![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Clinical Microbiology Reviews, April 2003, p. 242-264, Vol. 16, No. 2
0893-8512/03/$08.00+0 DOI: 10.1128/CMR.16.2.242-264.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Pediatrics Medical College of Wisconsin, Milwaukee, Wisconsin
SUMMARY INTRODUCTION VIROLOGY BASICS Structural Organization Viral Replication Interactions with the Environment Serotypes Growth in Cell Culture CPE Host Range Viral Antigens Viral Reagents EPIDEMIOLOGY AND CLINICAL FEATURES Clinical Syndromes Croup (acute laryngotracheobronchitis). Bronchiolitis. Pneumonia. Tracheobronchitis. Immunocompromised hosts. Neurologic disease. Other syndromes. Nosocomial infections. Economic burden. Morbidity and mortality. Mode of Transmission Pathogenesis Immunology Prevention and Treatment LABORATORY DIAGNOSIS Collection and Preparation of Clinical Specimens Serum Samples Virus Isolation Tissue culture. Isolation in animals. Detection and typing. Serologic Diagnosis Direct Examination for Viruses Genomic Detection REFERENCES
| SUMMARY |
|---|
 Top NextReferences |
|---|
| INTRODUCTION |
|---|
|
TopPreviousNextReferences |
|---|
|
(Portions of this article have been rewritten from reference 143a with permission.)
| VIROLOGY BASICS |
|---|
|
TopPreviousNextReferences |
|---|
|
vRNA, together with the N, P, and L proteins, form the nucleocapsid core of HPIV. The N binds to the vRNA (one N protein to six nucleotides), making a template that allows the L (RNA-dependent RNA polymerase) and P proteins to transcribe and eventually replicate the HPIV genome (43). The P protein of HPIV is probably a homotrimer (64). The surface glycoproteins (HN and F) interact with the M protein, which may direct their insertion and aggregation at specific cell membrane locations. The M protein also appears to play a role in attracting completed nucleocapsids to areas of infected cell membrane that will soon become viral envelope and may be involved in viral budding (51, 57, 209, 283).
The HN protein is found on the lipid envelope of HPIV and infected cells (136, 280, 351). There it most probably exists as a tetramer and functions in virus-host cell attachment via sialic acid receptors, suggesting that it has neuraminidase activity (important for virus release from cells). There are significant differences in the number of HN glycosylation sites between HPIV types and among strains within one type (139, 140, 143). This may be part of the strategy used by HPIV to escape immune detection. The terminal sialic acid sequences important for HN binding of HPIV are just beginning to be worked out (345). It appears that HPIV-1 HN is more limited in its binding than HPIV-3 HN, which may be important for host and tissue range. It is the binding of the HN protein to receptors on red blood cells that creates the well-recognized hemagglutination or hemadsorption of paramyxoviruses. Elegant work by Moscona and Paluso has demonstrated that the HN-cell receptor interaction is specific and complex. It involves both surface glycoproteins and varies between HPIV types. F protein-mediated cell fusion is affected by the affinity of the HPIV-3 HN to its receptor(s) (251). In addition, cell-to-cell fusion requires a minimum density of receptors which is greater than the density needed for virus membrane-cell membrane fusion (infection). The enzymatic removal of sialic acid receptors from HPIV can create persistently infected tissue cultures. This is one explanation for HPIV-3 persistence in vitro, but in vivo persistence may have additional mechanisms. Persistent infection with HPIV-2 appears to use a different mechanism (2).
As mentioned above, the HPIV F protein is integral in virus-host cell membrane fusion. It is this fusion of membranes which allows the viral nucleocapsid to enter and infect a host cell. Also, this protein is needed in membrane fusion between host cells (syncytial formation) and causes hemolysis. Initially an inactive precursor (F0) is made, which must be cleaved by an endopeptidase to yield the active F protein, which is composed of two disulfide-linked molecules (F1 and F2). The new N terminus on F1 is highly hydrophobic and is thought to make the first contact with the lipid membrane during virus-cell fusion. Furin and Kex2 have been proposed as the enzymes responsible for this proteolytic cleavage in humans (278), but trypsin is most frequently used in vitro. The host range and virulence of HPIV is strongly influenced by the enzymes that cleave the Fo precuror. The ability of the F protein to independently induce both fusion and hemolysis varies among the different HPIV types. HPIV-1, HPIV-2, and HPIV-3 in vitro require both HN and F for fusion and hemolysis (80, 252, 278, 395). The structure and location of the physical interactions between the HN and F proteins responsible for their functional interactions, including fusion promotion, oligomer formation, and cell surface expression, are still being determined (143, 275, 347, 397).
Two major subtypes of HPIV-4 (A and B) were discovered shortly after this virus was first identified 40 years ago. HAdI and neutralization assays could easily distinguish these subtypes, but complement fixation could not (36). During the same decade, several HPIV-2 strains were isolated that could be differentiated serologically from the type stain (272), and then about 10 years ago studies demonstrated significant antigenic variation between different HPIV-2 clinical isolates (302). At about the same time, strains of HPIV-1 were reported that could be separated from the type strain by ELISA, HI, and neutralization assays (143). Molecular analyses of all four types have demonstrated more antigenic and genetic heterogeneity than was initially appreciated (136, 139, 140, 143, 200, 201, 356, 362, 398). In fact, the data suggest that all four major HPIV serogroups (HPIV-1 to HPIV-4) have subgroups or populations that have unique antigenic and genetic characteristics. Even HPIV-4 subtypes A and B demonstrate this variability (201). The variability and changes seen in HPIV suggest an evolutionary pattern similar to that of influenza B virus. Polyclonal serologic testing can detect most HPIV strains using common "type" antigens, but subgroups of HPIV-1 (A, C, and D) and HPIV-3 have been reported with progressive antigenic change (143). In addition, HPIV-1 strains isolated over the last 12 years demonstrate persistent antigenic and genetic differences compared to the 1957 type strain, including differences between genotypes within the same epidemic and same geographic location (136, 139, 140, 143, 200). This progressive antigenic change (although slow) will cause standard reference sera raised to HPIV isolates from the 1950s, or antigen prepared from these same "type" strains, to not universally react in routine serologic assays in the future. Examples of this have already occurred, leading to failure of commercial diagnostic products. Investigators now often use more recent strains of HPIV as sources of antigen or genomic material.
