Respiratory Viruses Slide Set

Rhinoviruses

 

Common colds account for one-third to one-half of all acute respiratory infections in humans. they are responsible for a considerable proportion of morbidity and economic loss each year due to days lost from work. Rhinoviruses are responsible for 30% of common colds, coronaviruses for 10%, adenoviruses, enteroviruses, RSV, influenza, parainfluenza can also cause common cold symptoms indistinguishable form those caused by rhinoviruses and coronaviruses.

A. Properties

Belong to the family Picornaviridae
At least 100 serotypes are known
differ from enteroviruses in being acid-labile, having a high buoyant density in caesium chloride, and grow optimally at 33 to 35oC.
Naked virus, ssRNA consisting of 7200 bp that make up 30% of the mass of the virus particle.
Virus particle 28-32nm in diameter
Icosahedral capsid is made up of 60 copies of VP1, VP2, VP3, and VP4. Canyons are found on each of the icosahedral faces where cellular receptors bind.

Computer reconstruction of rhinovirus particle (Institute for Molecular Virology) 

B. Epidemiology

Rhinovirus infections occur worldwide. There are 2 seasonal peaks of infection: one major peak during late summer and early autumn, probably coinciding with the opening of schools, and a second peak in Spring. A number of serotypes may circulate simultaneously in a particular geographical area or community at any one given time. Some serotypes may persist in a particular community for several years. Other serotypes may disappear completely form a community. It has been postulated that the ecology of rhinoviruses include antigenic shift and drift. Rhinovirus serotypes are numbered in chronological order of discovery. In recent years, higher-numbered serotypes have replaced lower-numbered serotypes as the predominant strains circulating in the community in recent years. Some of the untypable strains submitted as new prototypes appear to be variants of lower-numbered strains.

Generally, it is estimated that an individual may suffer 2 to 5 episodes of colds per year. Infections are most common during early life and generally decline with an increase in age, probably due to the presence of antibodies against previously encountered strains. Peak excretion of rhinoviruses occurs during the acute phase of the illness. Close contact and crowding appears to increase the transmission of rhinoviruses. Rhinovirus appears to be transmitted mainly by the aerosol route, although mechanical transmission by contaminated fingers or fomites into the nasal epithelium or conjunctiva may also play a part.

C. Pathogenesis

The primary site of rhinovirus infection is in the nasal epithelium. Virus may be detected in the nasal washings of volunteers 24 hours after inoculation and reach a maximum peak by the second or third day. The titres then start to decline and the virus is usually undetectable by the fifth day. Symptoms of cold appears one day after inoculation and peak on the third or fourth day. It is uncertain whether rhinitis is due to the direct cytocidal effect of virus replication or through the release of mediators. Histamine have not been shown to play any role in the development of rhinitis. Kinins, however, are found in elevated quantities. Volunteers given kinins intranasally develop cold symptoms.

Following infection, a specific humoral response is found in both serum and nasal secretions. Serum-neutralizing antibodies do not appear until 14 days after infection and thus recovery is probably not mediated by antibodies. Serum antibodies remain elevated for many years and are probably responsible form protecting the person against reinfection. However, local neutralizing antibodies are lost after 2 years.
 

D. Clinical Symptoms

The common cold is characterized by rhinorrhoea, nasal obstruction, sneezing, sore throat and cough. There is little fever and systemic reactions are uncommon. The illness may last for a week or more. Otitis media and sinusitis may complicate rhinovirus infection in a small proportion of patients (<1%), usually in conjunction with a bacterial infection. The exact role of rhinoviruses in the development of otitis media is not clear. Rhinovirus infections also induce the onset of asthmatic attacks in atopic individuals. Very rarely, rhinovirus infections are associated with cough, chest pain, bronchiolitis and bronchopneumonia. The general opinion is that rhinoviruses are not a significant cause of croup, bronchiolitis , or viral pneumonia.

