3.9 Coronavirus

Dra. Bunyi I. Coronaviruses: overview a. Classification b. Historical background c. Viral structure d. Structural proteins e. Replication f. Epidemiology g. Pathogenesis h. Clinical manifestations i. Laboratory diagnosis j. Prevention and control

OUTLINE II. Severe acute respiratory (SARS) a. Etiology b. Epidemiology c. Pathogenesis d. Clinical presentation e. Specific diagnosis f. Treatment g. Prognosis h. Prevention

syndrome

CORONAVIRUSES: Overview

Human coronavirus OC43. Note the characteristic large, widely spaced spikes that form a “corona” around the virion.

Coronaviruses:  Large, enveloped RNA viruses (ssRNA)  Cause common colds in humans; also implicated in gastroenteritis in infants  Cause diseases of economic importance in domestic animals; cause persistent infections in their natural hosts (lower animals)  Identified as a novel cause of a worldwide outbreak of SARS in 2003  Difficult to culture from humans; human viruses are therefore more poorly characterized

 Spherical, 120-160 nm in diameter  Helical nucleocapsid  (+) ssRNA, nonsegmented  largest genome among the RNA viruses Genome  Capped and polyadenylated at the 3’ end  Isolated genomic RNA is infectious  2 glycoproteins, 1 phosphoprotein Proteins  Some viruses contain a third glycoprotein  Contains large, widely spaced, club/petalEnvelope shaped spikes  Cytoplasm Replication  Particles mature by budding into ER and Golgi  Causes colds and SARS Outstanding  Displays high frequency of characteristics recombination  Difficult to grow in cell culture  With widely spaced, petal-shaped, 20 nm long surface projections that give it the appearance of a solar corona  Labile to: heat, lipid solvents and acid pH Virion

STRUCTURAL PROTEINS  Viral envelope contains 2-3 glycoproteins:  Embedded in the envelope lipid bilayer Matrix protein (M)  Interacts with nucleocapsid Nucleocapsid  Phosphorylated nucleocapsid protein protein (N) Surface  Spike glycoprotein component (S)  Makes up the petal-shaped peplomers  Contains sequence closely related to influenza hemagglutinin Hemagglutinin  Found in some viruses including human esterase (HE) coronavirus (OC43)  Causes hemagglutination and has acetylesterase activity CORONAVIRUS REPLICATION

CLASSIFICATION  2 genera:  Coronavirus: primarily respiratory pathogens in humans  Torovirus: exclusive enteric pathogen in humans and animals; widespread in ungulates  The 2 genera are similar in:  Appearance in electron microscope  Replication strategy  The 2 genera in different in:  Size of RNA genome  Structural proteins  Morphology of nucleocapsid HISTORICAL BACKGROUND  1965: first report of isolation in humans (HCoV)  Coronavirus is confirmed as a frequent cause of the common cold in children and adults (2nd to rhinoviruses)  Contributor in lower respiratory infections (particularly, pneumonia) in children and adults, even in the elderly VIRAL STRUCTURE

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Virus attaches to receptors on target cells by glycoprotein spikes on the viral envelope (either by S or HE). The particle is internalized, probably by absorptive endocytosis. The S glycoprotein may cause fusion of the viral envelope with the cell membrane. Since CoV have (+)ssRNA, they can directly produce proteins and new genomes in the cytoplasm. Virus first synthesizes virus-specific RNA-dependent RNA polymerase, which then transcribes a minus strand RNA using the positive strand as a template.

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The minus strands serves as template to transcribe subgenomic positive RNAs used to synthesize all other proteins, as well as for the replication of new positive stranded RNA genomes. Protein N binds genomic RNA and protein M is integrated into the membrane of the ER. Assembled nucleocapsids with the helical twisted RNA then bud into the ER lumen and are encased with its membrane. The viral progeny are finally transported by the golgi vesicles to the cell membrane and are exocytosed into the extracellular space. Because HCoV (human coronavirus) do not grow well in cell cultures, details of viral replication have come from studies with mouse hepatitis virus, which is closely related to the human strain OC43 Replication cycle takes place in the cytoplasm of cells

EPIDEMIOLOGY  Occurs worldwide  May occur anytime of the year but mostly seen during midwinter and spring (or, in the Philippines, during cold months); associated with well-defined outbreaks  Individual serotypes typically predominate in one year  Asymptomatic and symptomatic infection occurs at all ages including the elderly due to transient nature of immunity  Mode of transmission: respiratory droplet  Antibodies to respiratory CoV appear in childhood, increase in prevalence with age and are found in more than 90% of adults  Reinfection with symptoms can occur after a period of one year  Coronaviruses are commonly associated with acute respiratory disease in the elderly, along with rhinoviruses, influenza virus, and RSV PATHOGENESIS  Replicate only in the upper respiratory tract and produce very minimal cytopathology  Repeated infections are common due to vast number of circulating serotypes CLINICAL MANIFESTATIONS  Common colds (remember that coronaviruses are 2nd to rhinoviruses as causative agents of common colds)  Precipitate acute asthma attacks  Lower respiratory tract disease (e.g. pneumonia) LABORATORY DIAGNOSIS  Using human embryo kidney or diploid cell lines Virus isolation  SARS virus was recovered from oropharyngeal specimens using Vero monkey kidney cells  Direct antigen detection technique  Nucleic acid detection  CoV antigens in cells in respiratory secretions may be detected using the ELISA test if a high-quality antiserum is available  Enteric CoV can be detected by examination of Virus detection stool samples by electron microscopy  PCR assays are useful to detect CoV nucleic acid in respiratory secretions and in stool samples  SARS virus RNA was detectable in plasma by PCR, with viremia most readily detectable between days 4 and 8 of infection  Complement fixation Serodiagnosis  Hemagglutination inhibition test  Neutralization

