaviruses

Coronaviruses carry single-stranded positive RNA. It is the largest viral RNA genome known.

Coronaviruses produce the middle-size or large spherical particles with external lipoprotein envelope. Viral nucleocapsid is of helical symmetry. Matrix intermediate layer scaffolds the outer coat of coronaviruses. Glycoprotein spikes surround viral envelope resembling sun crown.

Coronaviruses contain various proteins of nucleocapsid, matrix and outer coat that determine complex viral antigenic structure. Viruses of animal and human origin possess the number of common and specific antigenic determinants.

External glycoprotein spikes are responsible for viral absorption and penetration into the host cells. They display hemagglutinating activity.

The most studied is SARS coronavirus.

On the base of genomic analysis and electron microscopy data the main properties of SARS viruses were determined.

SARS virus has 4 major structural proteins – inner nucleocapsid N protein, membrane M and envelope E proteins, and S protein of receptor spikes.

Viral genome also encodes the number of viral enzymes including RNA-dependent RNA polymerase (replicase), helicase and proteases.

Multiple non-structural accessory proteins play a role of virulence factors for SARS virus. They stimulate apoptosis of infected cells, inhibit the expression of interferons of I type, grossly activate proinflammatory cytokines, and cause deep disorders of blood coagulation and fibrinolysis.

If cultured, coronaviruses are poorly adapted to laboratory animals and cell lines. Some strains can propagate in green monkey kidney cell cultures or Vero cells, and in suckling mice.

Virion Resistance

Coronaviruses are rather sensitive to external influences; ether, detergents and other disinfectants as well as heating above 56°C readily inactivate them.

SARS CoV is relatively stable in comparison with other coronaviruses. It maintains viability in feces and urine for 2-4 days at room temperature. It easily withstands cooling being stable at least for 3 weeks at low temperature range.

Heating at 56oС kills virus within 15 min. As other coronaviruses, it is sensitive to all conventional disinfectants.

Replication Cycle of SARS Coronavirus

SARS virus enters epithelial cells of upper respiratory tract via binding of spike S proteins to specific cell membrane receptor angiotensin- converting enzyme 2 (ACE 2). S-glycoprotein mediates absorption and subsequent fusion of virus particles with susceptible cells.

Uncoating is followed by translation of viral proteins from viral (+) RNA. Initial single polyprotein chain is produced that is eventually cut into mature proteins by viral protease.

Newly formed viral replicase enzyme activates genomic RNA replication. Viral genome is transcribed through intermediate minus RNAstrand that serves as the template for final positive sense RNA synthesis.

Viruses multiply in the cytoplasm of infected cells. After virion assemblage the viral particles are released out of the cells by budding that is promoted by M, E and N proteins.

The death of infected epithelial cells is caused by direct viral cytopathic effects as well as by immune cell lysis. The latter results from immune reaction against viral antigens expressed upon infected cells

Characteristics of Common Respiratory Coronaviral Infections

Coronaviral infections are transmitted by airborne route. Known before 2002 coronaviruses cause up to 30% of all common acute respiratory infections and some gastrointestinal disorders.

In adults respiratory infections are usually asymptomatic, or produce rhinitis and some other modest catarrhal manifestations. Secondary viremia leads to the infection of gastrointestinal tract resulting in diarrhea. Typically the infection course ceases in 5-7 days.

The immunity is mostly humoral and type-specific. Virus-neutralizing antibodies confer the resistance to reinfection with the same type virus.

Laboratory Diagnosis, Prophylaxis and Treatment of Coronaviral Infections

Cell culturing is rarely applied due to poor viral propagation in the cell lines.

For express analysis of nasal swabs or nasopharingeal washings immunofluorescence assay for viral antigens and PCR for identification of viral nucleic acid are elaborated.

Serological testing is commonly used in routine laboratory diagnosis of coronaviral infections. Paired sera tests (e.g. indirect hemagglutination assay, or ELISA) are most preferable; they determine the 4-fold rise of specific antibodies in patient’s serum.

Vaccination is not available for prophylaxis of coronaviral diseases. The infection needs only supportive treatment.

Epidemiology, Pathogenesis and Clinical Findings of SARS

At the end of 2002 the first cases of severe “atypical” pneumonia of unknown etiology have emerged in Guangdong Province of China. Theinfection rapidly spread throughout the Southeast Asia involving humans in Hong Kong, Vietnam, and then in Canada and Europe. As the infection demonstrated high mortality rate (near to 10%) and great communicability by airborne route, the World Health Organization (WHO) declared a global alert for this disease, designated as “severe acute respiratory syndrome”. The epidemic was curbed during 2003; it resulted in more than 8000 infection cases with 774 lethal outcomes.

Overall, SARS was regarded as the first threatening epidemic of XXI century.