E. Laboratory Diagnosis

Usually, a common does not require laboratory investigation. If required, the diagnosis is generally made by the isolation of the virus in a sensitive cell culture. Nasal washings are the best specimens and should be collected early in the disease when maximal titres of virus is excreted.

  1. Virus isolation - Rhinoviruses are best isolated in human embryo lung fibroblasts eg. MRC-5, or a sensitive continuous cell line such as Ohio HeLa. Samples should be inoculated into triplicates and rolled at 33oC. The virus CPE, which consists of the rounding of cells similar to that induced by enteroviruses should appear within 8 days of inoculation. The identity can be confirmed by acid lability tests. (pH3)
  2. Direct detection of rhinovirus antigen - an ELISA has been developed for the detection of rhinovirus antigen in nasal washings.
  3. Serology - virus neutralization tests remain the best method. ELISAs have been described.

 

F. Treatment_and_Prevention

Early attempts to prevent rhinovirus infections by vaccination have not been successful. The diversity of rhinovirus serotypes and the lack of cross-protection during reinfection with heterologus serotypes makes prevention by vaccination unlikely to succeed. Attention has therefore focused on the development of antiviral molecules such as interferons and synthetic anti- rhinovirus compounds which could be used therapeutically as well as prophylactically.

Prophylaxis - Natural and recombinant interferon have been shown to be effective in preventing both infection and illness when given intranasally in volunteers over short periods of time. However, prolonged administration resulted in considerable local cytotoxicity reactions eg. nasal irritation, ulceration and bleeding. It is clear that interferons can not be used for long periods, although they may considered for use over short periods eg. 1 week to prevent an infection within the family setting. Given this way, no side effects were reported in volunteers. A number of synthetic compounds have been developed which have potent anti-rhinovirus effects in vitro. One of these compounds, R61837 was shown to be effective in vivo when it significantly suppressed the appearance of colds in volunteers given this compound prophylactically.

Treatment - Clinical trials with interferons failed to modify the course of rhinovirus colds. A recent trial with R61837 again failed to modify the course of a clinical rhinovirus cold. Because of the multiplicity of serotypes, it would be very difficult to develop an effective vaccine against rhinoviruses. Therefore the pursuit of antiviral agents remains the best option.

 

Coronaviruses

 

Human coronaviruses were first isolated in the mid 1965 from volunteers at the Common Cold Unit. The coronaviridae are a monogeneric group of RNA-containing viruses that are associated with respiratory infections in animals, including pigs, cats, dogs, mice and chickens. The group was so named because of the crown-like projections on its surface. At present, at least 10 species are recognized, of which human coronavirus is one. The other are found in animals.

A. Properties

ssRNA enveloped viruses of pleomorphic morphology
60 to 220nm in diameter.
Positive stranded RNA; helical symmetry
characteristic club-shaped projections of 20nm, involved in neutralization, fusion and in some coronaviruses, haemagglutination.

Electronmicrograph of corona virus particles.

Some Human Coronavirus strains, namely 229E and serologically related strains, are generally readily propagated in human cell culture. However, other human coronavirus strains, namely OC43 and some serologically related strains, are considerably more difficult to propagate in cell culture. Three antigenic molecules are found in the virions ie. nucleocapsid, surface projection and transmembrane proteins. The NP antigens may be common to all coronaviruses, while the main antigenic determinants of individual viruses reside on the surface projections. The surface projection antigens are used for the serological grouping of coronaviruses. One avian and two mammalian serological groups have been established. Human coronavirus strains fall into each of the mammalian groups which are named the OC43 and 229E serological groups. It is not clear how closely related are the viruses within a serological group.
 

B. Epidemiology

Generally, human coronavirus infections occur during the winter and early spring but the peak period may vary by several months. The periodicity of infections caused by 229E and OC43 group viruses follows a complex pattern, although they usually cycle with an interval of 2 or 3 years. In general, high infection rates in any particular year are caused by either 229E or OC43 group viruses with only the occasional sporadic human coronavirus infection belonging to the other group. This pattern is observed throughout the world.

