Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Respiratory infections and coinfections: geographical and population patterns.

INTRODUCTION: Acute respiratory infections are the second cause of mortality in children younger than five years, with 150.7 million episodes per year. Human orthopneumovirus (hOPV) and metapneumovirus (hMPV) are the first and second causes of bronchiolitis; type 2 human orthorubulavirus (hORUV) has been associated with pneumonia in immunocompromised patients. OBJECTIVE: To define hOPV, hMPV and hORUV geographical distribution and circulation patterns. METHOD: An observational, prospective cross-sectional pilot study was carried out. Two-hundred viral strains obtained from pediatric patients were genotyped by endpoint reverse transcription polymerase chain reaction (RT-PCR). RESULTS: One-hundred and eighty-six positive samples were typed: 84 hOPV, 43 hMPV, two hORUV and 57 co-infection specimens. Geographical distribution was plotted. hMPV, hOPV, and hORUV cumulative incidences were 0.215, 0.42, and 0.01, respectively. Cumulative incidence of hMPV-hORUV and hMPV-hOPV coinfection was 0.015 and 0.23; for hOPV-hMPV-hORUV, 0.035; and for hORUV-hOPV, 0.005. The largest number of positive cases of circulating or co-circulating viruses occurred between January and March. CONCLUSIONS: This study successfully identified circulation and geographical distribution patterns of the different viruses, as well as of viral co-infections.

INTRODUCCIÓN: Las infecciones respiratorias agudas constituyen la segunda causa de mortalidad en los niños menores de cinco años, con 150.7 millones de episodios anuales. Entre los principales agentes etiológicos están Orthopneumovirus (hOPV) y metapneumovirus (hMPV) humanos como primera y segunda causa de bronquiolitis, respectivamente; Orthorubulavirus humano tipo 2 (hORUV) se ha asociado a neumonía en pacientes inmunocomprometidos. OBJETIVO: Definir patrones de distribución geográfica y de circulación de hOPV, hMPV y hORUV. MÉTODO: Se llevó a cabo un estudio piloto transversal prospectivo observacional. Se genotipificaron 200 aislamientos virales de pacientes pediátricos mediante transcripción inversa seguida de reacción en cadena de la polimerasa en punto final (RT-PCR). RESULTADOS: Se tipificaron 186 muestras positivas: 84 de hOPV, 43 de hMPV, dos de hORUV y 57 de coinfecciones. Se trazó la distribución geográfica. Las incidencias acumuladas de hMPV, hOPV y hORUV fueron de 0.215, 0.42 y 0.01, respectivamente. Las incidencias acumuladas de la coinfección de hMPV-hORUV y hMPV-hOPV fueron de 0.015 y 0.23; de hOPV-hMPV-hORUV, de 0.035; y de hORUV-hOPV, de 0.005. El mayor número de casos positivos de virus circulantes o cocirculantes se presentó entre enero y marzo. CONCLUSIONES: Fue posible identificar patrones de circulación y distribución geográfica de los diferentes virus, así como de las coinfecciones virales.

Respiratory infections and coinfections: geographical and population patterns / Infecciones y coinfecciones respiratorias: patrón geográfico y de circulación poblacional

Abstract Introduction: Acute respiratory infections are the second cause of mortality in children younger than five years, with 150.7 million episodes per year. Human orthopneumovirus (hOPV) and metapneumovirus (hMPV) are the first and second causes of bronchiolitis; type 2 human orthorubulavirus (hORUV) has been associated with pneumonia in immunocompromised patients. Objective: To define hOPV, hMPV and hORUV geographical distribution and circulation patterns. Method: An observational, prospective cross-sectional pilot study was carried out. Two-hundred viral strains obtained from pediatric patients were genotyped by endpoint reverse transcription polymerase chain reaction (RT-PCR). Results: One-hundred and eighty-six positive samples were typed: 84 hOPV, 43 hMPV, two hORUV and 57 co-infection specimens. Geographical distribution was plotted. hMPV, hOPV, and hORUV cumulative incidences were 0.215, 0.42, and 0.01, respectively. Cumulative incidence of hMPV-hORUV and hMPV-hOPV coinfection was 0.015 and 0.23; for hOPV-hMPV-hORUV, 0.035; and for hORUV-hOPV, 0.005. The largest number of positive cases of circulating or co-circulating viruses occurred between January and March. Conclusions: This study successfully identified circulation and geographical distribution patterns of the different viruses, as well as of viral co-infections.

