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IBV genotype QX causes suficient 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 ield strain (1148-A) to determine vaccines 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 ield. 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 inal 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 beneit by such comparative sequence data being mandatorily submitted to regulators prior to vaccine release following a successful registration process

Avian Metapneumovirus (aMPV) causes an upper respiratory tract infection in turkeys and in some other avian species. Based on genome sequence differences, four subtypes of aMPV (A, B, C and D) have been identified but subtypes A and B have an almost worldwide distribution. Nowadays control strategies are mainly based on vaccination and different commercial live vaccines, originating from both subtype A and B strains, are available. The not absolute coverage of animal population achieved by ordinary mass vaccination in poultry industry allows a prolonged circulation of vaccine viruses in bird populations. Diseases outbreaks have been reported due to reversion to virulence of vaccines; and multiple nucleotide mutations in the aMPV genome associated to this phenomenon have been identified. 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 were 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 vaccine derived strains obtained during experimental infections or in the field may clarify if reversion to virulence was due to selective pressure acting on a homogeneous but rapidly evolving vaccine population or to the selection of virulent subpopulations already present in the vaccine batches.

Live vaccines predominantly control avian metapneumovirus (AMPV) infection in the poultry industry but these can persist after application and revert to virulence. Sequencing of the G gene of the dominant subtype B VCO3 vaccinal strain identified a unique nucleotide substitution (A→G, nucleotide 91) which fortuitously introduced an MseI restriction endonuclease site within the amplicon obtained from a popular AMPV diagnostic RT- nested PCR. An Msel digestion protocol was developed then validated using Italian B subtype viruses of known field virus or vaccine origin.

Since 1996 a new Infectious Bronchitis virus (IBV) genotype, referred to as Q1, circulated in China and was reported for the Þ rst time in Italy in 2011, associated with an increase of mortality, kidney lesions and proventriculitis. During a 2013 Northern Italian outbreak of respiratory disease in a chicken ß ock, an IBV strain was detected by RT-PCR and characterized as Q1-like. The virus was isolated and named CoV/ 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, and showed most 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 that 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=0.001 with GenBank Accession Number CK 577410). 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 did not distinguish between these possibilities

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), where effective control benefits from a continuously updated knowledge of the epidemiological situation. Other real-time RT-PCRs have been published based on highly specific fluorescent-dye labeled probes but they have high initial cost, a 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 qRT-PCR for the detection of the two more prevalent AMPV subtypes (i.e subtype 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 TCID50/mL for both A and B subtypes. The high efficiency and linearity between viral titre and Cp displayed for both subtypes make it suited for viral quantification. Reaction conditions optimization and implementation of melting curve analysis guaranteed the high specificity of the assay. The stable Tm difference between the two subtypes, revealed 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.

A reverse genetics system is the only available method to introduce specific mutations into the AMPV genome. The virus has a negative sense genome but even transfecting a positive sense copy into AMPV susceptible cells will not lead to recovery of virus. This is because the virus polymerase is only able to copy and transcribe the RNA genome when as a component of the ribonuclear protein complex (RNP complex) which also includes the nucleocapsid (N) protein, phosphoprotein(P) and M2 protein. If plasmids coding for N, P, M2, the polymerase and a full length genome copy are transfected into suitable cells together, under the control of appropriate promoters, virus will be generated.To date no system for the AMPV subtype B had been reported. In the system described, plasmids for N, P, M2, the polymerase and the full genome were constructed under the control of the T7 promoter. Producing intact clones containing the virus polymerase gene proved highly technically demanding due to the issue of them containing sequences toxic to bacteria, thus leading to sections of the gene being spontaneously deleted. Nonetheless, once all clones had been generated and transfected into Vero cells previously infected with a fowlpox recombinant virus expressing T polymerase, AMPV was recovered.