C. Clinical Features

Human coronaviruses are responsible for 10 - 30% of all common colds. All age groups are affected, and infection rates have been shown to be uniform for all age groups. This is different from other respiratory viruses such as RSV, where there is a distinct decrease in infection rates with an increase in age. The incubation period is short, being 2 to 4 days. Infection may also be subclinical or very mild. There have been some reports of more severe lower respiratory tract involvement in young children and old people. Reinfection of individuals with the same human coronavirus serotype often occurs within 4 months of the first infection, suggesting that homologous antibodies are protective for only 4 months. Although many people have high antibody levels after infection, reinfection with the same or related strains is common. Antibodies to one human coronavirus group may not be protective against infection with viruses from another group. There are no differences in pathology observed between the OC43 and the 229E strains.

Other possible infections - coronavirus-like particles are often seen in the faeces of children and adults suffering from diarrhoea. These particles have a different morphological appearance to those seen in respiratory infections. As yet, there is no firm evidence associating the presence of these particles to diarrhoea. Also human coronavirus particles have been observed in tissue form patients suffering form multiple sclerosis, there is no evidence for an aetiological role.
 

D. Diagnosis and Treatment

Diagnosis of human coronavirus infections is not attempted in many routine laboratories. They have fastidious growth requirement in cell culture, and the conditions caused by them are of minor clinical significance. The routine diagnostic procedures comprises of cell culture and serology.

Virus isolation - 229E and related strains can be isolated in roller culture monolayers of human embryonic lung fibroblasts, such as W138 and MRC5 cells. A virus CPE of small, round, granular cells is seen throughout the monolayer. Isolates can be confirmed by virus neutralization tests. OC43 related strains usually cannot be grown in cell cultures. Isolation has to be performed on organ cultures of human embryonic tissue such as trachea.

Serology - virus neutralization are the most frequently used tests. HI, CF and ELISA tests have been used. Most of these tests are not carried out in routine diagnostic laboratories. There is little, if any, antibody cross-reaction between strains of 229E and OC43.

Direct detection of virus - indirect immunofluorescence and ELISAs have been developed to detect the presence of coronavirus antigen in nasal secretions with differing results. The usefulness of indirect immunofluorescence has still to be established.

No antiviral drugs against coronaviruses are available and little research is being taken to produce any. However, vaccines against certain animal coronaviruses are available.

 

SARS Virus
 

The SARS crisis of 2003 was an instance when vigorous international cooperation and intervention may have successfully prevented a global health crisis. It fully showed the value of the WHO influenza surveillance network. During the near pandemic between November 2002 and July 2003, with 8,096 known infected cases and 774 deaths (a mortality rate of 9.6%). Here are the key events of the crisis. Local outbreaks of SARS infections were reported in China, Hong Kong, Taiwan, Vietnam, Singapore, Philippines, Mongolia, Canada, and the USA. A number of dead-end sporadic cases were reported in other countries who received infected visitors from affected countries.

Identification of SARS. In early Feb 2003, Guandong province in China reported 305 cases and 5 deaths caused by atypical pneumonia of unknown cause. It later transpired that Guandong was already having similar cases as early as Nov 2006. On the 19th Feb 2003, the WHO influenza network activated emergency pandemic plans after receiving a report from Hong Kong confirming a case of Influenza H5N1 infection. This proved to one of the defining events in the control of the SARS outrbreak. On the 21st Feb, a Chinese medical professor came to Hong Kong to attend a relatives wedding. He stayed at a room on the 9th floor of the Metropole Hotel. Six people who stayed on the same floor of that hotel were infected and they carried the infection to the rest of Hong Kong, Vietnam and Canada. Therefore all the cases outside China could be traced to that event. In early March - Carlo Urbani identified SARS (Severe Acute Respiratory Syndrome) as a unique clinical entity in patients who had been infected by patient in a Vietnam hospital. That patient had previously stayed on the 9th floor of the Metropole hotel. WHO was put on alert. Sadly, Urbani himself later became infected and died.

