Subpopulations in aMPV vaccines are unlikely to be the only cause of reversion to virulence.

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

Reversion to virulence of a subtype B avian metapneumovirus vaccine: is it time for regulators to require availability of vaccine progenitors?

Empirically derived live avian metapneumovirus (AMPV) vaccines developed during the late 80s and early 90s have generally performed well in controlling turkey rhinotracheitis. Nonetheless, unstable attenuation was previously demonstrated in an AMPV subtype A vaccine. Until now this had not been investigated in subtype B vaccines due to lack of any similar availability of a vaccine progenitor or its sequence. The publication of the full genome sequence for the VCO3 vaccine progenitor facilitated a conclusive investigation of two AMPVs isolated from poults on a farm which had been vaccinated with VCO3 derived vaccine. Full genome sequencing of the isolates and their comparison to sequences of the vaccine and its progenitor, confirmed their vaccine origin. After determining the absence of extraneous infectious agents, one of these virus isolates was inoculated into 1-day-old turkeys in disease secure isolators and shown to cause disease with a severity similar to that caused by virulent field virus. This suggests that instability in live AMPV vaccines may be generalized and highlights the need for availability of vaccine progenitor sequences for the field assessment of all live viral vaccines.

High inter-species and low intra-species variation in 16S-23S rDNA spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool.

In order to investigate its value for phylogenetic analysis, species characterisation and diagnosis, the 16S-23S rDNA intergenic spacer regions (ISRs) of the type strain of 23 avian Mycoplasma species were amplified and the sequences determined. Also sequenced were the reference strains of Mycoplasma iowae serotypes J, K, N, Q and R and a number of field strains of Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma meleagridis and M. iowae. The ISRs demonstrated a high level of size variation (178-2488bp) between species and did not include tRNA genes. Phylogenetic analysis performed using the information conflicted with that based on the 16S rDNA and was therefore not helpful for phylogenetic studies. However, the ISR did appear to be of value for determining species since there was high inter-species variation between all 23 avian Mycoplasma species, and in addition there was low intra-species variation, at least in the four pathogenic species. It could also be very useful as additional information in the description of a new species and as a target for species-specific PCRs.

Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction.

A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

Avian pneumovirus infection in broiler chicks inoculated with Escherichia coli at different time intervals.

The effects of dual infection of 1-day-old broiler chicks with a chicken isolate of avian pneumovirus (APV) and a pool of pathogenic Escherichia coli strains were studied by supraconjunctival application of the bacteria simultaneously with the virus, or at 4, 7 or 11 days afterwards. When the agents were given together, the clinical disease was significantly more severe than that caused by the virus alone, but when the bacterium was given later the signs were less severe. None of the infections resulted in swollen head syndrome by 32 days. All mixed infections caused moderate to severe congestion in the turbinates, when birds were examined at 32 days of age, at which time no such lesions were present in birds having been infected with APV alone. E. coli was isolated from almost 100% of birds with mixed infections, while rates of those given only E. coli isolation varied between 56 and 67%. Furthermore, E. coli colony counts were consistently higher from mixed infection groups. Virus persistence in the choanal cleft was slightly prolonged in birds with the simultaneous mixed infection. Although the pool of E. coli included O2, O78 and O18 serotypes, only those of the O2 serotype and a small number of untypable strains were re-isolated from selected mixed and single E. coli-infected groups. Mixed APV and E. coli infection did not affect APV enzyme-linked immunosorbent assay antibody titres at 21 or 32 days. Thus, experimental infection of broiler chicks with APV and E. coli, simultaneously or at intervals afterwards, demonstrated a synergistic effect between the two agents, but none of the infection protocols caused swollen head syndrome.

Presence of avian pneumovirus type A in continental Europe during the 1980s.

Three isolates of avian pneumovirus (APV) were isolated in Germany during 1987 and 1988 from turkeys with clinical signs of turkey rhinotracheitis and one was isolated during 1990 from a broiler breeder flock with typical signs of swollen head syndrome. The isolates were typed using type-specific reverse transcriptase-polymerase chain reactions. The three isolates from turkeys were identified as type A, while the isolate from the broiler breeders was type B. During the late 1980s no APV live-virus vaccine was used in poultry flocks in Germany, which is indicative of the presence of both types at that time. Previous isolates detected from elsewhere in Europe during the 1980s had been only of type B.

Longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions.

In longitudinal studies, 13 flocks were swabbed twice each week for the life of the flock (up to 46 days). The swabs were analyzed by type-specific reverse transcriptase polymerase chain reactions. Massachusetts type vaccinal infectious bronchitis virus (IBVs), applied at the hatchery, were usually maximal during the first week, as expected and, notably, remained detectable for 3 to 4 weeks, occasionally longer. IBV of the 793/B type (also known as 4/91 and CR88) was detected in 11/13 flocks (85%). The time of first detection of 793/B varied over several weeks and was sometimes within the first week in low amounts, which increased gradually. In some flocks, detection of 793/B remained intermittent. IBV types D274 and D1466 were each detected once, in the same flock, for short periods, in low amounts, and in the presence of higher amounts of 793/B. In swabs from a further 30 broiler flocks, plus those already mentioned, there was an incidence for 793/B, D274 and D1466 of 79, 10 and 2%, respectively. Avian pneumovirus (APV) (avian or turkey rhinotracheitis virus) vaccines, applied at the hatchery or later, were either not detected or were detected only after a delay of 1 to 3 weeks. In five flocks that received no APV-A vaccine and two flocks that received only APV type A vaccine, field infection by APV type B was detected but only during the last week or so of life. In six flocks that had received an APV-B vaccine, no field APV-B, differentiated from vaccinal APV-B by restriction enzyme analysis, was detected. In swabs from 30 other flocks, the great majority of which had not been vaccinated against APV, the incidence of APV types B and A was 50 and 3%, respectively. The results show (a) that vaccinal IBV can be detected for several weeks, (b) the dominance of the IBV 793/B type and

Does IBV change slowly despite the capacity of the spike protein to vary greatly?

We have sequenced that part of the spike protein (S) gene which encodes the aminoterminal and most variable quarter (hypervariable region, HVR) of the S1 subunit of 28 isolates of the 793/B (also known as CR88 and 4/91) serotype of infectious bronchitis virus (IBV) and the whole of S1 for nine of them. The isolates were from France and Britain between the years 1985 (first isolation) and 1996. The maximum nucleotide and amino acid differences between the first isolate and the others were 4.1% and 7.6%, respectively, for the whole of S1 and 7.1% and 14.6%, respectively, in the HVR. Analysis within clearly recognisable subgroups suggested that even in the HVR the nucleotide mutation rate was only 0.3 to 0.6% per year. However, there was no evidence that mutations had become fixed in a progressive manner; this serotype did not appear to be evolving. Strains isolated several years apart could be more similar than those isolated in a given year. It is likely that the amino acid changes are largely at positions where amino acid differences are tolerated rather than as a consequence of immune pressure. Reasons for this conclusion are discussed.

The ectodomains but not the transmembrane domains of the fusion proteins of subtypes A and B avian pneumovirus are conserved to a similar extent as those of human respiratory syncytial virus.

The fusion glycoprotein (F(B)) gene of five strains of the B subtype of avian pneumovirus (APV; turkey rhinotracheitis virus) has been sequenced. The length of the F(B) protein was 538 amino acids, identical to that of the F protein of subtype A virus, with which it had 74% and 83% overall nucleotide and deduced amino acid identities, respectively. The F(B) and F(A) ectodomains had 90% amino acid identity, very similar to the 91% identity between the ectodomains of the F proteins of subtype A and B human respiratory syncytial virus (HRSV). As with HRSV, the F2 polypeptide was less conserved (83% identity) than F1 (94%). In contrast to the ectodomain, the transmembrane and cytoplasmic domains of the two APV subtypes were much less conserved (30% and 48% identity, respectively) than those of HRSV (92% and 87%, respectively). Comparisons within all the genera of the Paramyxoviridae (Pneumovirus, Morbillivirus, Paramyxovirus and Rubullavirus) show that low amino acid identity between F protein transmembrane domains is a feature of different species of virus rather than of strain differences. This may indicate that the two subtypes of APV have evolved in different geographical regions and/or different avian species. This is the first report of an F gene sequence from a subtype B APV.

