Ovine Respiratory Syncytial Virus Research Articles

Clinical and molecular identification study of ovine respiratory syncytial virus in sheep, Iraq

Objectives: The goal of this study is to use polymerase chain reaction (RT-qPCR) and sequencing to assess ovine respiratory syncytial virus (ORSV) infection in Awassi sheep in the middle and south of Iraq (Najaf, Babylon, Wassit, and Dhi-Qar), followed by phylogenetic analyses. Methods: From December 2020 to November 2021, a total of 205 nasal swab samples were taken from sheep of various ages and health situations in Najaf, Babylon, Wassit, and Dhi-Qar, Iraq. During the study, the clinical examinations, and vital clinical signs of disease were recorded. RT-qPCR was used to detect nonstructural protein one (NS1) of ORSV, while nested RT-PCR was used to amplify the NS1 protein gene for sequencing, ORSV's NS1 gene (306-bp). PCR products were sequenced, and MEGA 6 analytic software was utilized. The quantitative findings were given as the mean standard error of the mean, the chi-square test (X2), and T test were used to determine the significance of the observed variations. Results: A total of 205 nasal swabs were taken from Awassi sheep and ORSV had a molecular prevalence of 23.41 % in sheep.

Prevalence of Respiratory Syncytial Virus Infection in Sheep at Babylon Governorate, Iraq

The current study aimed to determine the occurrence of the ovine respiratory syncytial virus (ORSV) infection in sheep employing sandwich enzyme-linked immunosorbent assay (sandwich-ELISA) and look into some disease risk factors. We collected blood samples (n=460) from sheep of various ages and sexes in Babylon Governorate/Iraq. The epidemiological data obtained during the interview with the owners of the animals. The prevalence of ORSV infection at Babylon Governorate was 58.91% based on sandwich-ELISA. The risk factors related to ORSV infection included: sheep with 7months-2years old, imported animals, outdoor feeding, large herd size, in northern parts of the Governorate and winter and spring seasons. There was a significant elevation in RBC, Hb, PCV and a significant reduction in WBC and lymphocytes. There was a significant elevation in AST, CK, LDH, TP, globulin, and total bilirubin. At the same time, there was a significant reduction in albumin, creatinine, and glucose in the infected animals with ORSV. In conclusion, ORSV is prevalent in sheep at Babylon Governorate, with a higher prevalence of the disease in the northern parts of the Governorate. The present report is the first for detecting ORSV antigens in sheep at Babylon Governorate/Iraq.

Characterization of an experimental vaccine for bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins αVβ1, αVβ3, and α3β1. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

Bovine respiratory syncytial virus infection: immunopathogenic mechanisms

Bovine respiratory syncytial virus (BRSV) causes severe respiratory disease in young cattle. Much like the human respiratory syncytial virus, BRSV induces immunomodulation in the infected host, favoring a Th2 response. Several groups have demonstrated IgE responses to BRSV proteins during infection and particularly in response to vaccination with formalin-inactivated vaccine in the field and experimentally. Newer vaccine modalities that favor a shift to Th1 cytokine production have provided promising results. Infection with BRSV is a major contributor to the multi-pathogen disease, bovine respiratory disease complex. This review stresses the unique immunomodulatory aspects of BRSV infection, vaccination and its interaction with the host's immune system.

Immunization strategies for the prevention of pneumovirus infections

Pneumoviruses, which are viruses of the family Paramyxoviridae, subfamily Pneumovirinae, are pathogens that infect the respiratory tract of their host species. The human pneumovirus pathogen, human respiratory syncytial virus (RSV), has counterparts that infect cows (bovine RSV), sheep (ovine RSV), goats (caprine RSV) and rodents (pneumonia virus of mice). Each pneumovirus is host specific and results in a spectrum of disease, ranging from mild upper-respiratory illness to severe bronchiolitis and pneumonia with significant morbidity and mortality. Given the public health burden caused by human RSV and the concomitant agricultural impact of bovine RSV, these two viruses are considered as prime targets for the development of safe and effective vaccines. In this review, we describe the strategies used to develop vaccines against human and bovine RSV and introduce the pneumonia virus mouse model as a novel and invaluable tool for preclinical studies and new vaccine strategies.

The ovine respiratory syncytial virus F gene sequence and its diagnostic application.