Resumen Introducción: Las infecciones respiratorias agudas constituyen la segunda causa de mortalidad en los niños menores de cinco años, con 150.7 millones de episodios anuales. Entre los principales agentes etiológicos están Orthopneumovirus (hOPV) y metapneumovirus (hMPV) humanos como primera y segunda causa de bronquiolitis, respectivamente; Orthorubulavirus humano tipo 2 (hORUV) se ha asociado a neumonía en pacientes inmunocomprometidos. Objetivo: Definir patrones de distribución geográfica y de circulación de hOPV, hMPV y hORUV. Método: Se llevó a cabo un estudio piloto transversal prospectivo observacional. Se genotipificaron 200 aislamientos virales de pacientes pediátricos mediante transcripción inversa seguida de reacción en cadena de la polimerasa en punto final (RT-PCR). Resultados: Se tipificaron 186 muestras positivas: 84 de hOPV, 43 de hMPV, dos de hORUV y 57 de coinfecciones. Se trazó la distribución geográfica. Las incidencias acumuladas de hMPV, hOPV y hORUV fueron de 0.215, 0.42 y 0.01, respectivamente. Las incidencias acumuladas de la coinfección de hMPV-hORUV y hMPV-hOPV fueron de 0.015 y 0.23; de hOPV-hMPV-hORUV, de 0.035; y de hORUV-hOPV, de 0.005. El mayor número de casos positivos de virus circulantes o cocirculantes se presentó entre enero y marzo. Conclusiones: Fue posible identificar patrones de circulación y distribución geográfica de los diferentes virus, así como de las coinfecciones virales.

Full-genome sequencing and phylogenetic analysis of four neurovirulent Mexican isolates of porcine rubulavirus.

We report the complete genome sequences of four neurovirulent isolates of porcine rubulavirus (PorPV) from 2015 and one historical PorPV isolate from 1984 obtained by next-generation sequencing. A phylogenetic tree constructed using the individual sequences of the complete HN genes of the 2015 isolates and other historical sequences deposited in the GenBank database revealed that several recent neurovirulent isolates of PorPV (2008-2015) cluster together in a separate clade. Phylogenetic analysis of the complete genome sequences revealed that the neurovirulent strains of PorPV that circulated in Mexico during 2015 are genetically different from the PorPV strains that circulated during the 1980s.

Acute neurologic disease in Porcine rubulavirus experimentally infected piglets.

The objective of this study was to evaluate the clinical disease, humoral response and viral distribution of recent Porcine rubulavirus (PorPV) isolates in experimentally infected pigs. Four, 6-piglet (5-days old) groups were employed (G1-84, G2-93, G3-147, and G4-T). Three viral strains were used for the experimental infection: the reference strain LPMV-1984 (Michoacán 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacán (Mich/147/2013). Each strain was genetically characterized by amplification and sequencing of the gene encoding hemagglutinin-neuroamidase (HN). The inoculation was performed through the oronasal and ocular routes, at a dose of 1×106TCID50/ml. Subsequently, the signs were evaluated daily and necropsies were performed on 3 different days post infection (dpi). We recorded all micro- and macroscopic lesions. Organs from the nervous, lymphatic, and respiratory system were analyzed by quantifying the viral RNA load and the presence of the infectious virus. The presence of the viral antigen in organs was evidenced through immunohistochemistry. Seroconversion was evaluated through the use of a hemagglutination inhibition test. In the characterization of gene HN, only three substitutions were identified in strain Mich/147/2013, two in strain LPMV/1984 (fourth passage) and one in strain Qro/93/2013, with respect to reference strain LPMV-84, these changes had not been identified as virulence factors in previously reported strains. Neurological alterations associated with the infection were found in all three experimental groups starting from 3dpi. Groups G1-84 and G3-147 presented the most exacerbated nervous signs. Group G2-93 only presented milder signs including slight motor incoordination, and an increased rectal temperature starting from day 5 post infection (PI). The main histopathological findings were the presence of a mononuclear inflammatory infiltrate (lymphocytic/monocytic) surrounding the ventricles in the brain and focal interstitial pneumonitis with distention of the alveolar sacs in the lungs. PorPV and RNA distribution were identified in the organs of the nervous, lymphatic, and respiratory systems of the piglets analyzed at different times (days 5, 10, and 15 PI). The viral antigen was detected in the brain and lungs in most of the assessed groups. Seroconversion was evident in groups G1-84 and G2-93. Groups G1-84 and G3-147 were the most clinically affected by the experimental infection. Both strains were isolated in the state of Michoacán. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.

Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

Respiratory disease in growing pigs after Porcine rubulavirus experimental infection.

The aim of this study was to analyze the pathogenicity and distribution of Porcine rubulavirus (PorPV) in the respiratory tract of experimentally infected pigs. Nine 6-week-old pigs were infected with PorPV and examined clinically. Blood, nasal swab, and tissue samples were collected on different days post-infection (DPI). The humoral immune responses and viral loads were evaluated. The infected pigs exhibited an increase in the respiratory clinical signs. In addition, the excretion of PorPV was extended to 23 DPI in the nasal fluid. The distribution of PorPV in the respiratory tract tissues was extended until the end of the experiment; soft palate tonsil and lymph nodes exhibited high viral loads. The major microscopic lesions observed in the lungs corresponded to interstitial pneumonia and hyperplasia of the associated lymphoid tissue. In conclusion, PorPV infection causes a pneumonic disease characterized by a prolonged virus excretion and high viral load in the lymphoid tissues.

Efficacy of quantitative RT-PCR for detection of the nucleoprotein gene from different porcine rubulavirus strains.

Blue-eye disease is an emergent viral swine infection caused by porcine rubulavirus (PoRV). We have developed a qRT-PCR method to detect and quantify expression of the nucleoprotein gene for different PoRV strains. The limit of detection for this assay was 10(2) copies of synthetic RNA. Viral RNA from PoRV was detectable at a TCID50 of 0.01. Significant differences were observed between viral RNA quantification and virus titration results for nine PoRV strains. For nasal and oral swab samples that were collected from experimentally infected pigs, the qRT-PCR assay was more sensitive (87.1-83.9 %) for the detection of positive samples than methods involving isolation of virus. The implementation of highly sensitive assays that yield results quickly will be of great assistance in the eradication of PoRV from Mexico. We also believe that the newly developed qRT-PCR assay will help reduce the spread of this viral infection to other countries.

Identification of antigenic variants of the porcine rubulavirus in sera of field swine and their seroprevalence.

We sampled sera from 1013 non-vaccinated swine from four states in Mexico, Guanajuato, Jalisco, Michoacán and the Estado de Mexico, to analyse anti-porcine rubulavirus antibody titres against three different porcine rubulavirus isolates (PAC-4/1993, PAC-6/2001, and PAC-9/2003) using a hemagglutination inhibition assay. The results revealed that there were antigenic differences among the isolates assessed. In particular, the estimated correlation between the PAC-4/1993 and PAC-6/2001 (0.50) isolates and between the PAC-4/1993 and PAC-9/2003 isolates (0.56) displayed a moderate positive correlation. In contrast, there was a strong positive correlation between the PAC-6/2001 and PAC-9/2003 isolates (0.73). We also found that in the state of Guanajuato, PAC-4/1993 was the isolate that was most frequently identified; in Jalisco, the isolate was PAC-6/2001; and in Michoacán, the isolate was PAC-9/2003. By contrast, in the Estado de Mexico, all three isolates appeared to circulate with a low seroprevalence. In general, the analysed sera from the four states displayed a porcine rubulavirus serological prevalence ranging from 9% to 23.7%. These data indicate that there is not complete antibody cross-antigenicity among the three isolates, and the antigenic variations in the antibody response found in this study implies that the use of a monovalent vaccine would not generate complete protection against the different antigenic subtypes.

