Detection of antibody to bovine syncytial virus and respiratory syncytial virus in bovine fetal serum.

Batches of commercial fetal bovine serum, described by the suppliers as antibody-free, all contained antibody to bovine syncytial virus (BSV) when tested by indirect immunofluorescence. Antibody to bovine respiratory syncytial virus (RSV) was not detected in these sera. Twenty-four percent of individual fetal bovine sera contained antibody to BSV, and 14% contained antibody to RSV when tested by indirect immunofluorescence. BSV antibody titers in fetal sera from dams with high BSV antibody levels were variable but always higher than RSV antibody titers. Radial immunodiffusion studies with BSV-positive sera revealed the presence of immunoglobulin M (IgM), IgG, and IgA, but the quantity of these immunoglobulins was not directly related to the BSV antibody titers. The evidence suggests that the antibody present in fetal sera arose as the result of infection rather than from maternal transfer across the placenta.     

小发卡RNA抗呼吸道合胞病毒效应的研究

目的 为从基因水平抑制呼吸道合胞病毒（respiratory syncytial virus，RSV）的复制，针对RSV的M2-2基因构建小发卡结构状RNA（short hairpin RNA，shRNA）重组质粒，在细胞水平观察shRNA对RSV复制的影响。方法 将成功构建的针对RSV M2-2基因的重组质粒pshRNA8260转染HEp-2细胞，利用光镜观察pshRNA8260对RSV致HEp-2细胞病变效应（cytopathogenic effect，CPE）的影响并计算CPE抑制率，空斑形成实验检测RSV滴度变化。结果 成功构建了针对人RSV M2-2基因mRNA的pshRNA8260重组质粒，研究发现pshRNA8260能明显改善RSV所致的病变效应，降低RSV在细胞内复制的病毒滴度。结论 针对RSV M2-2基因的pshRNA82（g）重组质粒具有明显的特异性抗RSV效应的作用。     

Ultrastructural analysis of the interaction between F-actin and respiratory syncytial virus during virus assembly

During respiratory syncytial virus (RSV) infection there is a close physical interaction between the filamentous actin (F-actin) and the virus, involving both inclusion bodies and the virus filaments. This interaction appears to occur relatively early in the replication cycle, and can be detected from 8 h post-infection. Furthermore, during virus assembly we obtained evidence for the participation of an F-actin-associated signalling pathway involving phosphatidyl-3-kinase (PI3K). Treatment with the PI3K inhibitor LY294002 prevented the formation of virus filaments, although no effect was observed either on virus protein expression, or on trafficking of the virus glycoproteins to the cell surface. Inhibition of the activity of Rac GTPase, a down-stream effector of PI3K, by treatment with the Rac-specific inhibitor NSC23766 gave similar results. These data suggest that an intimate interaction occurs between actin and RSV, and that actin-associated signalling pathway, involving PI3K and Rac GTPase, may play an important role during virus assembly.     

Phenotypic analysis of local cellular responses in calves infected with bovine respiratory syncytial virus

Changes in lymphocyte subsets in the trachea, pulmonary tissue, bronchoalveolar lavage (BAL), peripheral blood and bronchial lymph node (BLN) of gnotobiotic calves infected with bovine respiratory syncytial virus (BRSV) were analysed by flow cytometry. Following BRSV infection, virus titres in the nasopharynx reached a peak between days 5 and 7 and infection was resolving from day 10. Although calves did not develop signs of clinical respiratory disease, there was evidence of gross pneumonia and histological changes typical of BRSV bronchiolitis, which were most extensive from day 710 of infection. Following BRSV infection there was a recruitment of CD8+ T cells into the trachea and lung, which peaked on day 10 after infection. Thus, there were approximately equal numbers of CD8+ and CD4+ T cells in the lung and trachea of uninfected calves, whereas by day 10 of infection, CD8+ cells outnumbered CD4+ cells by 3:1 in the lungs and 6:1 in the trachea of the infected calves. Although the increase in CD4+ T cells into the lungs was less marked than that of CD8+ T cells, changes in expression of CD45R, CD45RO, L-selectin and interleukin-2 receptors all suggested that CD4+ T cells were activated during BRSV infection. Changes in gamma delta T cells were not observed in BRSV-infected calves. There was a marked increase in B cells in the BLN after infection and BLN CD4+ T cells changed from the majority expressing L-selectin and CD45R in uninfected calves to a predominance of L-selectin- CD45R- CD45RO+ phenotype, 10 days after infection. In conclusion, CD8+ T cells constitute the major lymphocyte subpopulation in the respiratory tract of calves recovering from BRSV infection.     