Live vaccines predominantly control avian metapneumovirus (AMPV) infection in the poultry industry but these can persist after application and revert to virulence (Catelli et al., 2006; Catelli et al., 2010; Lupini et al., 2011). Sequencing of the G gene of the prevailing subtype B VCO3 vaccinal strain (Cecchinato et al., 2010) identified a unique nucleotide substitution (A→G, nucleotide in position 91, G gene), which fortuitously introduced an MseI restriction endonuclease site within the amplicon obtained from a popular AMPV diagnostic RT- nested PCR (Naylor et al., 1997). The study reports the development of a method which uses the restriction enzyme digestion of PCR products as a method for rapid differentiation between AMPV subtype B vaccine or vaccine-derived strains and field strains. Two Italian subtype B AMPV isolates and the VCO3 vaccine were used to test the restriction enzyme digestion protocol. The amplicons obtained from these strains by RT-nested PCR were incubated with restriction enzyme MseI for two hours at 65 °C and analyzed by agarose gel electrophoresis. After enzymatic digestion field strains remained uncut showing an amplicon of 360bp, while the vaccine strain was cut resulting in a smaller band of 320bp detectable by gel electrophoresis, and a band of 40 bp, not detectable (Figure 1). The developed protocol was subsequently used for a field epidemiological study. Forty-one AMPVs of subtype B, detected in Italy from 2001 to 2011, were collected. After RT-nested PCR and enzymatic digestion, 8 out 41 strains revealed a vaccine origin, while the others were field strains. The method herein described for rapid differentiation of vaccine and field strains of AMPV of subtype B is applicable in geographic areas where the specific vaccine included in the study is used and might be a good alternative to sequencing, which although provides further information, requires more time and higher costs.

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 3 recombinant viruses 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 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.

A virus was isolated in association with respiratory disease typical of turkey rhinotracheitis, from turkeys which had been vaccinated with a B subtype Avian Metapneumovirus (AMPV) vaccine. Sequencing of the virus showed that, as in a previous similar report, the virus had originated from a live A subtype vaccine. In this instance the disease was much later, at 50 days of age, and there had been no recent history of use of the vaccine. This may indicate that AMPV vaccines are able to circulate in the environment for longer than was previously envisaged

Un sistema di reverse genetics per AMPV è stato utilizzato per ottenere un virus ricombinante incapace di esprimere il gene SH che ha portato alla produzione su cellule Vero di un effetto citopatico anomalo caratterizzato da sincizi giganti. La sostituzione del gene SH col gene GFP ha portato alla produzione di un virus che ha prodotto gli stessi sincizi giganti. La simile dimensione della sequenza nucleotidica dei geni SH e GFP indica che l’alterazione dell’effetto citopatico causato dalla delezione del gene SH non è frutto della rimozione di un’unità trascrizionale. Inoltre per le sue proprietà fluorescenti, AMPV in grado di esprimere la proteina GFP può rivelarsi utile in studi di patogenesi.

A standardized RT-nested PCR of high sensitivity has been in use in our laboratory for some time to detect avian metapneumovirus subtype A and B. Until now we have avoided the use of positive control viruses (CV) because of the risk of contamination leading to sample false positives. The paper describes the production and testing of a modified virus which in our standard PCR produces RT-nested PCR amplicons of increased sizes compared to those generated from unmodified viruses, thus enabling cross contamination to the sample tested to be instantly detectable. A DNA copy of an AMPV subtype A genome was modified by site directed mutagenesis to introduce the subtype B G gene primer sequence (G9+B) at the equivalent position in the subtype A G gene. To increase amplicon sizes, an insertion was introduced between binding sites for the opposing primer pairs (outer pair G1+ and G6-, inner pairs G8+A and G5- or G9+B and G5-). PCR from the modified DNA produced nested amplicons of 463 and 556 bp compared to 268 for unmodified subtype A and 361 for unmodified subtype B viruses. Using reverse genetics the modified DNA generated a virus which, after RNA extraction, gave the same 463 and 556 bp amplicons in RT-nested PCRs. For further convenience virus was absorbed onto filter paper, dried and inactivated by microwave treatment, then stored in flip top tubes. RNA extracted from papers were henceforth used as reliable, efficient and distinguishable RT-nested PCR positive controls.

At the beginning of 2000 little information was available on circulation and molecular epidemiology of AMPV Italian poultry except for the data regarding the first Italian isolates made in the late 80s, which were shown to be B subtypes (Juhasz and Easton, 1994; Sperati Ruffoni L., personal communication). Moreover even though the vaccination was widely applied, disease still occurs occasionally in young as well older turkeys. This paper reports some studies performed in Italy since 2001 in order to understand better the epidemiology of AMPV and prophylactic problems encountered in the field.