Ruminant respiratory syncytial viruses (RSVs) are classified into 2 subgroups, ovine RSV and bovine RSV. Although ovine RSV infects cattle, its contribution to bovine respiratory tract disease has not been established, which is an important issue for vaccine development in cattle. Diagnosis by virus isolation or serology has low or variable sensitivity and/or specificity and polymerase chain reaction (PCR) has been recommended as a rapid and sensitive technique for RSV detection. A simple procedure has been developed to detect and identify bovine and ovine RSVs. First, the nucleotide sequence of the ovine RSV fusion (F) gene was determined and compared with representative strains of bovine RSV and human RSV subgroups A and B. The ovine RSV F gene has 85 and 72-73% nucleotide identity with those of bovine RSV and human RSV, respectively. The predicted amino acid sequence of the ovine RSV F gene has 94 and 83-84% amino acid identity with those of bovine RSV and human RSV, respectively. Then PCR primers targeting a specific F gene fragment of bovine and ovine RSV were designed. The primers represented bases 85-103 and the complementary sequence to bases 510-493 of the ovine RSV F gene. A similar PCR product (426 bp) was obtained on agarose gel electrophoresis from bovine RSV and from ovine RSV. The products, however, were unique to the parent virus and could be distinguished by EcoRI or MspI restriction endonuclease cleavage. EcoRI cleaved the ovine product into 2 bands (285 and 141 bp) but failed to affect the bovine RSV PCR product. However, MspI cleaved the bovine product into 2 bands (229 and 197 bp) but had no effect on the ovine product. Also, this assay did not amplify any PCR product with human RSV. The reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction enzyme digestion is a useful and practical approach for detection and differentiation of ruminant respiratory syncytial viruses.

Evolution of bovine respiratory syncytial virus.

Until now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an alpha helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines.

Rescue of a bovine respiratory syncytial virus genomic RNA analog by bovine, human and ovine respiratory syncytial viruses confirms the "functional integrity" and "cross-recognition" of BRSV cis-acting elements by HRSV and ORSV.

The nucleotide sequences of the 3' leader and 5' trailer regions were determined for genomic RNA of bovine respiratory syncytial virus (BRSV) strain A-51908. The leader and trailer sequences are '45' and '161' nucleotides in length, respectively. The functionality of BRSV leader and trailer sequences and their recognition by HRSV and ovine respiratory syncytial virus (ORSV) proteins were examined with a in vitro transcribed BRSV genomic RNA analog carrying the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of BRSV transcription signals. Upon transfection into BRSV, HRSV or ORSV infected cells, the BRSV minireplicons were 'rescued' such that the reporter gene was expressed, the minigenome was replicated and packaged into micrococcal nuclease resistant-infectious minireplicons. The passage of infectious minireplicons could be blocked by a polyclonal BRSV neutralizing antiserum. Bovine parainfluenza virus-3, a heterologous paramyxovirus was inactive in rescuing BRSV genomic RNA analog. Mutational substitution of the G residue at position 4 of leader sequence in the BRSV genomic RNA analog, with an A or U residue inhibited its transcription and replication, while replacement with a C residue had no significant effect on rescue. These results show that the cis-acting elements of BRSV are functional and are also recognized by the proteins of HRSV and ORSV. The helper virus complemented rescue system developed here will be useful for characterizing the cis-acting elements of BRSV.

Analysis of ruminant respiratory syncytial virus isolates by RNAse protection of the G glycoprotein transcripts.

Two different respiratory syncytial virus (RSV) radiolabeled probes were used to characterize the genetic heterogeneity of 25 ruminant RSV isolates by the ribonuclease protection assay. A 32P-radiolabeled antisense RNA probe was transcribed from cloned ovine and bovine RSV G glycoprotein genes and then hybridized with total RNA isolated from infected cells with various ruminant RSV isolates. The results of this study, along with previously published nucleotide sequence data of the ovine RSV G glycoprotein gene, suggest the presence of at least 2 ruminant RSV subgroups. One subgroup is represented by RSV isolated from respiratory disease outbreaks from calves and goats, and the other is represented by RSV isolated from sheep.

Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G

Peptides deduced from the central hydrophobic region (residues 158–189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

Sequence of the nucleocapsid protein gene of subgroup A and B avian pneumoviruses

The nucleocapsid protein (N) gene of two subgroup A and one subgroup B strains of avian pneumovirus has been cloned and sequenced. The gene of all three isolates comprised 1197 nucleotides (nt), which formed a single major open reading frame, potentially encoding a protein of 391 amino acid residues. The N gene of the two subgroup A isolates differed by only 1 nt but differed by 282 (24%) nt and 35 (11%) amino acids from the B isolate. The predicted protein was identical in length to that of human, bovine and ovine respiratory syncytial viruses, the amino acid identity being approximately 41% overall but with some regions of identity >90%.

Nucleotide sequence analysis of the non-structural NS1 (1C) and NS2 (1B) protein genes of bovine respiratory syncytial virus

The nucleotide and deduced amino acid sequences of the non-structural (NS) protein genes of bovine respiratory syncytial virus (BRSV) strain A51908 were determined. The NS1 and NS2 genes were 524 and 489 nucleotides long with single open reading frames, encoding polypeptides of 136 and 124 amino acids, respectively. Comparison of the NS1 gene of BRSV with the corresponding sequences of ovine respiratory syncytial virus (ORSV) and human respiratory syncytial virus (HRSV) subgroups A and B revealed 82, 67 and 65% identity at the nucleotide level, and 89, 69 and 68% identity at the amino acid level, respectively. The nucleotide identity of the NS2 gene of BRSV to those of ORSV and HRSV subgroups A and B was 80, 69 and 67% and the predicted amino acid identity was 87, 84 and 83%, respectively. The intergenic sequences of the NS1-NS2 and NS2-N gene junctions of BRSV were determined and found to be 13 nucleotides shorter and 29 nucleotides longer, respectively, than the corresponding regions of HRSV.

