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Human parainfluenza viruses (HPIVs) are a group of four distinct serotypes of enveloped single-stranded RNA viruses belonging to the paramyxovirus family. These viruses are closely associated with both human and veterinary disease. They are enveloped and are approximately 150-250nm in size, composed of negative sense RNA with a genome encompassing ~15,000 nucleotides.

The viruses can be detected via cell culture, immunofluorescent microscopy, and PCR. HPIV remains the second main cause of hospitalisation in children under 5 years of age suffering from a respiratory illness (only respiratory syncytial virus causes more hospitalisations for this age group).

Classification
The first HPIV was discovered in the late 1950’s. The taxonomic division is broadly based on antigenic and genetic characteristics, forming four major serotypes. These include:


 * Human parainfluenza virus type 1 (HPIV-1) (most common cause of croup)
 * Human parainfluenza virus type 2 (HPIV-2) (causes croup and other upper and lower respiratory tract illnesses)
 * Human parainfluenza virus type 3 (HPIV-3) (associated with bronchiolitis and pneumonia)
 * Human parainfluenza virus type 4 (HPIV-4) (includes subtypes 4a and 4b)

HPIV is closely related to a recently formed virus grouping, the 'megamyxoviruses' (Hendra and Nipah) and closely linked to the metapneumovirus. HPIV is divided within two genera: Respirovirus (HPIV-1 & HPIV-3) and the Rubulavirus (HPIV-2 & HPIV-4).

Viral Structure and Organisation
HPIV is characterised by being enveloped and containing single stranded negative sense RNA. It is interesting to note however, that non-infectious virions have also been reported with positive RNA polarity. The genome has in the region of 15,000 nucleotides and this is used to encode six key structural proteins.

The structural gene sequence is believed to be as follows: 3′-NP-P-M-F-HN-L-5′ (the protein prefixes and further details are outlined in the table below).

With the advent of reverse genetics, it has been found that the most efficient HPIV viruses (in terms of replication and transcription) have a genome total which is divisible by the number 6, leading to the formation of the “rule of six,” although exceptions to this have been found and its exact advantages are not fully comprehended.

Electrophoresis has shown that the molecular weight (MW) of the proteins for the four HPIV serotypes is similar (with the exception of the phosphoprotein, which shows significant variation). More information on the structural proteins can be found in the paramyxovirus protein section or here.

Viral Entry and Replication
Viral replication is initiated only after successful entry into a cell by attachment and fusion between the virus and the host cell lipid membrane. Viral RNA (vRNA) is initially associated with nucleoprotein (NP), phosphoprotein (P) and Large protein (L). The envelope is associated with a lipid bilayer which has specific viral glycoproteins upon it. The hemagglutinin-neuraminidase (HN) protein is involved with viral attachment and thus hemadsorption and hemagglutination. Furthermore, the fusion (F) protein is important in aiding the fusion of the host and viral cellular membranes, eventually forming syncytia. Initially the F protein is in an inactive form (F0) but can be cleaved by proteolysis to form its active form, F1 and F2, linked by di-sulphide bonds. Once complete, this is followed by the HPIV nucleocapsid entering the cytoplasm of the cell. Subsequently, genomic transcription occurs using the viruses own 'viral RNA-dependant RNA polymerase' (L protein). The cells own ribosomes are then tasked with translation, forming the viral proteins from the viral mRNA.

Towards the end of the process, (after the formation of the viral proteins) the replication of the viral genome occurs. Initially this occurs with the formation of a positive-sense RNA (intermediate step necessary for producing progeny) and finally negative-sense RNA is formed which is then associated with the nucleoprotein. This may then be either packaged and released from the cell by budding or it can be used for subsequent rounds of transcription and replication.

The observable and morphological changes which can be seen in infected cells includes the enlargement of the cytoplasm, decreased mitotic activity and 'focal rounding,' with the potential formation of multi-nucleate cells.

The pathogenicity of HPIV is mutually dependant on the virus having the correct accessory proteins which are able to elicit anti-interferon properties. This is a major factor in the clinical significance of disease.

Host Range
The main host (as the name suggests) remains human beings however, infections have been induced in other animals (both under natural and experimental situations) although these were always asymptomatic.

Clinical significance
In the USA it is estimated that there are 5 million children with lower respiratory infections (LRI) each year. Estimates have shown that HPIV-1, HPIV-2 and HPIV-3 have been linked with up to a third of these infections. Upper respiratory infections (URI) are also important in the context of HPIV, however are caused to a lesser extent by the virus.

For infants and young children it has been estimated that ~25% will develop ‘clinically significant disease.'

Repeated infection throughout the life of the host is not uncommon. Symptoms of later breakouts include upper respiratory tract illness, such as cold and a sore throat. The incubation period (time from the initial exposure to the virus to the onset of symptoms) for all four serotypes is 1 to 7 days. In immunosuppressed people, such as transplant patients, parainfluenza virus infections can cause severe pneumonia, which can be fatal.

HPIV-1 and HPIV-2 have been demonstrated to be the principal causative agent behind croup (laryngotracheobronchitis) which is a viral disease of the upper airway and is mainly problematic in children aged 6–48 months of age. Biennial epidemics starting in Autumn are associated with both HPIV-1 and 2 however, HPIV-2 can also have yearly outbreaks. ‎

HPIV-3 has been closely associated with bronchiolitis and pneumonia and principally targets those aged <1 year.

HPIV-4 remains infrequently detected. However, it is now believed to be more common than previously thought, but is less likely to cause severe disease. By the age of 10, the majority of children are sero-positive for HPIV-4 infection which may be indicative of a large proportion of asymptomatic or mild infections.

