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This study evaluated if, in field conditions, the infection with Avian Metapneumovirus (AMPV) and/or Turkey Hemorrhagic Enteritis Virus (THEV) was significant for the induction of episodes of colibacillosis in AMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. AMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques, and antibody titres were evaluated by ELISA. When AMPV of subtype B was detected, samples were subjected to a restriction endonuclease digestion protocol to discriminate between field and vaccine strains. Field strains were detected in all flocks at different ages of life, always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems do not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field AMPV infection is correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of AMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of AMPV.

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 molecular survey on Avian Metapneumovirus (aMPV) diffusion was performed from 2011 to 2013 in 122 turkey and 48 broiler farms, located in a densely populated poultry area of Northern Italy. Turkeys were all vaccinated at 1 day of age in the hatchery. aMPV was detected using RTnested PCR or qRT-PCR; both tests are able to detect and differentiate aMPV subtypes A and B. All samples but one resulted positive for aMPV subtype B confirming the high prevalence of this subtype in Italy. The majority of aMPV detections were of field origin, circulating mainly in 9 to 12- week-old turkeys and 5 to 7 week-old broilers, showing respiratory signs. The reasons for vaccine failure could be due to field virus changes occurred in key antigenic regions, which allow virus replication and disease in well-vaccinated birds. Strains of vaccine origin were detected in turkeys with a high prevalence during the first few weeks after vaccination with aMPV live vaccines. This confirmed the pattern previously seen where vaccine viruses were shown to persist on farm for 4-5 weeks. Uniquely in this study, vaccine derived strains were detected in older turkeys of up to 84 days of age. Moreover, sequence analysis of F and G protein genes of selected strains was performed.

Avian Metapneumovirus (AMPV) of subtype A or B affects mainly turkeys and chickens. However, also pheasants and guinea fowls are susceptible to natural and experimental infection. This study reports, for the first time, the molecular characterization of an AMPV of subtype B detected from guinea fowls during an outbreak of respiratory disease, which occurred in February 2012 in a flock located in a highly populated poultry area of Northern Italy. Nucleotide sequences of F and G protein genes of the strain detected were obtained and aligned against analogous gene sequences of AMPVs subtype B isolated from turkeys or chickens in Italy or deposited/published on GenBank. Phylogenetic analysis was carried out under distance criterion, with neighbour-joining as algorithm, using MEGA5 software. The trees constructed on the alignments of sequence data sets of the F and G protein genes showed one main cluster including all Italian strains isolated after 2001 and this was supported by a high bootstrap value. Inside this cluster, two sub-clusters were observed, one which included strains isolated between 2001 and 2004, and other that included strains isolated after 2007. The strain detected from guinea fowls was included in this last sub-cluster. These findings do not support genetic differences correlated to the host tropism and suggest that circulation of closely related AMPV strains occurs among different avian species in densely populated poultry areas.

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. 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.

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.

The current information on prevalence of avian metapneumovirus (AMPV) infection in layers is fragmentary and its true impact on egg production often remains unknown or unclear. In order to draw an epidemiological picture of AMPV spreading in layer flocks in Italy, a survey was performed on five pullet and eight layer farms, based on longitudinal studies or sporadic samplings. AMPV was detected by RT-PCR, and blood samples were collected for serology by AMPV ELISA. Where possible, occurrence of respiratory signs and drop in egg production were recorded. Only AMPV of B subtype was found in both pullet and layer farms. Surveys of pullets showed that most groups become infected prior to the onset of lay without showing clear respiratory signs. At point of lay these groups were serologically positive to AMPV. In two layer flocks, egg drops were observed and could be strongly linked in presence of AMPV infection. Results were correlated with AMPV vaccination programs applied to the birds. Only a vaccination program, including two live and one killed vaccines, was able to give complete protection from AMPV infection to the birds, while a single live vaccine application was not efficacious. The present survey provides preliminary but unique data on the epidemiology of AMPV infection in layers in Italy.

Direct diagnosis of avian Metapneumovirus (AMPV) infections rely on molecular techniques more than on virus isolation due to the fastidious nature of the virus. Six real-time reverse transcription PCR (RRT-PCR) protocols for the detection and differentiation of AMPV subtype A and B were developed in N (two tests), F, SH (two tests) and G genes. Five assays used SYBR Green I as detection system, and one molecular beacon probes. Specificity was evaluated using various AMPV strains, Newcastle disease, Infectious laryngotracheitis and Infectious bronchitis viruses. All tests were able to detect AMPVs and failed to detect non-AMPV viruses, and five out of six of them were also able to discriminate between AMPV A and B subtypes. Sensitivity was determined using serial dilutions of RNA from AMPV of both subtypes. The best results in terms of specificity and sensitivity was given by the RRT-PCR protocol designed in the SH gene. This new assay was later compared with a well-established molecular assay, an RT Nested-PCR based on the G gene