Cell-mediated immune responses of lambs to challenge with bovine respiratory syncytial virus.

The lamb is a good model to study the pathogenesis and immune responses to infections with respiratory syncytial virus (RSV) as lambs experimentally infected with bovine or human RSV may develop overt clinical disease. In the present study the development of cellular cytotoxic responses was studied in splenic, pulmonary and peripheral blood mononuclear cells obtained from lambs after primary and secondary infection with bovine RSV. Infection with bovine RSV was followed by the appearance of cytotoxic cells in the peripheral blood, the spleen and lung lavage fluids. These effector cells lysed virus-infected targets in a self-restricted manner. Depletion techniques revealed that cytotoxic activity was largely due to OvCD8+ cells. When effector cells obtained from primed lambs were stimulated with inactivated bovine RSV or with virus-infected cells in vitro, virus-specific cytotoxicity was significantly increased.     

Monoclonal antibodies to the fusion protein of bovine respiratory syncytial virus.

Five monoclonal antibodies specific for bovine respiratory syncytial virus were characterized by Western immunoblotting, radioimmunoprecipitation, and epitope mapping assays. The monoclonal antibodies were found to be specific for the fusion protein, and there were at least two antigen binding sites, one of which was neutralizing.     

Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein

Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.     

Comparison of caprine,human and bovine strains of respiratory syncytial virus

Summary A new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.     

Isolation,characterization, and pathogenicity studies of a bovine respiratory syncytial virus

Summary The isolation and characterization of a bovine respiratory syncytial (RS) virus is described. Serological studies indicate that bovine RS virus is widespread in Iowa cattle and that it is involved in some outbreaks of respiratory disease. Experimental infection in calves indicates that the virus can cause illness in calves, particularly those with serum neutralizing antibody.     

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus

Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus and the infections with human respiratory syncytial virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared by neutrophils whereas apoptosis was an important way of clearance of BRSV-infected epithelial cells. Neighboring cells, which probably were epithelial cells, phagocytized the BRSV-infected apoptotic cells. The number of both CD4(+) and CD8+ T cells increased during the course of infection, but the T cells were not found between the epithelial cells of the bronchi up until apoptosis was no longer detected, thus in the bronchi there was no indication of direct contact-dependent T-cell-mediated cytotoxicity in the primary infection.     

Immune responses of lambs experimentally infected with bovine respiratory syncytial virus and Pasteurella haemolytica.

The immune responses of lambs experimentally infected with bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica were compared with lambs infected with bovine RSV or Pasteurella haemolytica alone. Superinfection with P. haemolytica aggravated the reduction in the number of CD5+, CD4+ and LCA p220+ (B) lymphocytes caused by bovine RSV, but it had no effect on the neutralizing antibodies against P. haemolytica cytotoxin. Serum samples obtained from lambs experimentally infected with bovine RSV and P. haemolytica had significantly lower amounts of neutralizing antibodies to bovine RSV than those obtained from lambs infected with bovine RSV alone.     