o 8 guests who stayed on the same floor and 2 others from different floors acquired the disease o The outbreak of SARS in other countries occurred when the guests returned home o Secondary cases were reported in 2 Hongkong hospitals o Outbreaks in Singapore, Toronto and Hanoi were observed o As of June 4, 2003, WHO has received 8402 cases of SARS and 772 deaths from the infection ETIOLOGY  Human metapneumovirus was found by PCR in the bronchoalveolar lavage fluid and nasopharyngeal swabs  Novel coronavirus was isolated in Vero cell cultures of respiratory specimens in 5 patients: o This virus was considered as the causative agent of SARS o With high frequency of recombination o Arose from reassortment of genes between human and animal coronaviruses during a chance simultaneous infection EPIDEMIOLOGY  First severe and relatively easily transmissible new disease to emergy in the 21st century  High case fatality rate (9%)  Incubation period: 2 – 10 days  Most cases occur 2 – 7 days after exposure  Possible modes of transmission: o person to person via respiratory secretions o fecal-oral or fecal-nasal route o SUPERSPREADER: a patient who apparently has infected more than ten contacts (superspreaders have also been described for rubella, Ebola and TB) o Role of children (?) in the epidemiology of infection PATHOGENESIS  CoV tend to be highly species-specific; most of known animal CoV display a tropism for the epithelial cells of the respiratory or gastrointestinal tract  Primary site of pathology: respiratory tract (bilateral, patchy airspace consolidation in the lungs)  infiltration similar to that of bacterial infiltration by Staph. or Strep.  Signs of toxaemia include: o Lymphopenia o ↑ SGPT, SGOT o ↑ CPK o ↑ LDH

Children

PREVENTION AND CONTROL  No vaccine is available because of the high reinfection rate  Ribavirin has been used with success in some clinical trials SEVERE ACUTE RESPIRATORY SYNDROME (SARS)  Emerging infectious disease originating in Guandong, China in 2002  First appeared as an outbreak of atypical pneumonia; the first case was in Foshan city in mid-November  The Ministry of Health reported 305 cases, mostly affecting health care workers  SARS then spread to Hongkong: o First case was a 64 yo male physician from Guandong province who stayed in a hotel for one day

Adolescents

CLINICAL PRESENTATION  Less severe presentation with no or minimal systemic manifestations except fever  Signs and symptoms:  Fever (1-4 days PTA)  Coryza  Cough  Dizziness  (-) sore throat  Chills/rigor  Myalgia  Headache  None developed severe respiratory distress throughout the course of illness  Chest x-ray: evidence of pneumonia (with segmental or patchy consolidation)  similar to bacterial infection  Hematologic finding: leukopenia with absolute lymphopenia  Signs and symptoms:  Chills, rigor  Fever  Cough  Myalgia  Headache  Majority develop rapid respiratory distress and required O2 supplementation  Hematologic finding: lower leukocyte counts than children

SPECIFIC DIAGNOSIS A. CDC definition of a suspected case (as of March 2003):  Respiratory illness of unknown etiology with onset since February 1, 2003 and the ff. criteria:  Measured temperature > 38.0oC  ≥1 clinical findings of respiratory illness (e.g. cough, SOB, DOB, hypoxia, radiographic findings of either pneumonia or ARDS)  Travel within 10 days of onset of symptoms to an area with suspected or documented community transmission of SARS (Hongkong, Guangdong Province, China; Hanoi, Vietnam; Singapore)  Close contact (having cared for, having lived with, or had direct contact with respiratory secretions and/or body fluids of a patient suspected of having SARS within 10 days of onset of symptoms with either a person with a respiratory illness and travel to a SARS area or a person under investigation or suspected of having SARS B. Definitive etiologic diagnosis:  Identification of the coronavirus in respiratory specimens by RT-PCR or isolation of the organism in tissue culture  SARS was strongly suspected in a previously healthy patient with pneumonia and marked lymphopenia TREATMENT  No specific treatment and no vaccine  For the lower respiratory tract infection:  3rd generation cephalosporin (cefotaxime)  Oral clarithromycin (for possible bacterial infections)  Ribavirin (for associated viral infections)  Corticosteroid (anti-inflammatory) PROGNOSIS  Children: Good, though extent of lung damage is unknown  Adolescents/adults: Guarded PREVENTION  Effective hospital infection control practices and quarantine procedures outside hospitals  Use of full gear for hospital workers (gloves, gowns, goggles, N95 masks, etc.)  Isolate the patients, quarantine the exposed  Travel restrictions **end of trans**

Addendum to trans 3.3 (General Properties of Viruses): CULTIVATION OF VIRUSES  Growth of virus in animals is still used for the primary isolation of certain viruses and for studies of the pathogenesis of viral diseases and of viral oncogenesis.  The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus.  THREE BASIC TYPES OF CELL CULTURES: a. PRIMARY CULTURE: made by dispersing cells (usually with trypsin) from freshly removed host tissues; generally unable to grow for more than a few passages in culture b. SECONDARY CULTURE (DIPLOID CELL LINE): have undergone a change that allows their limited culture (up to 50 passages) but which retain their normal chromosome pattern c. CONTINUOUS CELL LINES: cultures capable of more prolonged – perhaps indefinite – growth that have been derived from diploid cell lines or from malignant tissues; with invariably altered and irregular numbers of chromosomes *end*

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