[CYCLIC VARIATIONS OF LARYNGOTRACHEITIS IN CHILDREN].

It was analyzed the incidences of laryngotracheitis (LT) in children aged 0 to 14 years in Vinnytsya between 1995 and 2008. It was studied seasonal and circadian rhythms of LT in children. The seasonal variations of LT are characterized by two-wave curve with peaks in October and March, and with a significant decrease in July and August. The incidences of LT in October and March exceed the incidences of LT in July and August in 2.6 times. Circadian variation of LT is characterized by peak at night. The incidences of LT at night exceed the incidences in the morning in 2.6 times. The total number of the incidences of LT in the evening and at night exceed the total number of the incidences of LT in the morning and in the afternoon in 1.7 times. The maximum of incidences of LT to minimum of incidences of LT per hour ratio is 5:1 in girls compared to 4:1 in boys.

Human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium.

Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.

A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling.

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-ß induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-ß induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.

Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates.

The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.

Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys.

Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32 degrees C and exhibited little productive replication at 37 degrees C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.

Local production of inflammatory mediators during childhood parainfluenza virus infection.

OBJECTIVE: To describe the clinical manifestations of parainfluenza virus (PIV) infection and to characterize biochemical markers of PIV disease severity. PATIENTS AND METHODS: We reviewed the medical records of 165 children who had a nasal wash culture positive for PIV at our institution between 1998 and 2008. Nasal wash samples were assayed for 26 inflammatory mediators using Luminex bead proteomics. RESULTS: A total of 153 patients, ages 2 weeks to 12 years, with single virus infection were included in our final analysis. Fifty-two patients were infected with PIV1, 19 with PIV2, 74 with PIV3, and 8 with PIV4. Lower respiratory tract infection (LRTI) was diagnosed in 67 (44%) patients, 21 (14%) had laryngotracheobronchitis, and 49 (32%) had an upper respiratory infection other than laryngotracheobronchitis. LRTI was diagnosed in 54% of patients infected with PIV3, 35% of those infected with PIV1, 26% of those with PIV2, and 50% of those with PIV4. Compared with uninfected control patients, PIV-infected patients had higher nasal wash concentrations of interleukin-6, CX-chemokine ligand 8 (CXCL8 or interleukin-8), CCL3 (macrophage inflammatory protein-1alpha), CCL4 (macrophage inflammatory protein-1beta), CXCL9 (monokine induced by interferon gamma), and CCL5 (regulated upon activation, normal T cell expressed and secreted (RANTES). Patients with LRTI, moderate or severe illness, and PIV 1 or 3 (respirovirus) infection had higher nasal wash concentrations of CXCL8 when compared with patients with upper respiratory infection, mild illness, or PIV 2 and 4 (rubulavirus) infection (P < 0.05). CONCLUSIONS: PIV infection causes a spectrum of illnesses associated with the expression and release of several proinflammatory mediators. Of note, elevated concentrations of CXCL8 in nasal wash samples are associated with more severe forms of PIV disease.

Human parainfluenza virus type 2 (HPIV2) induced host ADAM8 expression in human salivary adenocarcinoma cell line (HSY) during cell fusion.

BACKGROUND: The aim of the study was to investigate expression of ADAMs (A Disintegrin and A Metalloproteinase) of host cell origin during cell-cell fusion induced by human parainfluenza virus type 2 (HPIV2). RESULTS: Induction of host cell ADAM9 was observed in GMK cells, but the applicability of this model was restricted by lack of cross-reactivity of the anti-human ADAM8 antibodies with the corresponding green monkey antigens. HSG cells were not susceptible to HPIV2 virus infection. In contrast, in human parotid gland HSY cells, a natural host cell for paramyxoviruses, HPIV2 induced ADAM8 expression. ADAM8 staining increased dramatically over time from 7.9 +/- 3% at zero hours to 99.2 +/- 0.8% at 72 hours (p = 0.0001). Without HPIV2 the corresponding percentages were only 7.7% and 8.8%. Moreover, ADAM8 positive cells formed bi- (16.2%) and multinuclear cells (3.5%) on day one and the corresponding percentages on day three were 15.6% for binuclear and 57.2% for multinuclear cells. CONCLUSION: ADAM8, well recognized for participation in cell-to-cell fusion especially in osteoclast formation, is up-regulated upon formation of multinuclear giant cells after HPIV2 induction in HSY cells. The virus-HSY cell system provides a novel experimental model for study of the molecular mechanism of cell fusion events.

Vigilancia seroepidemiológica de los Parainfluenza virus humano (PIVh) tipos 2 y 3 en una población infantil seleccionada en Ciudad de La Habana / Seroepidemiological surveillance of human parainfluenza viruses (HPIVs) types 2 and 3 in an infantile population selected in Havana City

Se estudió la presencia de anticuerpos contra los virus Parainfluenza humanos tipos 2 y 3 en 1 793 sueros de una población infantil menor de 14 años de edad. Para el pesquisaje de las muestras clínicas se empleó como sistema la técnica de inhibición de la hemaglutinación. Del total de sueros analizados fueron positivos 1 382 (77,1 por ciento), de estos se confirmó la presencia de anticuerpos contra el tipo 2 en 320 (17,8 por ciento), para el tipo 3 en 334 (18,6 por ciento) y predominó la seropositividad simultánea en 805 (44,9 por ciento). Se constató la circulación de los virus Parainfluenza humanos durante todos los meses del año y en todos los grupos de edades analizados, con aumento en los porcentajes de positividad con la edad(AU)

Identification of amino acids essential for the human parainfluenza type 2 virus V protein to lower the intracellular levels of the STAT2.