The majority of investigators suppose SARS virus to originate from wild animal coronavirus via the chain of occasional mutations. In 2003 Chinese researchers found the virus causing severe acute respiratory syndrome in masked palm civet – a feliform mammal species eaten as a delicacy in China. This agent is regarded now as the animal virus, apparently related with SARS. Thus, the particular animal species is possible source for initial SARS infection.

Also it has been established that the long-term natural reservoirs for SARS virus are fruit bats.

Nevertheless, the sources of infection in verified SARS cases were sick humans. The disease is contracted predominantly via airborne route. Human-to-human transmission is common. Household contact and contacts in health-care settings are also ascertained as the important routes of transmission. Oral transmission of the disease is possible as well.

The healthcare workers and household members, who cared for patients with the disease, are regarded as the groups of highest risk of SARS acquisition. Special attention is to be paid on travellers returned from areas of infection outbreaks, especially from Southeast Asia.

Incubation period lasts from 1 to 7-10 days.

Specific cell membrane receptor for SARS virus angiotensin- converting enzyme 2 is present in high density on airways epithelium, endotheliocytes and enterocytes, which are the major primary targets for SARS infection.

The disease has sharp onset with fever and myalgia. The virus affects respiratory system causing severe lung damage. Multiple virulent accessory proteins of SARS virus cause cell death and provoke systemic inflammatory response. Hematogenous dissemination of virus involves gastrointestinal tract that results in diarrhea.

The fever may decline in a few days but on the 2-3 week of the illness many patients develop a secondary wave of fever with progressing respiratory failure. Since the viral replication slows down near the secondweek of the illness, the life-threatening disease progression largely ensues from the activation of host autoimmune reactions. About 20% of patients manifest respiratory distress syndrome (RDS) with alveolar pulmonary edema that can cause the lethal outcome.

Prognosis of SARS is deeply serious. Nevertheless, introduction of advanced methods of the disease treatmen substantially reduced the number of fatal outcomes.

The infection confers strong high-grade immunity maintained by specific antiviral antibodies. They render high virus-neutralizing activity.

Special Characteristics of MERS Infection Outbreaks

As mentioned above, first outbreak of Middle East respiratory syndrome (MERS), followed by successful isolation of its agent MERS coronavirus (MERS CoV) was registered in 2012 in Saudi Arabia.

According to WHO data, from 2012 to 2016 MERS infection spreaded from its intial focus in Middle East to Southeast Asia countries; the infection was also registered in Europe, United States, and African countries. On August, 2016 the total number of MERS cases was equal to 1791. Case-fatality ratio of MERS is extremely high – about 35%.

MERS virus and MERS disease to some extent are similar with SARS. Nevertheless, MERS infection demonstrates several distinct traits.

MERS CoV is zoonotic virus supposed to originate from local animals (mainly, Arabian camels). Humans become infected by alimentary route and after close contact with sick animals or humans (hospital-acquired infection).

MERS is manifested as severe systemic inflammatory disease that initially damages airways with development of respiratory distress syndrome and acute respiratory failure; this is followed by intestinal disorders and renal disfunction that may result in acute renal failure.

Current MERS epidemic is not completely controlled yet, as the separate cases of infection repeatedly occur nowadays, e.g. in Saudi Arabia. Nevertheless, MERS infection is not regarded as global epidemic threat primarily because of low human-to-human transmission rate.

Laboratory Diagnosis of SARS

Laboratory diagnosis of SARS infection is verified by PCR and serological reactions, e.g. by ELISA test.

For PCR virus-containing specimens are taken from nasopharingeal washings, feces, and occasionally from urine.

Laboratory handling of SARS clinical specimens should be performed in special biosafety facilities (BSL-2 – biosafety level 2).

The diagnosis of SARS is confirmed by PCR in case of at least two positive repeated tests.

For serological diagnosis ELISA test is elaborated. In patients the reaction is carried out with paired sera tests, where fourfold rise in antibody levels ultimately validates the infection. Healthy persons are negative for specific antibodies to SARS CoV.

Virus culturing is produced in most advanced specialized laboratories using various cell lines (e.g., Vero cells). Virus isolation is confirmed by PCR.

Principles of SARS Prophylaxis and Treatment

Global alert from SARS infection requires strict measures to prevent international spread of the disease. It needs professional and public awareness, heightened surveillance with rapid case validation, patient isolation and management. Additional measures should be directed to control travellers departing from the areas of disease outbreak.

International affiliation of scientific laboratories and institutions organized by WHO as Global Outbreak Alert and Response Network, joins collaborative forces to respond to SARS, MERS, or any other emerging infection threat.

Vaccine for SARS prevention is not yet elaborated; nevertheless, the availability of complete genomic sequence of SARS agent makes possible to design effective vaccines and antiviral agents.

Antiviral drug ribavirin is commonly used for treatment of SARS infection. To suppress immunopathological reactions aggravating the disease course steroid hormones (e.g. prednisolone or hydrocortisone) can be administered. The treatment of respiratory distress syndrome presumes the support of vital body functions including fluid resuscitation and artificial lung ventilation if required.