HPIV are isolated more easily in epithelial cell lines than in fibroblast cell lines. The addition of an exogenous protease (trypsin) to the cell culture medium facilitates virus recovery for some serotypes and strains of HPIV. All HPIV need to have their F0 protein cleaved and activated into F1 and F2 for efficient growth in cell culture. Cell lines vary in their ability to produce the apporgriate proteases. For example, HPIV-3 grows efficiently in PMK cells and to high titer in LLC-MK2 cells with serum-free medium, but the addition of trypsin allows increased infectivity in HEp-2 cells (311). HPIV-1, HPIV-2, and HPIV-4 demonstrate greater replication in Vero cells in the presence of trypsin (249, 323). HPIV-1 has a strict and specific requirement for trypsin or a trypsin-like protease in LLC-MK2 cells. HPIV-1, HPIV-2, and HPIV-3 all can be recovered in PMK cells (e.g., cynomologus and rhesus monkey cells) without trypsin (11). However, there is variability between species of PMK cells and the ability to efficiently grow HPIV because the addition of trypsin increases viral recovery in African green monkey PMK cells (138).
CPE PMK cells rarely show cytopathic effects (CPE) when infected with the majority of HPIV isolates. A small number of HPIV-2 and HPIV-3 clinical isolates may demonstrate focal rounding and destruction, elongated cells, and occasional syncytia on initial isolation. All HPIV demonstrate greater CPE on adaption to a particular cell line, with HPIV-3 being the most aggressive. Well-adapted strains of HPIV-3 can destroy more than 50% of a tissue culture monolayer by the third day.
There are numerous PIV closely related to HPIV that have adapted to other mammalian species. HPIV-1 has antigenic, genetic, and pathophysiologic homology to Sendai virus, which infects mice, hamsters, and pigs (47, 169, 293). Simian viruses 5 and 41 are related to HPIV-2 and infect primates (161, 243, 298). Another virus related to both HPIV-2 and simian virus 5 is canine parainfluenza virus (CP2), which causes croup and lower respiratory infection (LRI) in dogs (15, 282). Bovine PIV-3 has been associated with "shipping fever" in cattle, and is antigenically related to HPIV-3 (387). This virus or similar viruses may also infect horses, sheep, goats, water buffaloes, deer, dogs, cats, monkeys, guinea pigs, rats, and pigs (1, 91, 225, 290). Some PIV can infect nonmammalian species. A rubulovirus, Newcastle disease virus, infects poultry, penguins, and other birds and has been responsible for conjunctivitis in bird handlers and laboratory workers (3, 28, 264). There have been reports of human infections by some of the other nonhuman PIV, but these have not been well established (21, 161, 298, 387).
| EPIDEMIOLOGY AND CLINICAL FEATURES |
|---|
|
TopPreviousNextReferences |
|---|
Biennial fall epidemics are the hallmark of HPIV-1 and occur in both hemispheres (38, 232, 259). Reports from the United States have suggested that a minimum of 50% of croup cases are caused by this virus (69, 70, 232). During each HPIV-1 epidemic, an estimated 18,000 to 35,0000 U.S. children younger than 5 years are hospitalized (20, 69, 70, 116, 138, 232, 329, 388). Some of these children have bronchiolitis, tracheobronchitis, pneumonia, and febrile and afebrile wheezing. The majority of infections occur in children aged 7 to 36 months, with a peak incidence in the second and third year of life. HPIV-1 can cause LRI in young infants but is rare in those younger than 1 month. The full burden of HPIV-1 in adults and the elderly has not been determined, but several studies have shown this virus to cause yearly hospitalizations in healthy adults and perhaps play a role in bacterial pneumonias and deaths in nursing home residents (86, 87, 92, 231). HPIV-1, to HPIV-3 have all been found to occur at low levels in most months of the year, similar to RSV and influenza virus (142, 373).
HPIV-2 has been reported to cause infections biennially with HPIV-1 or alternate years with HPIV-1 or to cause yearly outbreaks (20, 75, 259). We see some HPIV-2 activity every year in Milwaukee, Wis. The peak season for this virus is fall to early winter. HPIV-2 causes all of the typical LRI syndromes, but in nonimmunocompromised or chronically ill children croup is the most frequent syndrome brought to medical attention. LRI caused by this virus has been reported much less frequently than with HPIV-1 and HPIV-3. This may be due to difficulties in isolation and detection. As many as 6,000 children younger than 18 years may be hospitalized each year in the United States because of HPIV-2 (Henrickson et al., submitted). About 60% of all HPIV-2 infections occur in children younger than 5 years, and although the peak incidence is between 1 and 2 years of age, significant numbers of infants younger than 1 year are hospitalized each year. HPIV-2 is often overshadowed by HPIV-1 or HPIV-3 infections, yet in any one year or location it can be the most common cause of parainfluenza LRI in young children.
