A comparison of AMPV subtypes A and B full genomes, gene transcripts and proteins led to reverse-genetics systems rescuing both subtypes.

Avian metapneumovirus (AMPV) infection of poultry causes serious disease in most countries and subtype A reverse-genetic (RG) systems have allowed a generation of viruses of known sequence, and proved useful in developments towards better control by live vaccines. While subtype B viruses are more prevalent, bacterial cloning issues made subtype B RG systems difficult to establish. A molecular comparison of subtype A and B viruses was undertaken to assess whether subtype A RG components could be partially or fully substituted. AMPV subtype A and B gene-end sequences leading to polyadenylation are, to our knowledge, reported for the first time, as well as several leader and trailer sequences. After comparing these alongside previously reported gene starts and protein sequences, it was concluded that subtype B genome copies would be most likely rescued by a subtype A support system, and this assertion was supported when individual subtype A components were successfully substituted. Application of an advanced cloning plasmid permitted eventual completion of a fully subtype B RG system, and proved that all subtype-specific components could be freely exchanged between A and B systems.

Rapid detection of subtype B avian metapneumoviruses using RT-PCR restriction endonuclease digestion indicates field circulation of vaccine-derived viruses in older turkeys.

Live vaccines predominantly control avian metapneumovirus (aMPV) infection in poultry flocks, but vaccine virus can be found for extended periods after application. The most frequently used aMPV vaccine in Italy, VCO3 subtype B, was shown to contain a unique Tru9I restriction endonuclease site within the amplicons produced by a commonly used aMPV diagnostic reverse transcriptase (RT)-nested polymerase chain reaction (PCR). Analysis of European and database logged subtype B aMPV sequences confirmed that the sequence occurred only in the VC03 vaccine. A subsequent RT-PCR restriction endonuclease study of field samples, collected from turkeys between 2007 and 2012, detected subtype B vaccine-derived strains in 12 of 90 samples tested that were collected from birds under 12 weeks of age.

A single polymerase (L) mutation in avian metapneumovirus increased virulence and partially maintained virus viability at an elevated temperature.

Previously, a virulent avian metapneumovirus, farm isolate Italy 309/04, was shown to have been derived from a live vaccine. Virulence due to the five nucleotide mutations associated with the reversion to virulence was investigated by their addition to the genome of the vaccine strain using reverse genetics. Virulence of these recombinant viruses was determined by infection of 1-day-old turkeys. Disease levels resulting from the combined two matrix mutations was indistinguishable from that produced by the recombinant vaccine, whereas the combined three L gene mutations increased disease to a level (P<0.0001) that was indistinguishable from that caused by the revertant Italy 309/04 virus. Testing of the L mutations individually showed that two mutations did not increase virulence, while the third mutation, corresponding to an asparagine to aspartic acid substitution, produced virulence indistinguishable from that caused by Italy 309/04. In contrast to the vaccine, the virulent mutant also showed increased viability at temperatures typical of turkey core tissues. The notion that increased viral virulence resulted from enhanced ability to replicate in tissues away from the cool respiratory tract, cannot be discounted.

A turkey rhinotracheitis outbreak caused by the environmental spread of a vaccine-derived avian metapneumovirus.

Avian metapneumovirus (aMPV) subtype A was isolated from 7-week-old turkeys showing respiratory disease typical of turkey rhinotracheitis. Comparison of the virus sequence with previously determined vaccine marker sequences showed that the virulent virus had originated from a licensed live subtype A aMPV vaccine. The vaccine had neither been in use on the farm within a period of at least 6 months nor had it been used on farms within a distance of approximately 5 km. Isolation of the virus and exposure to naive turkeys caused disease typical of a virulent aMPV field strain. The study shows that disease was caused by exposure to aMPV vaccine-derived virus that was present in the environment, and indicates that such virus is able to circulate for longer than was previously envisaged.