Important epidemiological factors that are associated with a higher risk of infection and mortality are those who are immuno-compromised and may be taken ill with more extreme forms of LRI. Associations between HPIV and neurologic disease are known, for example hospitalisation with certain types of HPIV has a strong association with febrile seizures. HPIV-4B has the strongest association (up to 62%) followed by HPIV-3 and 1.

HPIV has also been linked with rare cases of virally caused meningitis. and Guillain-Barré syndrome.

Overall, HPIV remains best known for its effects on the respiratory system and this appears to be where the majority of the focus has been upon. It should be noted that other reported symptoms of infection which are less well known have also been identified as aforementioned.

Airway Inflammation
The inflammation of the airway is a common attribute of HPIV infection. It is believed to occur due to the large scale up-regulation of inflammatory cytokines. Common cytokines which would be expected to be up-regulated include IFN–α, various interleukins (IL–2, IL-6) and TNF–α. Various other chemokines and inflammatory proteins are also believed to be associated with the common symptoms of HPIV infection.

Recent evidence seems to suggest that viral specific antibodies (IgE) may be responsible for mediating large scale releases of histamine in the trachea which are believed to cause croup. More detail on the pathways and interactions can be found here.

Immunology
The bodies primary defence against HPIV infection remains humoral immunity. This is mainly directed against surface proteins which can be found on the virus. In particular the proteins HN and F prove to be most immunogenic in terms of stimulating the immune system.

Most recently the importance of the cell mediated immune system has been scrutinised with reports that those who have defective adaptive immune systems are at a higher risk of severe infection.

Diagnosis
Diagnosis can be made in several broad ways:


 * Isolation and detection of the virus in cell culture.
 * Detection of viral antigens directly within bodily respiratory tract secretions using immunofluorescence, enzyme immunoassays or fluroimmunoassays.
 * Polymerase chain reaction (PCR).
 * Analysis of specific IgG antibodies showing a subsequent rise in titre following infection (using paired serum specimens).

Because of the similarity in terms of the antigenic profile between the viruses, hemagglutinin inhibition (HI) or hemadsorption inhibition (HAdI) processes are often used. Both complement fixation, neutralisation and enzyme linked immunosorbent assays – ELISA, can also be used to aid in the process of distinguishing between viral serotypes.

Morbidity and Mortality
Mortality caused by HPIV in more developed regions of the world remains rare. Where mortality has occurred, it is principally in the three core at risk groups (very young, elderly and immuno-compromised) however, long term changes associated with airway remodelling are believed to be a cause of morbidity. The exact associations between HPIV and diseases such as chronic obstructive pulmonary disease (COPD) are still being investigated.

In developing regions of the world, the highest risk group in terms of mortality remains pre-school children. Mortality may be as a consequence of primary viral infection or secondary problems such as bacterial infection. Predispositions, such as due to malnutrition or other deficiencies may elevate the chances of mortality associated with infection.

Overall, LRI causes approximately 25-30% of total deaths in pre-school children in the developing world and HPIV is believed to be associated with 10% of all LRI cases, demonstrating its significance.

Risk Factors
Numerous factors have been suggested and linked to a higher risk of acquiring the infection, inclusive of malnutrition, vitamin A deficiency, no breastfeeding during the early stages of life, environmental pollution and overcrowding.

Prevention
Despite decades of research, no vaccines currently exist. .

Recombinant technology however has been used to target the formation of vaccines for HPIV-1, 2 and 3 serotypes and has taken the form of several live-attenuated intranasal vaccines. Two vaccines in particular were found to be immunogenic and well tolerated against HPIV-3 in phase I trials however, HPIV 1 and 2 vaccine candidates remain less advanced.

Vaccine techniques which have been thus far employed against HPIV are not limited to intranasal vaccines, but also viruses attenuated by cold passage, host range attenuation, chimeric construct vaccines and also introducing mutations with the help of reverse genetics to achieve attenuation.

Maternal antibodies may offer some degree of protection against HPIV during the early stages of life via the colostrum in breast milk.

Medication
Ribavirin is one medication which has shown good potential for the treatment of HPIV-3 in recent in-vitro tests (in-vivo tests show mixed results). Rivavirin is a broadscale anti-viral and is currently being administered to those who are severely immuno-compromised, despite the lack of conclusive evidence for its use. Protein inhibitors and novel forms of medication have also been proposed to relieve the symptoms of infection.

Furthermore, antibiotics may be used if a secondary bacterial infection develops. Corticosteroids and nebulizers and also a first line choice against croup is breathing difficulties ensue.

Interactions with the Environment
Parainfluenza viruses last only a few hours in the environment and are inactivated by soap and water. Furthermore, the virus can also be easily destroyed using common hygiene techniques and detergents, disinfectants and antiseptics.

Environmental factors which are important for HPIV survival are pH, humidity, temperature and the medium the virus in found within. The optimal pH is around the physiologic pH values (7.4 to 8.0), whilst at high temperatures (above 37°C) and low humidity, infectivity reduces.

The majority of transmission has been linked to close contact in locations such as nosocomial infections, chronic care facilities and doctors surgery’s and occurs via aerosols, large droplets and also fomites (contaminated surfaces).

The exact infectious dose remains unknown.

Economic Burden
In the poorest regions of the world the result of many HPIV infections can be measured in terms of mortality. In the developed world where mortality remains rare, the economic costs of the infection can be estimated. Estimates from the USA are suggestive of a cost (based on extrapolation) of in the region of $200 million per annum.

External Reading
Parainfluenza Clinical Microbiology Review

Clinical Features of Human Parainfluenza Viruses (HPIVs)