Caracterización biológica de tres aislamientos naturales del Rubulavirus porcino (México)

Biological characterization of three natural isolates of the porcine rubulavirus (Mexico). Porcine rubulavirus (PoRV) produces a neurological and reproductive syndrome in pigs called the blue-eye disease, known only from Mexico. Several isolates were grouped by the main symptoms presented during outbreaks: a) neurotropic in piglets, b) broadly neurotropic in piglets and gonadotropic in adults, and c) gonadotropic in adults. We studied some biological properties of three strains, which fall in one of each virus group: La Piedad Michoacán (LPM) and Producción Animal Cerdos 1 (PAC1) and 3 (PAC3), respectively. The analyzed viral properties are mainly related with the trans-membrane hemagglutinin-neuraminidase (HN) and fusion (F) proteins, such as cytopathic effect, hemolysis, hemagglutinating (HA) and neuraminidase (NA) activities. In the infection assays PAC1 strain presented the highest fusogenicity level; however, the most cytolytic strain was PAC3. In addition, HA and NA activities and viral genome of PAC3 strain was detected in supernatants during cell infection earlier than in the other two strains, which shows that PAC3 virions release from the host cell earlier than LPM and PAC1. Experimental determination in purified viruses shows that PAC3 presented a higher HA and NA activities; however, PAC1 shows other interesting properties, such as a high thermostability of HN and differences about substrate profile respect to LPM and PAC3. Our data suggest that NA activity is associated with the virulence of RVP. Rev. Biol. Trop. 56 (2): 487-499. Epub 2008 June 30.

El Rubulavirus porcino causa un síndrome neurológico y reproductivo en cerdos, hasta ahora reportado sólo en México. Los virus aislados se agrupan de acuerdo con los síntomas principales observados durante los brotes en: a) neutrópicos en lechones, b) neurotrópicos en lechones/gonadotrópicos en adultos y c) gonadotrópicos en adultos. En este trabajo se estudiaron tres cepas: La Piedad Michoacán (LPM) y Producción Animal "Cerdos" 1 (PAC1) y 3 (PAC3), ubicadas respectivamente en cada grupo. Las propiedades estudiadas se relacionan principalmente con dos proteínas de la envoltura viral, la hemaglutinina-neuraminidasa (HN) y la proteína de fusión (F). Se cuantificaron el efecto citopático y las actividades de hemólisis, hemaglutinación (HA) y neuraminidasa (NA). En cultivo celular la cepa PAC1 presentó una mayor actividad fusogénica, sin embargo PAC3 presentó la mayor actividad citolítica. La cepa PAC3 fue la primera en ser detectada en sobrenadante de células infectadas (HA, NA y genoma), lo que muestra que sus viriones son liberados al medio antes que las otras dos cepas. PAC3 tuvo las actividades más altas de HA y NA, sin embargo, PAC1 presentó una mayor termoestabilidad en estas actividades de HN y un perfil de substrato algo distinto de los observados para LPM y PAC3. Estos datos sugieren que la actividad de NA está relacionada con la virulencia del RVP.

Molecular characterization of the hemagglutinin-neuraminidase gene of porcine rubulavirus isolates associated with neurological disorders in fattening and adult pigs.

"Blue eye disease" is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6-PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6-PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6-PAC9 viruses seems to be associated with their increased neurovirulence.

[Biological characterization of three natural isolates of the porcine rubulavirus (Mexico)]. / Caracterización biológica de tres aislamientos naturales del Rubulavirus porcino (México).

