Antigenic and molecular characterization of isolates of the Italy 02 infectious bronchitis virus genotype.

As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (dS/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype.     

Rapid detection and identification of avian infectious bronchitis virus.

A rapid and sensitive method for the detection and unambiguous typing of infectious bronchitis virus (IBV) is described. RNA was isolated from IBV-infected allantoic fluid and was transcribed into cDNA. This cDNA was amplified by the polymerase chain reaction. The polymerase chain reaction products were subsequently analyzed on an agarose gel. The presence of IBV-specific RNA in the allantoic fluid then allowed the amplification of a 438-bp DNA fragment from the nucleocapsid (N) gene. For the typing of IBV isolates, we used amplified double-stranded DNA as a template in a sequencing reaction. We report 360 bases of the N gene of 18 IBV isolates. The sequence of the N gene was different between serologically indistinguishable IBV strains and may be a valuable tool in epidemiologic studies. A phylogenetic tree that was based on the sequences obtained did not agree with trees that were based on other parts of the sequence, illustrating the high frequency of recombination between IBV strains.     

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.     

The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983

Between 1981 and 1983 10 isolations of infectious bronchitis (IB) virus were made from broiler chickens with respiratory infection and five from breeder chickens showing aberrant egg production. One isolate was serologically similar to an IB virus isolated in England in 1976; the remainder were related to three of the four serotypes of IB virus first isolated in The Netherlands (Davelaar et al., 1981). Neutralising antibody to each of the four Dutch serotypes of IB virus was widespread in the sera of commercial chickens in England, Wales and Scotland. Their possible role as causal agents of disease in the flocks from which they were isolated is discussed.     

The isolation and characterisation of six avian infectious bronchitis viruses isolated in Morocco

The first isolation and characterisation of infectious bronchitis (IB) viruses from poultry flocks in Morocco are reported. Five isolates designated D, E, F, H and M were related serologically to the Massachusetts serotype, while the sixth, isolate G, was found to be different from any previously reported serotype of IB virus. Neutralising antibodies to isolate G have been detected in sera collected from commercial flocks in Britain, although the virus has not been isolated. While all six isolates caused respiratory disease typical of IB in experimentally infected 3-week-old specified pathogen-free (SPF) chickens, isolate G was unusual in that it could be isolated from several parts of the alimentary tract for up to 21 days post inoculation, and from the duodenum up to 28 days. H120 vaccines protected chicks challenged with isolates E and F but not isolate G.     

Detection of avian leukosis virus subgroup J in chicken flocks from Malaysia and their molecular characterization.

Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.     

Antigenic variation in strains of avian infectious bronchitis virus

Summary Fifteen british field strains of IBV were compared using cross serum neutralization tests in embryonated eggs with seven standard reference strains of IBV. While the British field strains were considered to form a relatively homogeneous group considerable antigenic variation did occur. It was considered that it was not feasible at this time to describe accurately a serotype classification for IBV, similar to that described for other virus groups.     

Immune responses to structural proteins of avian infectious bronchitis virus.

Chickens were vaccinated with live and inactivated infectious bronchitis virus (IBV), and antibody responses to the individual structural proteins, S1, S2, M and N, followed by ELISA and western blotting. All four structural proteins elicited an antibody response in chicks vaccinated with either live or inactivated IBV. The S1, S2 and N proteins elicited similar titres of antibodies following vaccination with live IBV, whereas the M glycoprotein elicited significantly lower titres. Time of appearance and the course of development of the S1, S2 and N ELISA antibodies were similar, being first detected 2 weeks after vaccination and coincided with appearance of virus neutralizing antibodies. The M antibodies were first detected 4 weeks after vaccination. S1, S2, and N antibody titres were significantly higher in chicks vaccinated at 14 days of age than in chicks vaccinated at either 1 or 7 days of age, and reached maximum levels 4 weeks after the second vaccination. The S1, S2 and N proteins induced cross-reactive antibodies, whereas the M glycoprotein induced antibodies of limited cross-reactivity. Titres of cross-reactive N antibodies were higher than titres of cross-reactive S1 and S2 antibodies, which were similar. Epitopes on the N and S2 proteins that gave rise to cross-reactive antibodies showed the same degree of conservation, whereas the cross-reactive S1 epitopes were marginally less conserved. Vaccination with inactivated virus induced significantly lower antibody titres and at least three vaccinations were necessary for induction of S1, S2, N and M antibodies in all chicks. The S2 glycoprotein was the most immunogenic structural protein following vaccination with inactivated virus. All four proteins induced cell-mediated immune responses in chicks vaccinated with live IBV as determined by a delayed type hypersensitivity response.     