Discovery of SARS Virus. Initially, a number of agents were implicated as the causative agent, including chlamydia, metapneumoviruses, and influenza H5N1 but it soon became apparent that a new agent may be involved. The breakthrough came on 21st March when the Hong Kong university reported the isolation of an unknown virus in FRhk4 cells, and were able to demonstrate a rising antibody response against this virus by IF in patients with SARS. Furthermore, virus-like particles were seen in lung biopsies. On 22nd March, CDC reported the growth of a corona-like virus in Vero E6 cells. This was identified as a new coronavirus and PCR based diagnostic tests became rapidly available.

Properties or SARS Virus

The SARS virus is a novel coronavirus that did not belong to the previously known OC43 and 229E serogroups. It had a genome of 29,000 bases. It had some rather unusual virological aspects.

Epidemiology

 

Super-spreading Events. The SARS virus is not normally highly infectious but certain individuals have spread the virus to a large number of individuals. These individuals were already known as super-spreaders but the WHO now prefer to call them super-spreading events. In Hong Kong, 3 super-spreading events are known to have occurred.

Diagnosis

The initial diagnosis of SARS was clinical. According to the guidelines issued by the WHO, SARS may be suspected in a patient who has:
  • Any of the symptoms including a fever of 38 C (100.4 F) or more AND
  • Either a history of Contact (sexual or casual) with someone with a diagnosis of SARS within the last 10 days OR Travel to any of the regions identified by the WHO as areas with recent local transmission of SARS (affected regions as of 10 May 2003 that were parts of China, Hong Kong, Singapore and the province of Ontario, Canada).

A probable case of SARS has the above findings plus positive chest x-ray findings of atypical pneumonia or respiratory distress syndrome.

With the advent of diagnostic tests for the coronavirus probably responsible for SARS, the WHO has added the category of "laboratory confirmed SARS" for patients who would otherwise fit the above "probable" category who do not (yet) have the chest x-ray changes but do have positive laboratory diagnosis of SARS based on one of the approved tests (ELISA, immunofluorescence or PCR).

A battery of laboratory tests became rapidly available on the discovery of the SARS virus.

RT-PCR - This is the mainstay of diagnosis of SARS infection. A variety of specimens can be used including NPA (preferred), throat swabs, trachael aspirates, and faeces.

Virus Isolation - Vero E6 and FRhk4 cells may be used. However, the positivity rate is much lower than PCR and stringent Biosafety Level III facilities are required. Therefore, this is not recommended for small routine laboratories.

Serology - SARS virus infection may be confirmed by seroconversion or rising titres of antibodies. IFT and ELISAs are available but originally, whole virus antigen was used which required biosafety level III facilities. Because of the low predictive value of the first generation PCR assays, a serological diagnosis was often the only means of confirming a diagnosis of SARS.

Treatment

A number of treatments were tried initially including ribavirin and steroids. However there is little evidence to suggest that any therapies used during this period was effective. In 2004, it was reported that researchers in China had successfully produced a vaccine that induced antibodies in 24 out of 36 volunteers but more research will be needed to ascertain whether it would be effective.

Post Epidemic

Since July 2003, laboratory acquired cases of SARS had been reported in Singapore, Taiwan and China. These have occurred in Biosafety level III and IV laboratories. Sloppy practices and procedures were to blame rather than failings in the containment equipment. In Jan 2004, a 32 old male with diagnosed with naturally acquired SARS in Guandong, China. He infection was linked to contact with civet cats and the Chinese authorities promptly ordered the slaughter of 10,000 civet cats and related species of animals in the area.

 

Respiratory Viruses Slide Set