Analysis of the 16S to 23S rRNA intergenic spacer region of Mycoplasma synoviae field strains.

Mycoplasma synoviae has been associated with economic loss in the chicken and turkey industries. The molecular characterization of M. synoviae at strain level allows the analysis of relationships between strains that may be valuable in epidemiological investigations. In the present study, the intergenic spacer region (ISR) between the 16S and 23S rRNA genes was examined to see whether useful information about strains could be derived. M. synoviae has two copies of this region, which may not be exactly the same (intercistronic heterogeneity). Sequencing of the ISRs of 21 M. synoviae isolates and the type strain revealed that 19 of them had such heterogeneity so DNA cloning was performed where necessary. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and a dendrogram was constructed. The length of the ISRs varied between 305 and 309 base pairs. Apart from having extra A/Ts in poly-A or poly-T regions and the presence of a few polymorphisms, the sequences of the M. synoviae strains were similar. Based on phylogenetic analysis, the strains were assigned to 10 groups-taking into account that within each group the DNA similarity was 100%, while the lowest similarity between groups was 95.8%. The results were compared with those obtained with the vlhA gene, resulting in very similar M. synoviae groups. Although the ISR could be a good target for strain typing, as has been shown by others for Mycoplasma gallisepticum, the method may be too cumbersome for routine use with M. synoviae because of complications with intercistronic heterogeneity. However, if the ISR sequence information was to be combined with other mutation detection techniques it could increase the discriminatory power.

Two similar commercial live attenuated AMPV vaccines prepared by random passage of the identical field isolate, have unrelated sequences.

Since late '80 s Avian metapneumovirus subtype A causes sufficient disease in Europe for commercial companies to have started developing live attenuated vaccines. Here, two of those vaccines were fully consensus sequenced alongside their progenitor field strain (#8544). Sequences comparison shows that the attenuation of field strain #8544 was associated with no common substitutions between the two derived vaccines. This finding suggests that the attenuation of field viruses via serial passage on cell cultures or tissues is the result of a random process, rather than a mechanism aiming to achieve a specific sequence. Furthermore, field vaccination strategies would greatly benefit by the unambiguous vaccine markers identified in this study, enabling a prompt and confident vaccines detection.

Identification of IBV QX vaccine markers : Should vaccine acceptance by authorities require similar identifications for all live IBV vaccines?

IBV genotype QX causes sufficient disease in Europe for several commercial companies to have started developing live attenuated vaccines. Here, one of those vaccines (L1148) was fully consensus sequenced alongside its progenitor field strain (1148-A) to determine vaccine markers, thereby enabling detection on farms. Twenty-eight single nucleotide substitutions were associated with the 1148-A attenuation, of which any combination can identify vaccine L1148 in the field. Sixteen substitutions resulted in amino acid coding changes of which half were in spike. One change in the 1b gene altered the normally highly conserved final 5 nucleotides of the transcription regulatory sequence of the S gene, common to all IBV QX genes. No mutations can currently be associated with the attenuation process. Field vaccination strategies would greatly benefit by such comparative sequence data being mandatorily submitted to regulators prior to vaccine release following a successful registration process.

Development and evaluation of a diagnostic PCR for Mycoplasma synoviae using primers located in the intergenic spacer region and the 23S rRNA gene.

Mycoplasma synoviae (Ms) is an important pathogen of poultry, causing economic losses to this industry. Early and reliable diagnosis is a key to controlling the spread of this organism. In this study, a polymerase chain reaction with one primer based on the intergenic spacer region (ISR) was validated for detection of Ms. The ISR primer was paired with a general primer from within the 23S rRNA gene. The PCR primers were tested with the 22 other recognised avian Mycoplasma species to check the specificity and with 21 field isolates of Ms from various hosts and countries, and with several swab samples. The PCR appeared to be specific and sensitive. Four different sample preparation methods were compared for use in this PCR, and the amplification protocol was compared with three others, confirming the comparative sensitivity of the new PCR.