Young infants (younger than 6 months) are particularly vulnerable to infection with HPIV-3. Unlike the other HPIV, 40% of HPIV-3 infections are in the first year of life. Brochiolitis and pneumonia are the most common clinical presentations. Only RSV causes more LRI in neonates and young infants. HPIV-3 has caused outbreaks within neonatal intensive care units, and the natural history of this virus in the neonate is currently being studied (373). Approximately 18,000 infants and children are hospitalized each year in the United States because of LRI caused by HPIV-3. This virus causes yearly spring and summer epidemics in North America and Europe and is somewhat endemic, especially in the immunocompromised and chronically ill. Although the exact reasons for the different seasonality of the HPIV types is unknown, differences in ambient climate conditions have been proposed as one hypothesis (71, 116).
Only a small number of studies have reported on the isolation or epidemiology of HPIV-4 (36, 37, 46, 110, 191). These cases and data are distributed fairly equally between infants younger than 1 year, preschool children, and school age children and adults. Seroprevalence studies have demonstrated that 60 to 84% of infants have significant antibody levels after birth (presumably maternal in origin). These levels drop to 7 to 9% by 7 to 12 months of age and stay low for several years before increasing to about 50% by 3 to 5 years of age. Antibody levels to HPIV-4 continue to rise throughout childhood until approximately 95% of adults have antibody to HPIV-4 A and 75% have antibody to HPIV-4 B (110). Interestingly, the majority of HPIV-4 clinical isolates appear to be subtype B. All of the different respiratory tract syndromes can be caused by HPIV-4. Although hospitalization of infants and young children secondary to HPIV-4 LRI has been reported, serious disease is either rare or difficult to diagnose based on the seroprevelence (216).
Animal PIV have been reported on occasion to infect humans. The majority of evidence to support this hypothesis is serologic, and because of the antigenic relatedness of the respirovirus and rubulavirus genera, it is difficult to substantiate. However, closely related megamyxoviruses (Hendra and Nipah viruses), whose natural host appear to be bats, have recently caused serious epidemics of encephalitis in humans (53, 254, 265). Also, Newcastle disease virus clearly causes human disease and is discussed under "Host range." Other examples of PIV infecting outside of their usual host range include bovine PIV-3 in humans, HPIV-1 in mice, and mouse PIV-1 (Sendai virus) in African green monkeys (155, 182, 314). These infections are usually asymptomatic.
Croup (acute laryngotracheobronchitis). Children present with fever, a hoarse barking cough, laryngeal obstruction, and inspiratory stridor. The incidence peaks between 1 and 2 years of age and is more frequent in boys. In Milwaukee, we have found white children to have had a much higher incidence than black children (137). Approximately 10 to 25% of cases of LRI in children younger than 5 years presents as croup (depending on age). The causative agent has been identified in about 50% of croup cases (36 to 74%) (69, 70, 137, 216, 259). HPIV have made up between 56 and 74% of these cases (131), with HPIV-1 (26 to 74%) being the most frequent subtype (69). HPIV-2 and HPIV-3 average around 10% each as etiologic agents. Sampling of a national database (0.5% of total) and community-based estimates have suggested between 27,000 and 66,000 hospitalizations each year in the United States for croup (137, 232; Henrickson et al., submitted). In years when HPIV-1 is not epidemic, HPIV-2 has been found to cause croup outbreaks. Even during HPIV-1-epidemic years, HPIV-2 has been reported to cause >60% of the croup cases in an individual community (232). HPIV-3 has been demonstrated to cause severe croup in an adult (386). RSV, influenza A and B viruses, adenoviruses, rhinoviruses, and, in school age children, Mycoplasma have all been shown to cause croup. A recent retrospective review suggested that influenza virus croup was more severe than parainfluenza virus croup (284).
Bronchiolitis. The diagnosis of bronchiolitis is unique to pediatrics because of the size of infant's terminal airways. The predominant symptoms include fever, expiratory wheezing, tachypnea, retractions, rales, and air trapping. The peak incidence of bronchiolitis is in the first year of life (81% of cases occur during this period) and then dramatically declines until it virtually disappears by school age. This syndrome is diagnosed in approximately 25 to 30% of LRIs in childhood but makes up a larger percentage in the first year or two of life. At least 90% of cases of bronchiolitis are thought to be viral in origin, and a viral identification rate as high as 83% has been reported (70, 259). Hospitalizations for bronchiolitis have been steadily increasing over the last 20 years in the United States (322). All four types of HPIV can cause bronchiolitis, but HPIV-1 and HPIV-3 have been reported most commonly. Each of these two groups appears to cause 10 to 15% of cases of bronchiolitis in nonhospitalized children. However, in hospitalized children, HPIV-3 causes many more cases than HPIV-1 (three or four times as many) and is second only to RSV as a cause of bronchiolitis and pneumonia in young infants.