Dati epidemiologici sulla prevalenza di AMPV in allevamenti di tacchini da carne del Nord Italia hanno messo in evidenza come, dopo l’introduzione della vaccinazione di massa, il numero di positività per AMPV sia complessivamente diminuito negli anni in concomitanza con un aumento del numero di positività osservate in età avanzata (43-90 giorni di età), in gruppi vaccinati col medesimo sottotipo. Il successivo sequenziamento completo del gene di adesione (G) di alcuni isolati responsabili di focolai tardivi in gruppi vaccinati, ha permesso di distinguerli nettamente dal ceppo vaccinale e da tutti i ceppi di AMPV italiani isolati prima del 2001. Allo scopo di determinare se le mutazioni osservate nei ceppi “più recenti” siano state sufficienti al virus per eludere l’immunità vaccinale è stata eseguita un’infezione sperimentale in tacchini vaccinati, inoculandoli con un ceppo isolato nel 2004 o con un ceppo del 1987, e valutando la protezione mediante misurazione della sintomatologia clinica e dell’eliminazione virale.

Questo lavoro descrive l’utilizzo di metodiche di Reverse Genetics per la produzione di un virus geneticamente modificato in grado di generare nella RT Nested PCR standard precedentemente descritta, amplificati di dimensioni maggiori rispetto a quelli generati da virus non modificati. Tale virus, impiegato come controllo positivo, rende possibile evidenziare immediatamente eventuali contaminazioni.

Infection of turkeys and chickens with Avian Metapneumovirus (AMPV) leads to disease and serious economic losses in unprotected birds. In the late 1980s, live AMPV vaccines became available in Europe and these largely brought the disease under control. However disease has still been encountered and much of this remains unexplained. There is evidence that in some instances disease might be due to the involvement of another subtype or in some cases the disease may be caused by the vaccine itself. However many disease outbreaks remain unexplained especially in situations where they are observed after apparently successful vaccination with virus of the same subtype. Longitudinal studies were undertaken in Italian turkey farms and demonstrated that subtype B AMPV was frequently detected some period after subtype B vaccination. Sequencing showed that these later viruses were not derived from the previously applied vaccine, and have genome differences in key antigenic regions. An experimental infection of birds was carried out to discover whether the consistent mutations observed in the more recent Italian isolates might allow the viruses to avoid an immune response induced by the common subtype B vaccine, which has virus sequence close to AMPV strains isolated in Italy in the late 80s. The results of the study demonstrated that the more recent field viruses were able to overcome vaccine induced immunity and are consistent with the hypothesis that the field virus has changed in key antigenic regions in order to thrive within a groups of well vaccinated birds.

I vaccini vivi attenuati conferiscono una buona protezione ma la loro instabilità può portarli a riacquisire virulenza anche dopo un limitato numero di retropassaggi. Sperimentalmente è stato dimostrato che sono sufficienti 4-10 retropassaggi su animali sensibili. Uno studio successivo ha dimostrato come ciò può avvenire anche in allevamento. Animali vaccinati al primo giorno di vita in incubatoio con un sottotipo A hanno mostrato, dopo 3 settimane, una forma respiratoria durante la quale è stato isolato un AMPV sottotipo A. Il sequenziamento dell'intero genoma virale e l'identificazione di 9 nucleotidi marker vaccinali ne ha dimostrato l'inequivocabile origine vaccinale. In questo lavoro viene riportato un focolaio di TRT verificatosi in Italia nel 2003, dovuto ad AMPV sottotipo A di origine vaccinale che ha interessato tacchini di 7 settimane vaccinati con un sottotipo B.

Avian Metapneumovirus (AMPV) is a member of the Paramyxoviridae family and is responsible for Turkey Rhinotracheitis in turkeys which is an upper respiratory tract infection. This can cause severe disease and losses where secondary pathogens exacerbate the situation. An AMPV reverse-genetics system have been developed which enables specific mutations to be introduced into the virus genome and the subsequent phenotypic consequences determined (Naylor et al., 2004). This system was used to show that infection with a recombinant virus, unable to express the SH gene, resulted in the production of unusually large syncytia in Vero cells. It is possible that this was due to a change in the pattern of genome transcription resulting from the loss of a transcription unit. In this work we replace the SH gene with GFP which is a gene of similar size, and in which expression can be confirmed by fluorescence of its gene product, in suitable UV irradiation.