Molecular investigation of a full-length genome of a Q1-like IBV strain isolated in Italy in 2013.

Since 1996 a new Infectious Bronchitis virus (IBV) genotype, referred to as Q1, circulated in China and was reported for the first time in Italy in 2011, associated with an increase of mortality, kidney lesions and proventriculitis. During northern Italian outbreak of respiratory disease in a broiler flock in 2013, an IBV strain was detected by RT-PCR and characterized as Q1-like based on partial S1 sequence. The virus was isolated and named γCoV/Ck/Italy/I2022/13. All coding regions of the isolate were sequenced and compared with 130 complete genome sequences of IBV and TCoV, downloaded from ViPR. This showed the highest identity with a Chinese strain CK/CH/LDL/97I (p-distance=0.044). To identify potential recombination events a complete genome SimPlot analysis was carried out which revealed the presence of possible multiple recombination events, but the minor parent strains remained unknown. A phylogenetic analysis of the complete S1 gene was performed using all complete S1 sequences available on ViPR and showed the isolate clustered with an Q1-like strain isolated in Italy in 2011 (p-distance=0.004) and a group of Chinese Q1-like strains isolated from the mid 90's (p-distance equal or higher than 0.001). It could be hypothesized that the isolate descended from the Italian 2011 Q1-like strain or was the result of a separate introduction from China through commercial trade or migratory birds; but the data currently available does not distinguish between these possibilities.

Design, validation, and absolute sensitivity of a novel test for the molecular detection of avian pneumovirus.

This study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). Polymerase chain reaction (PCR) diagnostic tests were designed for the detection of nucleic acid from an A-type APV genome. The objective was selection of PCR oligonucleotide combinations, which would provide the greatest test sensitivity and thereby enable optimal detection when used for later testing of field materials. Relative and absolute test sensitivities could be determined because of laboratory access to known quantities of purified full-length DNA copies of APV genome derived from the same A-type virus. Four new nested PCR tests were designed in the fusion (F) protein (2 tests), small hydrophobic (SH) protein (1 test), and nucleocapsid (N) protein (1 test) genes and compared with an established test in the attachment (G) protein gene. Known amounts of full-length APV genome were serially diluted 10-fold, and these dilutions were used as templates for the different tests. Sensitivities were found to differ between the tests, the most sensitive being the established G test, which proved able to detect 6,000 copies of the G gene. The G test contained predominantly pyrimidine residues at its 3' termini, and because of this, oligonucleotides for the most sensitive F test were modified to incorporate the same residue types at their 3' termini. This was found to increase sensitivity, so that after full 3' pyrimidine substitutions, the F test became able to detect 600 copies of the F gene.

Avian metapneumoviruses expressing Infectious Bronchitis virus genes are stable and induce protection.

The study investigates the ability of subtype A Avian metapneumovirus (AMPV) to accept foreign genes and be used as a vector for delivery of Infectious bronchitis virus (IBV) QX genes to chickens. Initially the GFP gene was added to AMPV at all gene junctions in conjunction with the development of cassetted full length DNA AMPV copies. After recombinant virus had been recovered by reverse genetics, GFP positions supporting gene expression while maintaining virus viability in vitro, were determined. Subsequently, either S1 or nucleocapsid (N) genes of IBV were positioned between AMPV M and F genes, while later a bivalent recombinant was prepared by inserting S1 and N at AMPV MF and GL junctions respectively. Immunofluorescent antibody staining showed that all recombinants expressed the inserted IBV genes in vitro and furthermore, all recombinant viruses were found to be highly stable during serial passage. Eyedrop inoculation of chickens with some AMPV-IBV recombinants at one-day-old induced protection against virulent IBV QX challenge 3 weeks later, as assessed by greater motility of tracheal cilia from chickens receiving the recombinants. Nonetheless evidence of AMPV/IBV seroconversion, or major recombinant tracheal replication, were largely absent.