Biological characterization of three natural isolates of the porcine rubulavirus (Mexico). Porcine rubulavirus (PoRV) produces a neurological and reproductive syndrome in pigs called the blue-eye disease, known only from Mexico. Several isolates were grouped by the main symptoms presented during outbreaks: a) neurotropic in piglets, (b) broadly neurotropic in piglets and gonadotropic in adults, and (c) gonadotropic in adults. We studied some biological properties of three strains, which fall in one of each virus group: La Piedad Michoacán (LPM) and Producci6n Animal Cerdos 1 (PAC1) and 3 (PAC3), respectively. The analyzed viral properties are mainly related with the trans-membrane hemagglutinin-neuraminidase (HN) and fusion (F) proteins, such as cytopathic effect, hemolysis, hemagglutinating (HA) and neuraminidase (NA) activities. In the infection assays PAC1 strain presented the highest fusogenicity level; however, the most cytolytic strain was PAC3. In addition, HA and NA activities and viral genome of PAC3 strain was detected in supernatants during cell infection earlier than in the other two strains, which shows that PAC3 virions release from the host cell earlier than LPM and PAC1. Experimental determination in purified viruses shows that PAC3 presented a higher HA and NA activities; however, PAC1 shows other interesting properties, such as a high thermostability of HN and differences about substrate profile respect to LPM and PAC3. Our data suggest that NA activity is associated with the virulence of RVP.

[Clinical and epidemiological manifestations of parainfluenza infection in hospitalized children]. / Características clínicas y epidemiológicas de la infección por virus parainfluenza en niños hospitalizados.

BACKGROUND: Human parainfluenza viruses (hPIV) are a common cause of respiratory illness of children but published data on clinical characteristics of hPIV infection in South America is scarce. OBJECTIVE: To review the clinical presentation and epidemiological features of hPIV in a series of hospitalized children in Chile. PATIENTS AND METHODS: Retrospective review of clinical charts from all pediatric admissions with a diagnosis of respiratory disease (between January 2001 to December 2004) at the Catholic University Hospital, Santiago, Chile. Nasopharyngeal secretions were tested for hPIV in children admitted with suspected respiratory viral infections. RESULTS: A total of 3,043 respiratory admissions were recorded during the study period; 64 children (2.1%) were hPIV positive. Average age was 13 months (range: lm to 12y) and 77%> were younger than 2 years. HPIV-2 was the most common type identified (47%). A seasonal trend was noted for serotypes hPIV-2 and 3. Acute wheezing (40%o) and pneumonia (30%) were the most common clinical diagnosis in hPIV positive children and 17%> hPIV positive children (44%> for hPIV-1) were associated with laryngitis. All hPIV positive bronchiolitis were due to serotypes hPIV-2 and 3. CONCLUSION: hPIV can cause respiratory disease requiring hospitalization; serotypes hPIV-2 and 3 displayed a seasonal trend. Although hPIV is an uncommon cause of severe respiratory infecion requiring hospitalization in children, it should be considered in the differential diagnosis of laryngitis, bronchiolitis and pneumonia, especially in younger children.

Características clínicas y epidemiológicas de la infección por virus parainfluenza en niños hospitalizados / Clinical and epidemiological manifestations of parainfluenza infection in hospitalized children

Los virus parainfluenza del ser humano (VPIh) son patógenos importantes de enfermedad respiratoria en niños; pese a ello, existe escasa información publicada en Sudamérica dirigida a caracterizar esta infección. Objetivo: Describir las manifestaciones clínicas y epidemiológicas específicas de los VPIh en niños hospitalizados. Pacientes y Métodos: Se revisaron todas las hospitalizaciones respiratorias (HR) efectuadas en el Hospital de la Pontificia Universidad Católica, Santiago, Chile, durante el período 2001-2004 y sus respectivos estudios virales obtenidos de secreciones nasofaríngeas en aquellos con sospecha de infección viral. Resultados: Se identificaron 3.043 HR siendo 64 (2,1 por ciento) VPUrh La edad promedio fue 13 meses (rango: 1 m-12 a) siendo 77 por ciento) de edad inferior a dos años. VPIh-2 fue el serotipo prevalente (47 por ciento), observándose una tendencia estacional para los serotipos 2 y 3. Las presentaciones más frecuentes fueron sibilancias asociadas a virus (40 por cientoo) y neumonía (30 por ciento). Todas las bronquiolitis se presentaron asociadas a VPIh serotipos 2 y 3. Sólo 17 por ciento de los hospitalizados por VPIh+ (44 por ciento VPIh-1) desarrollaron laringitis. Conclusión: Virus parainfluenza humano puede ser responsable de HR en niños, mostrando una tendencia estacional VPIh-2 y el serotipo 3. Aunque son poco frecuentes como causa de HR, confirmamos su participación como etiología específica de laringitis, bronquiolitis y neumonía, especialmente en niños pequeños.