Alteration of leukocyte populations in calves concurrently infected with bovine respiratory syncytial virus and bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection altered leukocyte populations in calves that were reflected by depression of T, BoCD4+, and BoCD8+ lymphocytes in the thymus and depression of B lymphocytes in Peyer's patches (PP). The present study was based on mononuclear leukocyte preparations from eighteen 9- to 12-month-old crossbred calves that were each exposed to either bovine respiratory syncytial virus (BRSV), BVDV, or BRSV and BVDV concurrently, or served as mock-infected controls. Peripheral blood leukocytes were collected on postinfection days (PID) 0, 2, 4, 6, and 8, and cell populations from thymus, spleen, mesenteric lymph node, and PP were collected at necropsy on PID 9. The leukocytes were analyzed using flow cytometry for lymphocyte subpopulations expressing antigens specific for BoCD2, BoCD4, BoCD8, BoWC1, lambda light chain of bovine immunoglobulin, BoCD11b and major histocompatibility complex (MHC) class II. Concurrent BRSV and BVDV infections caused exaggerated alterations in leukocyte populations with a greater percentage of T-lymphocytes harvested from the PP. Alterations in the leukocyte populations in lymphatic tissues and in peripheral circulation due to BVDV infection may be an important mechanism for causation of clinically severe diseases of the respiratory and digestive tracts during concurrent BRSV and BVDV infections.     

Respiratory syncytial virus, pneumonia virus of mice, and influenza A virus differently affect respiratory allergy in mice

BACKGROUND: Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses. OBJECTIVE: In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge. RESULTS: Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice. CONCLUSION: The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.     

Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein

The nucleotide sequence of the gene encoding the major nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) has been determined. The N mRNA is 1196 nucleotides long with a single, large open reading frame. The derived polypeptide has 391 amino acids corresponding to a calculated molecular weight of 42,600 Da. This is in agreement with the molecular weight of 43,000 Da determined for the BRSV N protein by SDS-polyacrylamide gel electrophoresis (PAGE). Comparison of the nucleotide sequence of BRSV N gene with the sequence of the N gene of human respiratory syncytial virus (HRSV) revealed a homology of 80.7%. There is a 93.3% homology at the amino acid level between the N proteins of BRSV and HRSV. The 5'- and 3'-terminal untranslated sequences that are conserved among HRSV mRNAs were also identified in the N mRNA of BRSV. The results indicate that the N genes are highly conserved in the bovine and human strains of respiratory syncytial virus.     

Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus

Bovine respiratory syncytial (BRS) virus is an important cause of serious respiratory illness in calves. The disease caused in calves is similar to that caused by human respiratory syncytial (HRS) virus in children. The two viruses, however, have distinct host ranges and the attachment glycoproteins, G, have no antigenic cross-reactivity. The fusion glycoproteins, F, of the HRS and BRS viruses, however, have some antigenic cross-reactivity. To further compare the BRS virus and HRS virus fusion proteins, we determined the nucleotide sequence of cDNA clones to the BRS virus F protein mRNA, deduced the amino acid sequence, and compared these sequences with the HRS virus F protein sequences. The BRS virus F mRNA was 1899 nucleotides in length and had a single major open reading frame which could code for a polypeptide of 574 amino acids with an estimated molecular weight of 63.8 kDa. Structural features predicted from the amino acid sequence included an NH2-terminal signal sequence (residues 1-26), a site for proteolytic cleavage (residues 131-136) to generate the disulfide-linked F1 and F2 subunits, and a hydrophobic transmembrane anchor sequence (residues 522-549). The nucleic acid identity between the BRS virus and the HRS virus F mRNA sequences was 71.5%. The predicted BRS virus F protein shared 80.5% overall amino acid identity with the HRS virus F protein with 89% identity in the F1 polypeptide but only 68% identity in the F2 polypeptide. The position and number of the cysteine residues in the F1 and F2 polypeptides were conserved among all F proteins. However, BRS virus F protein had only three potential N-linked carbohydrate acceptor sites in comparison to four or five for the HRS viruses. A difference in the extent of glycosylation between the BRS and HRS virus F2 polypeptides was shown to be responsible for differences observed in the electrophoretic mobility of these proteins. A cDNA containing the complete open reading frame of the BRS virus F mRNA was inserted into the thymidine kinase gene of vaccinia virus and following homologous recombination, a recombinant virus containing the BRS virus F gene was isolated. The BRS virus F protein was expressed in recombinant virus infected cells as demonstrated by immunoprecipitation and was transported to and expressed on the surface of infected cells as shown by indirect immunofluorescence.