Avian metapneumovirus (AMPV) causes an upper respiratory tract infection in turkeys leading to turkey rhinotracheitis. In other avian species, including chickens, it is also involved in the aetiology of multifactorial diseases such as swollen head syndrome. Sensitivity of wild birds to AMPV and their role in maintaining and spreading the virus to poultry is still a matter of debate. Recently the sensitivity of pigeons to AMPV of subtype A or B has been claimed, based on very limited PCR detections from wild or experimentally infected birds. In order to have conclusive evidence regarding the sensitivity of pigeons to AMPV of subtype B and its role in spreading the virus to turkeys, two experimental trials were planned in secure isolation conditions. In trial 1 two isolators were modified to host turkeys and pigeons in the same environment, separated only by a net. Drinkers were shared between groups. 16 Pigeons were infected with AMPV and housed in one isolator with naïve turkeys. Similarly turkeys were infected and housed with naïve pigeons. Additional turkeys and pigeons were kept in different isolators as uninfected controls. Post-infection clinical signs, virus shedding and immune response were assessed for three weeks. In trial 2, commercial two-weeks old turkeys were divided in two groups of ten and housed in two different isolators. Birds in isolator A were challenged as previously described. Four days post-infection, five 7 weeks old naïve pigeons were introduced in the isolator A and kept with the infected turkeys for 24 hours, then removed, sprayed with 0,5 % of Wirkon S solution. After 10 minutes, pigeons were rinsed with water, dried, and introduced in the isolator B, where 10 naïve turkeys were housed. Clinical sign were monitored for 10 days. Pigeons were found refractory to AMPV experimental infection and neither able to spread the virus to naïve turkeys. Our paper shows that pigeons are highly unlikely to play any relevant role in the environmental spread of subtype B AMPV. Pigeons are not biological vector or reservoir species for AMPV subtype B.

Avian metapneumoviruses (AMPV) of subtype B dominate over other subtypes on Italian poultry farms in northern Italy. AMPVs from the Veneto region of Italy between 1987 and 2007 were sequenced in their attachment (G) and fusion (F) protein genes, together with other subtype B AMPVs from other parts of Western Europe, collected prior to 1994. All viruses in the survey had very similar predicted F sequences whereas the predicted G protein sequences found in Italy from 2001 were distinctly different from those found in pre 1994 viruses. Nonetheless pre 1994 Italian AMPVs were more similar to post 2000 AMPVs than other pre 1994 viruses from other parts of Europe, thereby showing that the later viruses had probably evolved from early Italian viruses. The occurrence of the later variants followed introduction of the mass administration with a single subtype B vaccine and its G protein sequence placed it clearly in the pre 1994 cluster. The possibility of the vaccine having driven field virus evolution in order to evade immune pressure cannot be excluded.

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.

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

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.

The first avian pneumovirus (APV) outbreak, caused an 18.4% mortality, was observed in Croatia in a broiler flock in 1989. The APV outbreak in the laying turkeys was detected in 1990. About 10% of the birds were affected by a mild form of the disease. A 3-week decrease of the egg production and hatchability was recorded in the period between weeks 5 and 18 of lay. The APV infection in turkeys was monitored systematically in the turkey flocks in the period between 1997 and 2002. Sera samples were randomly collected from female and male breeding and fattening turkeys suspected to be infected with APV. Ten to 20 sera samples were taken from a flock. The turkey age ranged from 1 day to 47 weeks and turkeys were not vaccinated against APV. The levels of APV antibodies were determined by commercially available ELISAs (Avisure, Vetoquinol SA, France). A differential diagnosis including Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma meleagridis (MM), avian influenza virus (AIV) and Newcastle disease virus (NDV) was carried out. In 1997 the presence of antibodies against APV was confirmed in only 1.3% of sera of turkeys with the clinical signs and the pathological findings characteristics for respiratory diseases. Suspected were 2.9% of analyzed sera. An increase of antiAPV positive (13.3%) and suspected (16.7%) sera was detected in 1998. The antiAPV positive rate tended to increase and a considerably higher number of antiAPV positive sera amounting to 90.7% and 4.7% suspected sera was found in 1999. Following the introduction of the rigorous non-specific preventive measures for respiratory disease, 5.6% antiAPV positive and 7.2% suspected sera were detected in 2000. The number of antiAPV (48.3%) and suspected (12.6%) sera increased in 2001. In the period between January and March 2002, 10.9% of analyzed sera were antiAPV positive and 5.0% were suspected. The presence of APV antibodies was identified more frequently in sera of older than younger birds as well as in female rather than in male bird sera. Turkey breeder hens infected by the APV had a drop in egg production accompanied by the decrease of hatchability and the reduction of egg shell quality. In some cases the presence of the antibodies against other avian respiratory pathogens was found. All birds submitted to the serological analysis were vaccinated against NDV and the NDV antibodies in their sera were probably generated by the vaccine.

A recent outbreak of infectious bursal disease (IBD) in vaccinated broilers in Croatia with generally no increased mortality, watery diarrhoea and significantly reduced body weight gains is described. Experimental infection of fully susceptible 30-day-old broilers with IBD virus (IBDV) isolated from affected chickens resulted in no mortality and mild clinical signs but body weight gains were significantly reduced (52.9 grams and 83.6 grams per day in susceptible and immune birds, respectively), during the two weeks after contact infection. Rapid seroconversion was noted in unvaccinated birds. Induced transcription of interferon alpha and gamma of susceptible broilers was significantly reduced on the 7th and 14th day after exposure to isolated virus, whereas induction in broilers vaccinated for IBDV was not reduced. Infection of 4-week-old SPF chickens with the virus resulted in severe disease with 60% mortality.