Background: Human parainfluenza viruses (hPIV) are a common cause of respiratory illness of children but published data on clinical characteristics of hPIV infection in South America is scarce. Objective: To review the clinical presentation and epidemiological features of hPIV in a series of hospitalized children in Chile. Patients and Methods: Retrospective review of clinical charts from all pediatric admissions with a diagnosis of respiratory disease (between January 2001 to December 2004) at the Catholic University Hospital, Santiago, Chile. Nasopharyngeal secretions were tested for hPIV in children admitted with suspected respiratory viral infections. Results: A total of 3,043 respiratory admissions were recorded during the study period; 64 children (2.1 percent) were hPIV positive. Average age was 13 months (range: lm to 12y) and 77 percent> were younger than 2 years. HPIV-2 was the most common type identified (47 percent). A seasonal trend was noted for serotypes hPIV-2 and 3. Acute wheezing (40 percento) and pneumonia (30 percent) were the most common clinical diagnosis in hPIV positive children and 17 percent> hPIV positive children (44 percent> for hPIV-1) were associated with laryngitis. All hPIV positive bronchiolitis were due to serotypes hPIV-2 and 3. Conclusion: hPIV can cause respiratory disease requiring hospitalization; serotypes hPIV-2 and 3 displayed a seasonal trend. Although hPIV is an uncommon cause of severe respiratory infecion requiring hospitalization in children, it should be considered in the differential diagnosis of laryngitis, bronchiolitis and pneumonia, especially in younger children.

Identification of potential B cell epitope determinants by computer techniques, in hemagglutinin-neuraminidase from the porcine rubulavirus La Piedad Michoacan.

Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.

Semen alterations in porcine rubulavirus-infected boars are related to viral excretion and have implications for artificial insemination.

Porcine rubulavirus (PoRV), also known as blue eye disease (BED) of swine, causes respiratory and reproductive problems in pigs at several developmental stages. To study the effect of PoRV infection on semen production, five boars were infected with 1 x 10(6) TCID(50)/ml of PoRV strain PAC-3 and evaluated for 59 days post inoculation (DPI). Infected boars developed reproductive tract pathology that included swelling of the testes and epididymides. Analysis of the semen showed that the infection had little effect on semen production in four animals, but semen from one boar showed severe alterations in sperm concentration, motility, and morphology. When motility was analyzed in BTS-diluted semen after 24, 48, or 72 h, alterations were detected in all boars. Furthermore, viral antigen was detected in semen, the seminal plasma fraction, or sperm fraction from all boars. These results showed that PoRV is excreted via semen and, therefore, artificial insemination is a potential route of dissemination.

Blue eye disease porcine rubulavirus (PoRv) infects pig neurons and glial cells using sialo-glycoprotein as receptor.