Recombinational histories of avian infectious bronchitis virus and turkey coronavirus

Phylogenetic analysis of complete genomes of the avian coronaviruses avian infectious bronchitis (AIBV) and turkey coronavirus (TCoV) supported the hypothesis that numerous recombination events have occurred between these viruses. Although the two groups of viruses differed markedly in the sequence of the spike protein, the gene (S) encoding this protein showed no evidence of positive selection or of an elevated mutation rate. Rather, the data suggested that recombination events have homogenized the portions of the genome other than the S gene between the two groups of viruses, while continuing to maintain the two distinct, anciently diverged versions of the S gene. The latter hypothesis was supported by a phylogeny of S proteins from representative coronaviruses, in which S proteins of AIBV and TCoV fell in the same clade.     

Co-infections of chickens with avian influenza virus H9N2 and Moroccan Italy 02 infectious bronchitis virus: effect on pathogenesis and protection conferred by different vaccination programmes

ABSTRACT

Since the emergence of low pathogenic avian influenza (LPAI) H9N2 viruses in Morocco in 2016, severe respiratory problems have been encountered in the field. Infectious bronchitis virus (IBV) is often detected together with H9N2, suggesting disease exacerbation in cases of co-infections. This hypothesis was therefore tested and confirmed in laboratory conditions using specific-pathogen-free chickens. Most common field vaccine programmes were then tested to compare their efficacies against these two co-infecting agents. IBV γCoV/chicken/Morocco/I38/2014 (Mor-IT02) and LPAI virus A/chicken/Morocco/SF1/2016 (Mor-H9N2) were thus inoculated to commercial chickens. We showed that vaccination with two heterologous IBV vaccines (H120 at day one and 4/91 at day 14 of age) reduced the severity of clinical signs as well as macroscopic lesions after simultaneous experimental challenge. In addition, LPAI H9N2 vaccination was more efficient at day 7 than at day 1 in limiting disease post simultaneous challenge.

RESEARCH HIGHLIGHTS

• Simultaneous challenge with IBV and AIV H9N2 induced higher pathogenicity in SPF birds than inoculation with IBV or AIV H9N2 alone.

• Recommended vaccination programme in commercial broilers to counter Mor-IT02 IBV and LPAIV H9N2 simultaneous infections: IB live vaccine H120 (d1), AIV H9N2 inactivated vaccine (d7), IB live vaccine 4-91 (d14).

     

Phylogenetic analysis of avian infectious bronchitis virus strains isolated in Japan

Summary To define the origin and evolution of recent avian infectious bronchitis virus (IBV) in Japan, a genetic analysis was performed. By phylogenetic analysis based on the S1 gene including the sequence of the hypervariable regions, IBV isolates in Japan were classified into five genetic groups, which included two already-known groups (Mass and Gray). Among them, three major genetic groups were associated with the recent outbreaks of IB in Japan. One group is indigenous to Japan and could not be placed within the known existing groups in other countries. The remaining two groups, which have emerged recently, are related to isolates in China and Taiwan.     

Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells

Summary Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles.Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles.With 10 Figures     

A clearance test to assess protection in chickens vaccinated against avian infectious bronchitis virus

A clearance test is described that is designed as a model for the quantitative assessment of protection in chickens vaccinated against avian infectious bronchitis virus (IBV). This is based on the ability of a vaccinating strain to have induced within 3 weeks an immunity sufficient to inhibit the replication of a challenge strain in the upper respiratory tract by the 4th day subsequent to challenge. A median protective dose (PD(50)) was determined for each of 4 vaccinating strains (H120, H52, D41 and Doorn 274), and chickens were vaccinated with 20 PD(50) of one of these intranasally. Challenge strains were administered intratracheally 3 weeks later and assays of residual challenge virus in the trachea as well as the kidneys and oviducts were made after a further 4 days. Observations were also made on tracheal ciliary activity and histopathological changes. The H120 and H52 strains were efficient in clearing antigenically related challenge strains, but the H120 strain was less so in the case of the Doom 274 strain and D41 strain. The Doom 274 strain was effective against the heterologous strains examined with the exception of the T strain. The D41 strain was generally effective in protecting against all the strains selected for challenge, and its candidature as a broad-spectrum vaccine strain is endorsed accordingly. A small number (6%) of unvaccinated chickens had virus in low titre in the kidneys or oviducts after challenge with some strains. Virus was not detected in these organs of vaccinated birds.