Molecular cloning and sequence analysis of the phosphoprotein, nucleocapsid protein, matrix protein and 22K (M2) protein of the ovine respiratory syncytial virus

Respiratory syncytial viruses (RSVs) cause serious respiratory tract disease in infants and children worldwide and similar disease in calves. Strains have been isolated from other ruminant animals such as sheep and goats, but these viruses have not been characterized at the molecular level. In this study, we report the cloning and sequencing of four structural genes coding for the phosphoprotein, nucleocapsid (N) protein, matrix (M) protein and 22K protein of an ovine RSV strain. Comparisons of these sequences with those of bovine and human RSV show that the M and N proteins are the most conserved between ruminant RSV strains and the N protein is the most conserved protein between human and ruminant RSV strains. The attachment G glycoprotein and the small hydrophobic protein are the most divergent proteins among human and ruminant RSV subgroups.

Nucleotide and predicted amino acid sequence analysis of the ovine respiratory syncytial virus non-structural 1C and 1B genes and the small hydrophobic protein gene.

Respiratory syncytial virus (RSV) infects humans and cattle causing serious respiratory tract disease in both. The genome of the human and bovine RSV (HRSV and BRSV) codes for two non-structural and eight structural proteins. RSV has also been isolated from naturally infected sheep, but the genome of the ovine RSV (ORSV) has not been characterized and nor has the virus host range been identified. Here we report on the cloning and sequencing of the two non-structural 1C and 1B genes and of the small hydrophobic (SH) protein gene of the ORSV. The nucleotide identity of the ORSV 1C gene to those of subgroups A and B of HRSV was 70% and 65% respectively whereas the predicted amino acid identity was 68% and 69% respectively. The ORSV 1B gene had a 70% and 72% nucleotide identity with that of subgroups A and B of the HRSV respectively, and 79% predicted amino acid identity with both HRSV subgroups. The identity level of these two ORSV 1C and 1B genes to those of the BRSV could not be determined since these two BRSV genes have not been sequenced. The SH protein of RSV is a structural protein expressed on the surface of infected cells. In common with HRSV and BRSV subgroups, the ORSV SH had the three proposed domains with the C-terminal domain least conserved among the viruses. In the latter domain, the ORSV SH gene had a nucleotide identity of 68 to 69% and 47 to 51% with those of the BRSV and HRSV, respectively, and a predicted amino acid identity of 56% and 33 to 47% with those of BRSV and HRSV, respectively. Defining the genes and their products should help determine which genes and gene products are most suitable for use as diagnostic tools or as vaccine candidates.

Nucleotide Sequence Analysis of the Ovine Respiratory Syncytial Virus G Glycoprotein Gene

Respiratory syncytial virus (RSV) infects humans and animals including ruminants. Among the 10 genes coded for in the viral genome, the putative attachment glycoprotein G gene has been the most variable among strains. Human RSV have been divided to two subgroups based on immunological and base sequence data on the attachment glycoprotein G and its gene, respectively. It has been suggested that similar antigenic diversity also exists among bovine RSV (BRSV) isolates. In this study, we report on the cloning and sequencing of the G glycoprotein from an ovine RSV (ORSV) strain originally isolated from a naturally infected sheep with rhinitis. This ORSV G glycoprotein gene had greater identity to the BRSV G gene than to the human RSV G gene. ORSV G gene and its encoded protein shared 70 and 62% nucleotide and amino acid identity to the equivalent gene and encoded protein, respectively, of BRSV but, in contrast, only 50-55% and 21-29% identity, respectively, to equivalent sequences of the HRSV strains. The relationship of the ORSV to other RSV subgroups and the possibility that ORSV could be a subgroup of the ruminant RSV is discussed.

Characteristic differences in reverse transcription-polymerase chain reaction products of ovine, bovine, and human respiratory syncytial viruses.

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of approximately 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were no amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.

Subacute effects of respiratory syncytial virus infection on lung function in lambs

We examined the effects of ovine respiratory syncytial virus (RSV) infection on lung mechanics, lung histology, and airway reactivity in lambs. Nine lambs were inoculated with ovine RSV and seven control lambs with normal saline or viral media. Serum neutralization titers were obtained prior to and 3 weeks post-inoculation (PI). Open lung biopsies were performed 1 and 3 weeks PI. Lung mechanics including dynamic compliance (Cdyn), resistance of the lung (RL), and functional residual capacity (FRC) were measured 2 and 6 weeks PI using a plethysmograph. Airway reactivity to aerosolized carbachol, citric acid, and histamine was determined 2 and 6 weeks PI. Most RSV and control lambs were asymptomatic after inoculation. Control lambs had significantly greater average daily weight gain by the third week after inoculation. Seven RSV lambs tested had a fourfold or greater rise in serum neutralization titers, while two control lambs had a fourfold increase. At 2 weeks PI, RSV lambs had significantly lower FRC and higher RL. At 6 weeks RL remained significantly elevated in the RSV lambs. Airway reactivity was not increased in the RSV group. This animal model is useful for studying the effects of RSV infection on lung growth and lung function over time.