Avian metapneumovirus RT-nested-PCR: a novel false positive reducing inactivated control virus with potential applications to other RNA viruses and real time methods.

Using reverse genetics, an avian metapneumovirus (AMPV) was modified for use as a positive control for validating all stages of a popular established RT-nested PCR, used in the detection of the two major AMPV subtypes (A and B). Resultant amplicons were of increased size and clearly distinguishable from those arising from unmodified virus, thus allowing false positive bands, due to control virus contamination of test samples, to be identified readily. Absorption of the control virus onto filter paper and subsequent microwave irradiation removed all infectivity while its function as an efficient RT-nested-PCR template was unaffected. Identical amplicons were produced after storage for one year. The modified virus is likely to have application as an internal standard as well as in real time methods. Additions to AMPV of RNA from other RNA viruses, including hazardous examples such HIV and influenza, are likely to yield similar safe RT-PCR controls.

Avian metapneumovirus M2:2 protein inhibits replication in Vero cells: modification facilitates live vaccine development.

Throughout the world, avian metapneumovirus (AMPV) infection of subtype A is principally controlled by two live vaccines both derived from UK field strain #8544. Improvements of those vaccines by use of reverse genetics technology was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based reverse genetics system. A systematic reverse genetics based genome modification of a DNA copy of #8544, employing sequence data from a Vero grown, #8544 derived, live vaccine; was used to determine mutations required to facilitate virus recovery and replication in Vero cells. This identified a single coding substitution in the M2:2 reading frame as responsible. Furthermore, ablation of M2:2 was found to elicit the same outcome. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The study shows that M2:2 modification of field virus #8544 will enable research leading to improved vaccines. This may have more general application to other AMPV field strains.

Charged amino acids in the AMPV fusion protein have more influence on induced protection than deletion of the SH or G genes.

Modifications to F, G and SH genes of an avian metapneumovirus (AMPV) field isolate were made by reverse genetics and their virulence and protective capacity were tested in young turkeys. Infection of one-day-old turkeys with a subtype A AMPV neither caused disease nor stimulated detectable protection against subsequent virulent challenge. While serial passage of this virus in tracheal tissue increased virulence, protection stimulated remained moderate. Substitution of the fusion protein from a protective AMPV very minimally increasing virulence but dramatically increased induced protection; and this was associated with five amino acid substitutions all involving charged amino acids which computational analysis predicted to affect protein surface properties but not immunodominant helper T-lymphocyte antigenic sites. When SH or G genes were deleted, viruses caused no disease but still conferred full protection to the majority of turkeys. In the case of the SH deletion, shed virus post-inoculation was undetectable. Partial SH deletions were found to confer protection related to the length of SH open reading frame remaining. Removal of both SH and G genes together produced a virus conferring negligible protection. We conclude that the characteristics of the AMPV fusion protein are important in inducing protection while the SH and G genes under investigation played a lesser role.

Field avian metapneumovirus evolution avoiding vaccine induced immunity.

Live avian metapneumovirus (AMPV) vaccines have largely brought turkey rhinotracheitis (TRT) under control in Europe but unexplained outbreaks still occur. Italian AMPV longitudinal farm studies showed that subtype B AMPVs were frequently detected in turkeys some considerable period after subtype B vaccination. Sequencing showed these to be unrelated to the previously applied vaccine. Sequencing of the entire genome of a typical later isolate showed numerous SH and G protein gene differences when compared to both a 1987 Italian field isolate and the vaccine in common use. Experimental challenge of vaccinated birds with recent virus showed that protection was inferior to that seen after challenge with the earlier 1987 isolate. Field virus had changed in key antigenic regions allowing replication and leading to disease in well vaccinated birds.

Avian metapneumovirus (AMPV) attachment protein involvement in probable virus evolution concurrent with mass live vaccine introduction.