The V protein of SV41 targets STAT1, while a specific loss of STAT2 is induced by the hPIV2 V protein. We established HeLa cells constitutively expressing various chimeric proteins between the hPIV2 and SV41 V proteins, and which STAT (STAT1 or 2) was expressed in these cells was analyzed. Both the P-V common domain and the V specific domain of hPIV2 V protein are necessary for STAT2 lowering. The internal domain (aa145-173) containing a large number of nonidentical amino acids between hPIV2 and SV41 does not direct STAT tropism, and the regions necessary for STAT2 lowering are discontinuous. The N-terminal domain (aa1-104) and the internal domain (aa126-196) of the hPIV2 V protein do not determine STAT tropism. HeLa cells expressing A105E or H108P show distinct expression of STAT2, but do show low expression or a loss of STAT1, indicating that the amino acid residues 105 and 108 of the hPIV2 V protein are essential for STAT2 lowering. Interestingly, there is an important amino acid(s) in the region (aa121-125) for STAT2 lowering, and the presence of either amino acid residue 123 or 125 of the hPIV2 V protein is necessary for lowering of STAT2. In addition, HeLa cells expressing S216D or 1217R expressed STAT2, but no STAT1, indicating that the amino acid residues 216 and 217 of the hPIV2 V protein are indispensable for STAT2 lowering. HeLa/hPIV2V cells and HeLa/S104/P are resistant to IFN-beta, while they are sensitive to IFN-gamma. On the other hand, HeLa/SV41V, HeLa/S216D, and HeLa1217R cells are resistant to both IFNs. Intriguingly, HeLa/A105E and HeLa/H108P cells were found to be sensitive to IFN-gamma.

The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5.

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.

Selective STAT protein degradation induced by paramyxoviruses requires both STAT1 and STAT2 but is independent of alpha/beta interferon signal transduction.

The alpha/beta interferon (IFN-alpha/beta)-induced STAT signal transduction pathway leading to activation of the ISGF3 transcription complex and subsequent antiviral responses is the target of viral pathogenesis strategies. Members of the Rubulavirus genus of the Paramyxovirus family of RNA viruses have acquired the ability to specifically target either STAT1 or STAT2 for proteolytic degradation as a countermeasure for evading IFN responses. While type II human parainfluenza virus induces STAT2 degradation, simian virus 5 induces STAT1 degradation. The components of the IFN signaling system that are required for STAT protein degradation by these paramyxoviruses have been investigated in a series of human somatic cell lines deficient in IFN signaling proteins. Results indicate that neither the IFN-alpha/beta receptor, the tyrosine kinases Jak1 or Tyk2, nor the ISGF3 DNA-binding subunit, IFN regulatory factor 9 (IRF9), is required for STAT protein degradation induced by either virus. Nonetheless, both STAT1 and STAT2 are strictly required in the host cell to establish a degradation-permissive environment enabling both viruses to target their respective STAT protein. Complementation studies reveal that STAT protein-activating tyrosine phosphorylation and functional src homology 2 (SH2) domains are dispensable for creating a permissive STAT degradation environment in degradation-incompetent cells, but the N terminus of the missing STAT protein is essential. Protein-protein interaction analysis indicates that V and STAT proteins interact physically in vitro and in vivo. These results constitute genetic and biochemical evidence supporting a virus-induced, IFN-independent STAT protein degradation complex that contains at least STAT1 and STAT2.

The V protein of human parainfluenza virus 2 antagonizes type I interferon responses by destabilizing signal transducer and activator of transcription 2.

Type I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3 gamma/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery.

Isolation of a cytopathogenic virus from a case of porcine reproductive and respiratory syndrome (PRRS) and its characterization as parainfluenza virus type 2.

From a lung of a fetus of a breeding sow showing PRRS-like symptoms a viral agent could be isolated. It was characterized as an enveloped, hemagglutinating RNA virus. Ultrastructural examination of purified virus revealed paramyxovirus-like pleomorphic virions of approx. 200 nm in diameter. The helical nucleocapsids were about 18 nm in diameter. The virus was found to be antigenically related to simian virus 5 (SV5) a prototype strain of parainfluenza virus type 2, but not to bovine respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 3, and Newcastle disease virus as determined by western blot analysis.

Laringotraqueobronquitis, CRUP Viral: controversias en la terapia

La laringotraqueobronquitis o crup viral es una de las entidades clínicas más frecuentes y clásicas de la patología respiratoria pediátrica. La presente revisión evalúa, a la luz de las más recientes publicaciones sobre el tema, el papel terapéutico de medidas como la aerosolterapia, las nebulizaciones de epinefrina racémica, adrenalina y fenilefrina, la administración parenteral de glucocorticoides y el empleo de una escala de puntaje para evaluar la severidad del compromiso del paciente y la respuesta al tratamiento.