Avian Pneumovirus infection in Italy is reported from its first appearance in 1987 to present day. The B type virus has been found in different geographical regions but more recently, A type virus has also been detected. To establish the identity and heterogeneity among the APV strains that circulated in Italy from 1987 to 2004, the nucleotide sequences of whole Fusion protein gene were determined and compared with European APV field strains and commonly available vaccine strains.

Since the 1980s a number of live attenuated type A and B Avian Pneumovirus (APV) vaccines have been developed in Europe and these have generally afforded good protection. All have been produced by repeated passages of field virus in a laboratory culture system to achieve various degrees of attenuation. They are widely used in commercial growing turkeys and to prime future layers and breeders. When tested under experimental conditions, these empirically derived vaccines were shown to be fully protective whilst not causing detectable disease themselves. However, they have not performed as well when used in the field and unstable attenuation has been considered to be a possible factor. This paper describes the first evidence of reversion of an APV vaccine in the field. First, systematic evidence of extended vaccine virus persistence in the field arose from longitudinal studies performed in the UK in 1995. Secondly, from an outbreak of Turkey Rhinotracheitis (TRT) in an Italian turkey flock previously vaccinated at day old, we isolated an APV which proved to be a vaccine derivative by sequencing. Finally, in order to determine whether vaccine virus or a derivative of increased virulence had been isolated, the virus was applied to 1 day old poults in secure isolation conditions. The vaccine derivative virus was shown to be able to cause disease with a severity similar to that caused by virulent field virus.

Avian Pneumovirus (APV) infection in Italy is reported from its first appearance in 1987 to present day. The B type virus has been found in different geographical regions but more recently, A type virus has also been detected. To establish the identity and heterogeneity among the APV strains that circulated in Italy, the nucleotide sequences of the Fusion (F) gene were determined and compared with European APV field strains and commonly available vaccine strains. Six B type and one A type Avian Pneumovirus strains isolated in Italy from 1987 to 2004, were grown in chicken embryo tracheal organ cultures (TOC). RNA was extracted from TOC medium and three overlapping RT-PCRs, covering the entire F gene, were performed for further sequencing of the strains. Phylograms for the ~1600 bp sequence were constructed with the MEGA package, version 3.1. As expected, the 6 B type isolates were clustered with the previously published European APV B sequences. Within the group, significant sub clustering was apparent, as the more recent isolates (2001-2004) formed one cluster clearly separated from viruses isolated in the former decade (100% bootstrap).

The first study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). PCR diagnostic tests were designed for the detection of nucleic acid from an A-type avian pneumovirus 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 to an established test in the attachment (G) protein gene. Known amounts of full-length avian pneumovirus 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, with 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. In the second study a new RT-nested PCR, able to detect and distinguish A and B APV types was designed in the SH protein gene and tested showing good sensitivity for both types.

APV infection in Italy is reported from its first appearance in 1987 to present day. Serological, virological and molecular testing have been applied during one-off and longitudinal studies of turkeys and chickens. The B type virus has been found in different geographical regions but more recently, A type virus has also been detected. Material was further compared by means of PCR and sequencing of sections of selected genes. Diagnostic and prophylactic problems encountered in the field are also considered.

This study describes attempts to measure and increase sensitivity of molecular tests which would be later used to detect Avian pneumovirus (APV) in field material. PCR diagnostic tests were designed for the detection of nucleic acid from an A type avian pneumovirus genome. The objective was selection of PCR oligonucleotide combinations which would provide the greatest test sensitivity, to 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 DNA copies of the same A type full length APV genome. Four new nested PCR tests were designed in the fusion protein [2 tests] (F), small hydrophobic protein (SH) and nucleocapsid (N) protein genes and compared to an established test in the attachment protein (G) 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, with the most sensitive being given by the established G test, which proved able to detect 8000 copies of G gene. The established G test contained predominantly pyrimidine residues at their 3’ termini and because of this, oligos 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 F gene. An RT-nested PCR test was then used to test limited field material. In this an RT step preceeded PCR testing which was identical to that used for DNA above. The modified F test again proved more sensitive than the established G test.