Avian metapneumoviruses detected in Northern Italy between 1987 and 2007 were sequenced in their fusion (F) and attachment (G) genes together with the same genes from isolates collected throughout western European prior to 1994. Fusion protein genes sequences were highly conserved while G protein sequences showed much greater heterogeneity. Phylogenetic studies based on both genes clearly showed that later Italian viruses were significantly different to all earlier virus detections, including early detections from Italy. Furthermore a serine residue in the G proteins and lysine residue in the fusion protein were exclusive to Italian viruses, indicating that later viruses probably arose within the country and the notion that these later viruses evolved from earlier Italian progenitors cannot be discounted. Biocomputing analysis applied to F and G proteins of later Italian viruses predicted that only G contained altered T cell epitopes. It appears likely that Italian field viruses evolved in response to selection pressure from vaccine induced immunity.

Identification of two regions within the subtype A avian metapneumovirus fusion protein (amino acids 211-310 and 336-479) recognized by neutralizing antibodies.

The fusion (F) protein of a subtype A AMPV was expressed in sections in Escherichia coli. Six genome sections were selected which encoded the majority of the protein. These were cloned then expressed from a His tag expression plasmid and, following purification on nickel columns, identities were confirmed by Western blot analysis. The interactions of each fragment with AMPV neutralizing antisera were determined. Purified fragments were mixed with AMPV sera raised against A-C subtypes by a natural route, in order to determine any reduction in their neutralizing capacities. Two fragments covering regions of the F ectodomain reduced neutralizing capacities of both subtype A and B antisera to a highly significant degree (p<0.001) while no effects were seen with subtype C antiserum. Previous studies of similar viruses had identified neutralization as being associated with equivalent F regions. Findings are likely to be useful in guiding future vaccine design.

Avian metapneumovirus SH gene end and G protein mutations influence the level of protection of live-vaccine candidates.

A prototype avian metapneumovirus (AMPV) vaccine (P20) was previously shown to give variable outcomes in experimental trials. Following plaque purification, three of 12 viruses obtained from P20 failed to induce protection against virulent challenge, whilst the remainder retained their protective capacity. The genome sequences of two protective viruses were identical to the P20 consensus, whereas two non-protective viruses differed only in the SH gene transcription termination signal. Northern blotting showed that the alterations in the SH gene-end region of the non-protective viruses led to enhanced levels of dicistronic mRNA produced by transcriptional readthrough. A synthetic minigenome was used to demonstrate that the altered SH gene-end region reduced the level of protein expression from a downstream gene. The genomes of the remaining eight plaque-purified viruses were sequenced in the region where the P20 consensus sequence differed from the virulent progenitor. The seven protective clones were identical, whereas the non-protective virus retained the virulent progenitor sequence at two positions and contained extensive alterations in its attachment (G) protein sequence associated with a reduced or altered expression pattern of G protein on Western blots. The data indicate that the efficacy of a putative protective vaccine strain is affected by mutations altering the balance of G protein expression.

Demonstration of loss of attenuation and extended field persistence of a live avian metapneumovirus vaccine.

A live A type avian metapneumovirus (AMPV) vaccine which had been shown to be highly protective and short lived in experimental conditions was found to persist for longer periods in the field and to be associated with disease. Previously other factors such as possible secondary pathogens and management considerations had made it impossible to conclude whether the observed disease was a result of an increase in the vaccine virulence. In this study, an AMPV was isolated from poults on a farm which had been vaccinated with the same live A type vaccine. Full sequencing of the isolate, the vaccine and the vaccine progenitor confirmed its vaccine origin and further showed that generation of the vaccine had only involved nine substitutions of which three coded for amino acid changes. The isolated virus was inoculated into 1-day-old turkey poults in disease secure isolators and shown to cause disease with a severity similar to that caused by virulent field virus. Only two coding mutations were associated with this reversion to virulence.

Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability.

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.