Manejo actual de la laringotraquebronquitis en niños / Present management of the laryngel-tracheal-bronchitis in children

Las enfermedaes respiratorias infantiles constituyen la causa más frecuente de consulta al pediatra. Las características anatómicas de la vía aérea, la predisposición alérgica y las condiciones epidemiológicas hacen al niño más vulnerable al contagio con agentes virales. La laringotraqueobronquitis es una enfermedad de etiología viral y su curso normal dura aproximadamente una semana, descartando el tratamiento antibióticvo, en la mayoría de los casos. La evaluación médica es el método de disgnóstico más importante. Las medidas generales de tratamiento, como ambiente tranquilo, estímulos mínimos y reducción de estrés y dolor, son factores que favorecen la regresión espontánea de la obstrucción de la vía aérea

Infection of cultured human tracheal epithelial cells by human parainfluenza virus types 2 and 3.

Despite growing information of the effects of human respiratory virus infection on airway physiology, little information is available on the mechanisms of pathology and pathophysiology in these infections. The human respiratory pathogens, parainfluenza virus types 2 and 3 (hPIV2, hPIV3, respectively), clinically cause laryngotracheobronchitis (infection of the large proximal airways). In order to examine the pathobiology of these viruses in airway cells of human origin, we exposed primary cultures of human tracheal epithelial cells. Primary cultures of human tracheal epithelial cells were readily infected by these agents: cells exposed to hPIV2 and hPIV3 expressed viral antigens (demonstrated by indirect immunofluorescence assay), produced infectious virus, and demonstrated cytopathic effects (including early syncytium formation). Peak viral titers of 2 x 10(7) plaque-forming units per milliliter were obtained, similar to titers from permissive CV-1 cells. Trypan blue staining and direct cell counts demonstrated no difference in the viability of the control and infected cells until the infected cells began to detach from the culture substrate. However, infected cells release significantly more LDH than control cells by 48 h following infection at a multiplicity of infection of 1 virus/target cell. This system provides a model for studying the effects of infection of the human tracheal epithelium by human respiratory viral pathogens without confounding interactions with other cell and tissue types.

In vitro effect of ascorbic acid on infectivity of herpesviruses and paramyxoviruses.

Suspensions of herpes simplex virus types 1 and 2, cytomegalovirus, and parainfluenzavirus type 2 were inactivated within 24 h when treated at 37 degrees C with 1 mg (5.05 mM) of copper-catalyzed sodium ascorbate per ml. The infectivity titer of respiratory syncytial virus was reduced substantially after 24 h but required 48 h for inactivation. Under these conditions, inactivation of these viruses was also successfully achieved with 5.68 mM catalyzed ascorbic acid. Copper (Cu2+), when added with the ascorbate solution at 5 micrograms/ml (0.022 mM), exhibited a catalytic effect on the inactivation of these viruses. The rate of inactivation was affected by the incubation temperature, time of exposure, and the virus concentration. Ascorbate concentrations as high as 10 mg/ml (50.5 mM) demonstrated only a minimum increase in effect on viral inactivation. The loss of infectivity did not alter either the hemagglutination or complement fixation qualities of the antigens.

Respiratory infection and airway reactivity.

The data reviewed demonstrate that viral and mycoplasma infections induce a spectrum of functional abnormalities in airways. Acute virus infections cause wheezing illnesses in both children and adults. Changes in peripheral airway function during infection with similar organisms are observed in other subjects, usually normal adults. The pathogenesis of these responses is unclear. Pathologic data do show that infection with these pathogens damages the airway epithelium. These changes appear to increase permeability of the respiratory epithelium to protein antigens and consequently may contribute to increased frequency of attacks in asthmatic subjects. In addition, increased mucosal permeability may enhance delivery of inhaled drugs to effector sites in airway walls to induce exaggerated bronchoconstrictor responses in clinical challenge situations. Whether changes in the epithelium during infection, inducing greater antigen entry into the interstitium, results in subsequent development of specific allergy is not known and requires further study.

Studies on the pathogenicity of human-origin parainfluenza virus in the brain of mice.

The pathogenicity of parainfluenza type 2 (croup-associated) virus known to cause infections of the respiratory tract in the early life of man was studied in the brain of mice. One- to 4-day-old sucklings and 4-week-old mice were inoculated intracerebrally with the virus. The virus multiplied in sucklings, but not in adults. Most mice inoculated intracerebrally with the virus appeared healthy. Histological examination showed minimum inflammatory changes, although moderate hydrocephalus developed in three of twenty-one sucklings by 6 weeks post-infection. Immunofluorescent study in sucklings showed viral antigens in ependymal lining cells and choroid plexus epithelium during the first two weeks, and parenchymal cells for more than two months. Virus specific antibody response was observed in adults, but not in sucklings. One interesting finding was that viral antigens persisted in six out of 11 suckling brains for one to two months.