Pneumonia. Pneumonia is classicly diagnosed by the presence of fever and rales and evidence of pulmonary consolidation on physical examination or X ray. Pneumonia is diagnosed in 29 to 38% of children hospitalized with LRI and in 23% treated as outpatients (20, 70, 173, 190, 259). However, since several of these classic LRI syndromes decrease with age, pneumonia causes 83% of hospitalizations of children with LRI older than 5 years. The peak incidence for pneumonia is in the second and third years of life. Recently, at least one-third of pneumonia hospitalizations have been in children with chronic diseases (115; Henrickson et al., submitted). Viruses have been shown to cause up to 90% of these LRI, especially in the first year (66, 70, 156, 205, 262, 270), and this percentage decreases to approximately 50% by school age (70, 173, 205). After 9 to 10 years of age, viruses cause a decreasing but still significant amount of pneumonia in immunocompetent individuals. The percentage of pneumonias with a documented viral etiology eventually declines to about 12% by adulthood (74, 76, 122, 231). HPIV-1 and HPIV-3 each cause about 10% of outpatient pneumonias, but, similar to bronchiolitis, HPIV-3 causes a larger percentage of cases in hospitalized patients. Pneumonia can be caused by both HPIV-2 and HPIV-4, but the incidence of disease is not well described. HPIV-1 infection has been associated with secondary bacterial pneumonias in the elderly (92).
Tracheobronchitis. Patients with lower respiratory signs and symptoms who do not fit well into the above three syndromes often receive a diagnosis of tracheobronchitis. The most common symptoms include cough and large-airway noise on auscultation (rhonchi), but patients also may have fever and URI. About 20 to 30% of children with LRI receive this diagnosis. The incidence is lower in the first 5 years of life, but tracheobronchitis is fairly evenly diagnosed throughout school age and adolescence. Viral agents make up the majority of etiologies in children (70). More than 25% of the agents identified to cause tracheobronchitis have been HPIVs (HPIV-3 is more common than HPIV-1 or HPIV-2). Several studies have recorded tracheobronchitis as the most common diagnosis in patients with HPIV-4 infections. Also, this diagnosis is used more commonly in patients with chronic diseases (Henrickson et al., submitted).
Any single HPIV can cause more than one of these LRI syndromes to occur simultaneously or progressively in the same child. In 5 to 20% of LRI cases, two viruses can be detected and may be associated with more severe disease (76). HPIV routinely cause otitis media, pharyngitis, conjunctivitis, and coryza (common cold). These URI can occur singly or in any combination with the above-mentioned LRI. Otitis media has been shown to be associated with viral respiratory tract infections in 30 to 60% of cases. Viruses may work synergistically with bacteria to initiate otitis media or prolong symptoms, and occasionally they are found to be the only cause of disease (50). HPIV-3 is the most frequently reported HPIV associated with otitis media (388). HPIV have been found in 1% of middle ear effusions and in 2% of nasopharyngeal secretions in children with acute otitis media (310).
Immunocompromised hosts. Immunocompromised children and adults appear to be particularly succeptible to developing sever and fatal LRI with HPIV. HPIV-2 has caused giant-cell pneumonia in severe combined immunodeficiency syndrome (SCIDS) (180) and has been found along with HPIV-3 in SCIDS (68, 98, 143, 219, 236) and acute myelomonocytic leukemia (377) and after bone marrow transplantation (BMT) (380). It is common for more than one pathogen to be found in these patients on autopsy, but HPIV alone is found in two-thirds of the cases. Persistent respiratory tract infection and excretion of HPIV-1, HPIV-2, and HPIV-3 have been described in SCIDS (98, 143, 180), with HPIV-3 in a child with DiGeorge syndrome after a thymic transplant (17), and in human immunodeficiency virus-infected children (176, 194, 195). The natural history of HPIV in human immunodeficiency virus-infected children is still largely unknown, but severe disease appears uncommon until they are significantly T-cell deficient (239). HPIV-3 has been associated with acute rejection episodes in renal and liver transplant recipients (67, 146). Solid-organ transplant and BMT patients develop HPIV URI and LRI, including consolidated and interstitial pneumonia (7, 214, 220, 374, 380, 381). HPIV-3 has been the most frequent HPIV type isolated from immunocompromised patients with LRI and is a common cause of fever and neutropenia in children with cancer (9, 224, 266). This virus is persistently shed from BMT patients for up to 4 months without accumulating any mutations (399). Between 5 and 7% of adult BMT and leukemia patients become infected with HPIV-1, HPIV-2, and HPIV-3 (9, 214, 224, 266, 381); approximately 24 to 50% develop pneumonia and 22 to 75% of these will die. A retrospective study found that 41% of pediatric BMT patients with HPIV infection (47% of viral respiratory infections) developed pneumonia and 6% died (224). HPIV has been shown to cause serious LRI and death (8%) in lung transplant recipients up to 5 years after transplantation (367). HPIV-3 has been reported to cause parotitis in children with SCIDS and "common variable" hypogammaglobulinemia (63). Immunocompromised patients have been found to have HPIV infection in other nonrespiratory sites including cerebrospinal fluid (CSF), pericardial fluid, white blood cells, and liver (16), as well as in postmortem cultures of the myocardium and liver (93).
Neurologic disease. For many decades, PIV have been associated with acute and chronic neurologic disease. Children hospitalized with HPIV have had significant problems with febrile seizures. HPIV-4B appears to have the greatest association (up to 62%), followed by HPIV-3 (17%) and HPIV-1 (7%) (75). HPIV-1 strains were isolated from two patients with multiple sclerosis (213, 242), but evidence of antibody to HPIV-1 in the serum or CSF of MS patients has been lacking (31, 211). One report demonstrated that 2 of 25 CSF samples from MS patients had low positive HI titers to HPIV-1 but 3 of 25 had positive titers to measles virus (12). Similarly, virus cultures and electron microscopy of other MS patients failed to find evidence of HPIV-1. However, HPIV-1 and Sendai virus have been detected using PCR in tissue from the ofactory bulbs of mice (247, 248). Animal PIV related to HPIV-2 have been found in patients with MS and subacute sclerosing panencephalitis (118, 307). Two closely related paramyxoviruses (Hendra and Nipaha viruses) have been shown to cause severe encephalitis and death in children and adults (53, 254, 265). HPIV-3 has been isolated from the CSF of a patient with Guillain-Barré syndrome (309) and adults with demyelinization syndromes (370) and has occasionally been shown to cause meningitis in children (8, 370, 385) and adults (370). HPIV-1, HPIV-2, and HPIV-3 have been isolated from the respiratory tract of children with acute encephalitis (23). In addition, several diseases have been linked to HPIV-3 by serology, including encephalitis, ventriculitis, and cluster headaches in an adult (24, 321). All of these data suggest that some strains of HPIV may have neurotropism in certain hosts. Until more studies are done to clarify the role HPIV plays in human neurologic disease, this possibility will be vastly overshadowed by their significant contribution to respiratory disease.