Pig neural cells express glycoproteins with sialylated N-linked oligosaccharide chains (SNOC) which are used by the porcine rubulavirus (PoRv) as receptors. Pig neuronal or glial cell cultures were employed to investigate (a) whether PoRv infects such cells using a molecule expressing SNOC, and (b) the role of viral envelope glycoproteins in establishing the infection. Enriched neuronal or glial cell cultures were exposed to PoRv and infection was detected immunocytochemically. Neuronal cultures prepared from neonatal pigs were treated enzymatically to eliminate sialic acid or N-linked oligosaccharide chains. Primary neural cultures were exposed to anti-HN or anti-F preincubated with PoRv to study the role of the viral glycoproteins. In enriched cultures, PoRv infected neurons and glial cells, and sialic acid expressed in N-linked oligosaccharide chains appeared to play a central role in infection. It was concluded that HN and F viral glycoproteins are required to infect neurons and glial cells.

Uptake of porcine rubulavirus (LPMV) by PK-15 cells.

BACKGROUND: The porcine virus denominated La Piedad Michoacan Virus (LPMV) is a member of the family Paramyxoviridae and is the cause of a disease in pigs present only in Mexico. The disease is characterized by meningoencephalitis and respiratory distress in young pigs, epididymitis and orchitis in boars, and reproductive failure and abortion in sows. METHODS: The cytopathology, morphology, and distribution of the hemagglutination neuraminidase (HN) and nucleoprotein (NP) proteins of LPMV were investigated following inoculation into PK-15 cells. The cytopathic effect was characterized by cytoplasmic vacuolation and the formation of syncytia and cytoplasmic inclusion bodies. RESULTS: In immunofluorescence assays using a monoclonal antibody (MAb) against the HN protein at 5-60 min post-infection (early infection), a diffuse immunofluorescence was observed near the cell membrane and adjacent to the nuclear membrane. At 24 h post-infection (late infection), a dust-like immunofluorescence was observed throughout the cytoplasm. LPMV-infected cells incubated with the MAb against the NP protein showed punctate cytoplasmic fluorescence during the early stages of infection. At the late infection stage, these fluorescent particles became larger and were seen predominantly in the cytoplasm of syncytia. This pattern was also apparent by immunohistochemical labeling and immunogold electron microscopy. The latter technique revealed that HN protein was diffusely distributed throughout the cytoplasm. When using the MAb against the NP protein, nucleocapsid organization was the most prominent feature and resulted in the formation of cytoplasmic inclusion bodies visible by light and electron microscopy. Immunogold labeling of purified nucleocapsids was shown by electron microscopy. Virus particles and nucleocapsids were morphologically similar to members of the Paramyxoviridae family. CONCLUSIONS: The morphologic characteristics of the virions and the distribution patterns of the HN and NP proteins in PK-15 infected cells indicate that the mechanisms of LPMV replication are generally similar to those of the members of the Paramyxoviridae family.

Comparative evaluation of the CD4+CD8+ and CD4+CD8- lymphocytes in the immune response to porcine rubulavirus.

The porcine immune system is unique in the expression of CD4+CD8+ (double-positive, DP) lymphocytes. These cells have been associated with immunological memory due to their gradual increase with age, the expression of memory phenotype and their ability to respond to recall viral antigen. This work analyzes the biological function of CD4+CD8- and CD4+CD8+ lymphocytes in the immune response to porcine rubulavirus (PRv). CD4+CD8- cells isolated from pigs 3 weeks after infection with porcine rubulavirus proliferated in response to homologous virus and generated lymphoblasts which were predominantly of the CD4+CD8+ phenotype, whereas stimulation with mitogen induced proliferation but did not switch the phenotype. CD4+CD8- lymphocytes isolated after 10 weeks of infection proliferated in response to phytohemagglutinin (PHA) but did not proliferate in response to homologous virus and did not change their phenotype, whereas CD4+CD8+ lymphocytes proliferated in response to PHA and to viral antigen. The cytokine profile of both lymphocyte populations showed the presence of IL-2 and IL-10 transcripts, quantitation demonstrated that CD4+CD8+ cells expressed mainly IL-10, whereas CD4+CD8- lymphocytes expressed primarily IL-2. Our results show that CD4+CD8- lymphocytes in the early phase of porcine rubulavirus infection can be converted to double-positive cells expressing IL-10 in an antigen-dependent manner, and that CD4+CD8- T-cells late in infection do not acquire CD8.