Failure of maternal antibodies to protect young turkey poults against challenge with turkey rhinotracheitis virus.

Groups of turkey poults with high levels of maternal antibodies (MA+) to turkey rhinotracheitis virus (TRTV) were challenged with virulent TRTV at 1, 5, and 10 days of age. A maternal antibody-free group (MA-) was also challenged at 1 day of age. Before each challenge, levels of maternal antibodies to TRTV were measured by enzyme-linked immunosorbent assay. Clinical signs were scored for each group. Unchallenged poults showed no signs. Respiratory signs in poults infected at 10 days of age resembled those seen in MA- birds infected at 1 day of age but both were more severe than in MA+ birds infected at 1 day of age, when the maternal antibodies were highest. However, overall, the presence of high levels of maternal antibodies did not prevent the development of clinical disease.

Demonstration of a virulent subpopulation in a prototype live attenuated turkey rhinotracheitis vaccine.

A prototype live attenuated turkey rhinotracheitis (TRT) vaccine, which was known to cause occasional disease in young poults following multiple back passage, was tested for the presence of a virulent subpopulation by a novel combined in vitro and in vivo screening technique. When vaccine was inoculated at high titres into chick embryo tracheal organ cultures, 17% of aliquots were found to cause ciliostasis, and when these aliquots, in turn, were inoculated into 1-day-old poults, approximately one-quarter caused clinical disease. Removal of the subpopulation by plaque purification led to viruses which had reduced tendency to revert to virulence but remained protective. The technique proved valuable in identifying virulent subpopulations in specific prototype TRT vaccines. The principle may have more general application.

A simple method for immunofluorescence staining of tracheal organ cultures for the rapid identification of infectious bronchitis virus.

A method is described for the simple application of immunofluorescence (IF) staining for the identification of infectious bronchitis virus (IBV) in tracheal organ cultures (TOC). It involves the application of antiserum and fluorescent antiglobulin to unfixed TOC. In TOC inoculated with high titres of virus, specific fluorescence was observed in less than 24 h. IF staining almost always occurred before ciliostasis. Following experimental infection of chicks with IBV, virus could sometimes be detected in material from tracheal or cloacal swabs within 24 h of inoculation of TOC.

Effect of cyclophosphamide immunosuppression on the immunity of turkeys to viral rhinotracheitis.

Turkey poults, free of antibodies to turkey rhinotracheitis (TRT) virus were treated with cyclophosphamide on days 1, 2 and 3 after hatching and vaccinated by eyedrop when 10 days old with a Vero cell-attenuated preparation of TRT virus. No ELISA antibodies to TRT virus developed in the sera of these poults but they were as resistant to virulent virus challenge 21 days later as vaccinated groups which were not cyclophosphamide-treated but produced humoral antibodies. Following challenge with virulent virus at 31 days old cyclophosphamide-treated unvaccinated poults developed a more severe clinical response than untreated birds and had higher virus titres in tracheal swabs. The findings show that the respiratory tract of turkeys may be resistant to TRT despite the absence of ELISA antibodies in the serum.

Exacerbation of Mycoplasma gallisepticum infection in turkeys by rhinotracheitis virus.

Groups of 1-day-old turkey poults from a parent flock free of antibodies to turkey rhinotracheitis virus (TRTV) and the pathogenic mycoplasmas, were infected by eyedrop with virulent TRTV, with Mycoplasma gallisepticum (Mg) or with both agents together. Dual infection resulted in increased morbidity compared with those groups given single infections. The presence of the Mg in the dual infection had no apparent effect on the pathogenesis of the virus, but the virus caused the Mycoplasma to be more invasive. Mg infection caused a transient depression in TRTV ELISA antibody titres at 29 days post-inoculation. At 14 days post-infection Mg haemagglutination inhibition (HI) and rapid serum agglutination (RSA) titres were higher (P <0.01) in the mixed infection group compared with those infected with Mg alone, but there was no significant difference between ELISA antibody titres of these two groups.