Other syndromes. Full-term and premature infants have developed apnea and bradycardia during HPIV infection (235, 241, 321, 328). Two children with adult respiratory distress syndrome were infected with HPIV-1 (157). HPIV-3 has been implicated in supraglottitis and bronchiolitis obliterans (120, 285). HPIV parotitis has been diagnosed in otherwise healthy children infected with HPIV-1 and HPIV-3 (26, 402), in immunocompromised children (381), and in a patient with cystic fibrosis (34). Croup caused by HPIV has been associated with bacterial tracheitis (83, 218). Exacerbations of nephrotic syndrome in children have been linked to viral respiratory infections in general (226). Nonrespiratory tissues are rarely infected with HPIV. However, both immunocompromised patients and children with croup have had documented viremia and disseminated infection (308). Similarly, children and adults with acute non-A, B, C hepatitis have had paramyxovirus-like nucleocapsids found in their liver, giant cells, and syncytia on tissue examination (286). It is possible that HPIV may play a role in this disease. HPIV-3 has been cultured from a 37-year-old man with myopericarditis, and other HPIV types have been diagnosed serologically in patients with myocarditis or peridcarditis (383). Elevated levels of antibodies to HPIV-1 have been reported in patients with systemic lupus erythematosus and Reiter's syndrome (287). However, the differences between the mean antibody titers of the controls and the subjects were only one or two dilutions. Recent work using reverse transcription-PCR (RT-PCR) has demonstrated no evidence of mumps virus P or HN genes in patients with inflammatory bowel disease (168). There does not yet appear to be a clear link established between HPIV and inflammatory or connective tissue disease. There is a relationship between infection with common respiratory pathogens and myalgias and rhabdomyolysis. HPIV-3 has been cultured from a child who died with rhabdomyolysis (359). Also, HPIV-2 has been serologically linked to myalgias and myoglobinuria in an adult (273). HPIV-3 has been cultured from throat and rectal cultures in an adult patient with bloody diarrhea, but other now common gastrointestinal pathogens were not sought (M. D. Aronson, D. Kaminsky, and C. A. Phillips, Letter, Ann. Intern. Med. 81:856-857, 1974). Likewise, respiratory tract symptoms were found in 11% of young children diagnosed with rotavirus gastroenteritis, but the etiology of the respiratory symptoms was not determined (30). No clear role for HPIV in gastroenteritis has been determined.
Nosocomial infections. Doctors' offices, hospitals, and chronic care facilities are institutions where respiratory viruses are frequently transmitted between patients. The populations at greatest risk for HPIV infection are young preschool children, the immunocompromised, and the elderly (399). Children infected with HPIV-3 will transmit this virus to a minimum of 20% of uninfected control children residing on the same ward (258). About one-third of these will develop mild respiratory symptoms, but some will experience serious LRI and even death (75, 258). Serious sequelae are most common in patients with underlying medical problems. The mean length of hospitalization was increased by many days, even for those with only mild symptoms, because of unnecessary tests and therapies due to their new signs and symptoms. If it is not practical to isolate all children admitted to a particular institution with signs of respiratory infection, then strict handwashing between patients must be enforced and high-risk patients must be cohorted away from these possibly infected patients. In addition, strict rules to limit patient exposure to visitors or staff with respiratory symptoms may help to decrease nosocomial HPIV infections.
Economic burden. The socioeconomic conditions within a country are the biggest determinants of the ultimate cost of HPIV infection. In undeveloped regions, the cost is in human lives. In the United States, mortality is unusual and the cost of these viruses is seen mostly in health care utilization, hospitalization, lost productivity for parents, and, perhaps, long-term respiratory tract sequelae in children. A parent's lost time both from work and home secondary to HPIV infections has not been quantitated but is considerable. Extrapolations based on hospitalizations a decade ago indicate that the yearly cost of HPIV-1 and HPIV-2 infection could exceed $186 million nationally (138). Another estimate based on a computer generated sampling of a national data base (0.5%) for croup caused by HPIV-1 resulted in an estimate of $30 million per croup epidemic for this subset of disease caused by this one virus (232). Separate and independent extrapolations using more recent data from Children’s Hospital of Wisconsin (CHW) suggest that the yearly cost of hospitalizations for HPIV infections in the United States exceeds $200 million (Henrickson et al., submitted). In addition, this estimate does not take into account URI caused by these viruses, the fact that HPIV-3 causes longer and more costly stays than HPIV-1 or HPIV-2, or the economic burden of infections in adults and immunocompromised patients.
Morbidity and mortality. Children who were tested years after having croup have demonstrated increased bronchial reactivity (221). Similarly, children with LRI caused by RSV have been shown to have chronic pulmonary abnormalities including decreased gas exchange (128, 187), restrictive lung disease (144, 341), obstructive lung disease, and hyperreactivity (128, 144, 187, 357). Even adults have been reported with long-term lung disease following HPIV LRI (124, 277). These viruses clearly cause acute inflammatory changes directly to airways and also are capable of inducing responses in the immune system (e.g., humoral, cellular cytokine production and interaction with allergens) that lead to acute pulmonary changes (357). The exact role of HPIV or other common respiratory viruses in chronic respiratory illness (e.g., asthma and chronic obstructive pulmonary disease is still being determined. This includes looking at the question of biased selection of susceptible or at-risk individuals (phenotype expression) in long-term follow-up studies. However, persistent changes in lung mechanics and hyperresponsiveness have been demonstrated in animal models of PIV LRI (295, 333), suggesting that HPIV LRI may lead to chronic pulmonary damage in some hosts.
As stated above, mortality induced by HPIV is unusual in developed countries and is seen almost entirely in young infants, the immunocompromised, and the elderly. However, the preschool population in developing countries has considerable risk of HPIV-induced death. Whether by primary viral disease or by facilitating secondary bacterial infections in malnourished children, LRI causes 25 to 30% of the deaths in this age group and HPIV causes at least 10% of the LRI (22).
When HPIV infects a cell, the first observable morphologic changes may include focal rounding and increase in size of the cytoplasm and nucleus. HPIV decreases host cell mitotic activity as soon as 24 h after inoculation (204). Other changes that can be observed include single or multilocular cytoplasmic vacuoles, basophilic or eosinophilic inclusions, and the formation of multinucleated giant cells (60, 204). These giant cells (fusion cells) usually occur late in infection and contain between two and seven nuclei. Paramyxoviruses are known to induce apoptosis in tissue culture cells (134). The focal tissue destruction caused by HPIV is usually mild and rapidly repaired and, in many infections, may not even be detectable. Infection in immunocompromised hosts is an exception where giant-cell pneumonia can lead to death. Disease severity has been correlated with HPIV shedding in children (127) but not in adults (352). Species-specific down regulation of mouse smooth muscle receptors have been reported following Sendai virus infection (198). However, virus-induced pathologic features and hyperresponsiveness still occurred in rats even without down regulation. Airway hyperresponsiveness in guinea pigs has been associated with sensory neuropeptide depletion (305).
Persistent infection with active viral replication has been demonstrated both in vitro (tissue culture) and in vivo (human infection) (2, 81, 100, 112, 124, 125, 169, 252, 256, 261). Persistent infections with HPIV do not seem to cause morphologic changes or symptoms (e.g., HPIV-2) (2). However, genetic alterations in HPIV and host cells have been reported (81, 112, 261), as well as decreased numbers of host cell surface sialic acid HN receptors (251). The immune response, or lack thereof, in an infected patient clearly affects the clearance of HPIV and the development of persistent infection (67, 236, 377). It is not known what percentage of HPIV infections lead to persistent infection or how persistence is maintained in human hosts.
Both the HN and F proteins have been shown to be involved with cell membrane fusion, but this appears to be virus (type) specific, with some paramyxoviruses needing only the F protein (162, 251). Furthermore, fusion may demonstrate different kinetics depending on whether it takes place virus to cell or cell to cell (251). Certainly, HPIV host range and virulence are determined in part by the ability of host epithelial cells to enzymatically activate the F protein. Also, the neuraminidase activity of the HN protein may play a role in altering muscarinic receptors, leading to vagus-induced bronchospasm, and can cause HPIV-infected cells to resist infection when challenged with HPIV (viral interference) (103, 104, 154).
The immune mechanisms and pathways involved in local cell injury and tissue damage are largely unknown. HPIV-specific immunoglobulin E (IgE) and histamine may contribute to disease severity in infants (379). During acute infection in guinea pigs, increased numbers of inflammatory cells can be found and airway hyperreactivity and decreased bronchoalveolar cellular superoxide production can be demonstrated (94, 95, 306). Rats with antibody to formalin-inactivated HPIV-3 showed enhanced pulmonary inflammation on challenge. In addition, HPIV-3 induced greater peribronchiolar lymphocyte aggregation and histologic change in one species than in another (290). In closely related hosts, an individual's genetically programmed immunologic response may play an important role in HPIV pathogenesis.
HPIV does not need antibody to directly activate the initial components of complement (366). Complement may interfer with virus-host cell receptor binding. Viral neutralization (in vitro) has been demonstrated by the terminal attack complex of complement directly disrupting the virus membrane. HPIV-1 and other respiratory viruses have been shown to have procoagulant activity that may play a role in cardiovascular disease (368). Also, PIVs have been shown to enhance the appearance of pulmonary edema in hypoxic rats, suggesting that they may be a cause of high-altitude pulmonary edema (39).
Secretory IgA plays an important but not fully defined role in protection against natural infections with HPIV. After natural infection with HPIV, most children and adults develop measurable levels of this antibody (160, 330, 378, 392). This has been shown to be correlated with disease prevention and amelioration in adults (330, 352). However, in infants, secretory IgA levels did not correlate with the ability of nasal secretions to neutralize infection or ameliorate disease (392). Cytotoxic T-lymphocyte responses appear to be important in the clearance of virus from the lower respiratory tract during infections with HPIV-3 (135, 158, 193) and mouse PIV-1 (152, 153, 185). Alpha-beta T cells appear to play a larger role in cell-mediated viral cleance than do gamma-delta or natural killer cells, although all may be involved (73, 152, 228). The human cytotoxic T-lymphocyte response has been shown to be directed to determinants on the HN, P, and NP proteins of HPIV-1 and HPIV-3 (55, 65). However, the viral epitopes important in stimulating this line of defense are still largely unknown. More recently, the cytokines produced by CD4 and CD8 T cells have been found during acute LRI caused by Sendai virus in a mouse model. Interleukin-2 (IL-2), gamma interferon, tumor necrosis factor, IL-6 and IL-10 were all detected in the lungs (245). Similarly, human peripheral blood leukocytes stimulated by Sendai virus have been shown to produce macrophage inflammatory protein 1 alpha, monocyte chemotactic protein 1, tumor necrosis factor alpha, IL-6, and IL-8 (163). Interferon may inhibit HPIV-3 primary transcription (52), and likewise HPIV-3 may inhibit interferon-mediated pathways in T cells, including major histocompatibility complex class II antigen expression (108). Another inflammatory mediator induced by Sendai virus is intercellular cell adhesion molecule 1, which has been shown to be responsible for neutrophil and lymphocyte migration into the lung (334).
One of the many inflammatory mediators produced by HPIV-3-stimulated T-cells is IL-10. This cytokine causes T cells to decrease their proliferation response and helps protect them from virus-mediated apoptosis (327). Children with URI caused by HPIV-3 produce IL-11, which correlates with clinical bronchospasm (84). No long-lasting immunity to URI caused by HPIV ever develops, and infections continue throughout life.
HPIV-3 and bovine PIV-3 are antigenically related, and bovine PIV-3 induces resistance in rats and primates to challenge with HPIV-3 (364). In human trials, seropositive adults did not produce significant immune responses to bovine PIV-3, but seronegative volunteers responded quite well to this Jennerian vaccine (54, 62). Vaccine studies of young infants and children have demonstrated the same promising results (182). Further studies evaluating the safety and efficacy of bovine PIV-3 when given as multiple doses and simultaneously with routine childhood immunizations are under way (210). A new approach using recombinant bovine PIV-3 with human F and HN genes and human RSV G and F genes has induced good antibody titers and protection in hamsters and rhesus monkeys (129, 319, 320). Sendai virus is antigenically related to HPIV-1. In another attempt to use the classic Jennerian approach, Sendai virus was tested in primates, in which two doses protected against challenge by HPIV-1 (155).
Antigenically and genetically stable attenuated strains of HPIV-3 have been developed by cold adaption (CA). The stability of these CA vaccine strains is enhanced because of multiple markers of attenuation in tissue culture (19). The initial work with these CA strains showed poor infectivity in older children and adults but protection in weanling hamsters and chimpanzees (54, 61). In addition, evidence of reversion toward the wild type was reported in low-passage strains (54). Higher-passage CA HPIV-3 strains demonstrated immunogenicty and attenuation in younger infants, but symptoms still developed (18). Finally, after further attenuation, a passage of CA HPIV-3 was found that demonstrated good infectivity, immunogenicity, attenuation (no diseases), and stability (78, 183). In addition, CA HPIV-3 appears to be more immunogenic then BPIV-3. CA strains of HPIV-1 and HPIV-2 have also been developed, and initial investigation has demonstrated attenuation in tissue culture and animal models.
Protease activation mutants represent another approach to developing attenuated strains of HPIV. Infection with HPIV requires that the F protein be cleaved by a trypsin-like protease. HPIV strains that are resistant to trypsin cleavage but sensitive to cleavage by another protease may be attenuated in humans (318). Protease activation mutants of Sendai virus have protected mice from lethal challenge infections (230, 349).
The envelope glycoproteins (HN and F) of HPIV-3 have been cleaved from whole virus to create subunit vaccines that have demonstrated efficacy in rats, lambs, mice, and hamsters (4, 246, 300, 303). Many different routes of immunization have been tried, including subcutaneous, intramuscular, and intranasal. No human trials have yet been reported. Subunit vaccines created by cloning HN and F in vaccinia virus and baculovirus vector systems have been used to immunize rats and primates (338, 340, 365). Viral shedding and peak titer were both reduced on subsequent challenge. Reverse genetics are now being used to develop systems able to evaluate current live HPIV-3 vaccines in terms of specific mutations and hopefully introduce specific mutations into wild-type strains in the future (77, 151). An example of this is the production of a recombinant HPIV-3 that contains the HN and F for HPIV-1 and the specific CA mutations found in the L gene of HPIV-3 (348). This vaccine has been found to be protective in hamsters. Sometime over the next decade, one or more of these approaches may produce the first licensed HPIV vaccine.
Agents found to have in vitro antiviral activity against paramyxoviruses include neuraminidase inhibitors (e.g., zanamivir), protein synthesis inhibitors (puromycin), nucleic acid synthesis inhibitors, benzthiazole derivatives, 1,3,4-thiadiazol-2-ylcyanamide, carbocyclic 3-deezaadenosine (Cc3Ado), ascorbic acid, calcium elenolate, and extracts of Sanicula europaea leaves (113, 123, 179, 222, 240, 263, 289, 312, 332, 382, 389-391). None of these compounds or drugs have yet found clinical applications. Potent inhibitors of HPIV syncytium formation in tissue culture have been found by screening hundreds of small synthetic peptides. These peptides, which are to a specific domain on the F protein, may provide a novel approach to the development of antiviral therapies for HPIV (114, 206, 281). Amantadine did not decrease URI disease severity in adult volunteers challenged with HPIV-1 even though it has some anti-HPIV activity at very high concentrations (330). Similarly, ribavirin has both in vitro and in vivo activity against HPIV (32, 326). Furthermore, there have been anecdotal reports of decreased HPIV shedding and clinical improvement when infected immunocompromised patients were treated with aerosolized and oral ribavirin (41, 42, 227, 336). However, a recent review at Fred Hutchinson Cancer center demonstrated that established HPIV-3 pneumonias responded poorly to aerosolized ribavirin (267). Currently there are no antiviral drugs with proven clinical efficacy against HPIV.
There have been several studies demonstrating that nonspecific immunostimulators can protect against challenge infections with paramyxoviruses (e.g., dihydroheptaprenol, N-acetylglucosaminylmuramyl tri- or tetrapeptides, imiquimod, and polyriboinosinic and polyribocytidylic acids) (149, 166, 167, 342). A portion of this protective effect is due to stimulation of endogenous cytokines. Animals have been protected by the direct administration of cytokines (107, 167). Interferon (alpha and gama), human granulocyte colony-stimulating factor, and human IL-1ß are a few of the cytokines that have been tested (400). Numerous studies, as summarized in a recent meta-analysis, have demonstrated that oral or systemic steroids are effective at improving symptoms of croup as early as 6 h after treatment (331).
Protection against RSV and other respiratory pathogens has been accomplished by giving high-titer pooled immunoglobulin (56, 325). LRI caused specifically by HPIV may be decreased (40, 288, 350), but little has been reported concerning the use of immunotherapy in HPIV disease. Combined immunotherapy and steroids have been demonstrated to decrease pulmonary virus titer and inflammation in a cotton rat model (291). Immunotherapy may offer a possible theraputic option in patients with severe disease until safe and effective vaccines and antivirals are available.
| LABORATORY DIAGNOSIS |
|---|
|
TopReferences |
|---|
HPIV lose infectivity rapidly when the temperature rises above 4 to 8°C. Specimens (swabs or 2- to 4-ml nasal wash aspirates) should be collected and placed in viral transport medium (2 to 3 ml), either veal infusion broth or minimum essential medium supplemented with some protein source (not serum) such as 0.5% bovine serum albumin. The transport medium should contain antibiotics and antifungal agents to decrease contamination and be buffered to yield a pH of 7.5 to 80 after addition of the clinical sample. Ideally, the specimen in transport medium should be kept at 4°C until tissue culture inoculation. If a delay of more than 24 h is anticipated, the specimen should be frozen. Centrifugation at 1,000 x g prior to inoculation is also helpful in removing debris.
It is rare to have to process nonrespiratory specimens for HPIV. CSF can be directly plated onto tissue culture cells. If it is going to be frozen, transport medium should be added 1:1 prior to freezing. Transport medium with extra antibiotics can be used to dilute stool specimens or for rectal swabs. Standard transport medium can be used to dilute tissue homogenized in a dounce prior to testing.
Isolation in animals. Eggs are a very poor growth medium for HPIV. It is only after many repeated passages and much presumed antigenic change that HPIV can be detected in embryonated eggs. Only three isolates of HPIV-2 have ever been reported to have undergone primary isolation in eggs (369).
Detection and typing. CPE is rarely demonstrated during primary isolation of HPIV in tissue culture, and so this is not a reliable or useful method to detect positive cultures. HAd of guinea pig red blood cells directly onto the tissue culture monolayer is the most common method to detect the presence of HPIVs. The respiroviruses are quickly and easily detected using HAd. Most HPIV-1 isolates are detected by day 4 (138), and HPIV-3 takes only a little longer (36, 37, 145). However, the rubulaviruses are much slower. Only 35% of HPIV-2 isolates are HAd positive by day 7 (138), and HPIV-4 may take 3 weeks or longer (36). Interestingly, the neuramindase of HPIV-4 appears to be temperature sensitive because this virus hemadsorbs better at room temperature or 37°C while all of the other serotypes react well at 4°C (37, 110).
IF is the most rapid and accurate method currently available to detect and type HPIV in tissue culture (109, 344, 372). HPIV isolates are detected much faster using this technique than using to HAd, and IF detects positive cultures that never develop HAd. This is especially true for HPIV-2 (138). In addition, further typing of HPIV is unnecessary when IF detection is used. This can save time and money. Tissue culture isolation of HPIV is being done less and less. However, high-risk populations may continue to have a need for this method. A typical testing plan (all year round) would be as follows. IF detection is performed on all inoculated PMK tissue culture cells on days 2 or 3, 4 or 5, and 10 and on MK2 cells on days 14 and 21. A MAb pool reagent could be used such that only a single test had to be done per culture (344). Also, the addition of centrifugation (e.g., shell vial) to the above IF schedule may speed HPIV detection by 1 day or more (296). If desired, HAd could be performed first and then IF could be performed only on those that were positive. It is important to remember that primary screening with HAd will miss some HPIV. Other useful methods of detecting and typing HPIV in tissue culture include HAdI, HI, complement fixation, and neutralization (36, 45, 136). These methods are currently more useful for research purposes since they are time-consuming and costly compared to IF (except HI). On primary isolation, most strains of HPIV do not release enough free virus into the medium to react in the HI assay, limiting the usefulness of this relatively easy assay in clinical virology. Until commercial MAb IF reagents became widely available, HAdI was the method of choice for HPIV serotype identification. This meant that each identification took an additional 3 to 5 days after the culture was identified as positive (37). Although tissue culture is the current "gold standard," the new molecular assays like PCR are rapidly replacing this time-honored method in clinical viro
