V. Listorti

Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype's disappearance

Abstract Over a period of almost two years, broilers chickens on several hundred Italian farms, were monitored for infectious bronchitis virus. Detections were genotyped using a hypervariable region of the gene coding for the S1 segment of the spike protein. A range of genotypes were detected which comprised QX, Q1, Mass, D274 and 793B. Sequences of 793B viruses detected in chickens, vaccinated with either of the two commonly used 793B type vaccines were almost identical to sequences of one or other of these vaccines. This strong indication of vaccine association led to the withdrawal of live 793B vaccine use on all of the farms of the study. Except for one sample collected soon after 793B vaccination ceased, it was no longer possible to detect 793B vaccine on these farms. It appears that field 793B strains have disappeared from the region of Italy tested thus obviating any need for current vaccine protection against 793B.

Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B

In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10−0.41 median infectious dose/ml and 101.15 median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were <0.2. Standard curves, generated either using the serial dilution of an RNA suspension or RNA extracted from the serial dilution of titrated viral suspensions as templates, exhibited good linearity (R 2>0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

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.

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

Abstract 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 90s (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.

A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B

SUMMARY 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. RESUMEN Un método de transcripción reversa y PCR en tiempo real para la detección de Metapneumovirus aviares subtipos A y B sensible, reproducible y económico. El uso de PCR en tiempo real es cada vez mayor en el diagnóstico de enfermedad infecciosas, debido a su sensibilidad, especificidad y rapidez de detección. Estas características la hacen especialmente adecuada para el diagnóstico de las infecciones virales, como metapneumovirus aviares (aMPV), en donde se facilita el control mediante la constante actualización de la situación epidemiológica. Otras metodologías de transcripción reversa y PCR en tiempo real han sido publicadas con base en sondas altamente específicas marcadas con colorante fluorescentes, pero tienen alto costo inicial, su validación es compleja, y muestran una marcada susceptibilidad de acuerdo con la variabilidad genética de su secuencia blanco. Con esto en mente, se ha desarrollado y validado un método de RT-PCR cuantitativo basado en SYBR Green I para la detección de los dos subtipos aMPV más prevalentes (por ejemplo, los subtipos A y B). El ensayo demostró una sensibilidad analítica comparable con la de un método de RT-PCR en tiempo real publicado previamente y con capacidad de detectar ARN equivalente a aproximadamente 0.5 dosis infecciosas para ambos subtipos A y B. La alta eficiencia y linealidad entre la titulación viral y el punto de cruce para ambos subtipos muestra que el método es adecuado para la cuantificación viral. La optimización de las condiciones de reacción y la aplicación de análisis de la curva de fusión garantiza la alta especificidad del ensayo. La diferencia de temperatura de fusión estable entre los dos subtipos indicó la posibilidad de subtipificación a través del análisis de la temperatura de fusión. Estas características hacen de este ensayo una herramienta sensible, específica y rápida, lo que permite la detección simultánea, la cuantificación y la discriminación de metapneumovirus subtipos A y B.

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.

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.

Gamma and Deltacoronaviruses in quail and pheasants from Northern Italy

&NA; In view of the restricted knowledge on the diversity of coronaviruses in poultry other than chicken, this study aimed to investigate the genetic diversity of coronaviruses in quail, pheasant, and partridge from two regions of Northern Italy. To this end, pools of tracheal and cloacal swabs from European quail (Coturnix Coturnix) and intestinal tract from pheasants (Phasianus Colchicus) and partridge (Perdix Perdix) flocks, with or without enteric signs, were collected during 2015. Avian coronavirus (Gammacoronavirus) was detected in quail not vaccinated against Infectious Bronchitis Virus (IBV) and in pheasants vaccinated with an IBV Massachusetts serotype. Based on DNA sequences for the gene encoding the S protein, the avian coronaviruses detected in the quail and pheasant are related to the IBV 793B and Massachusetts types, respectively. However, RNA‐dependent RNA polymerase (RdRp) analyses showed the susceptibility of quail also to Deltacoronaviruses, suggesting that quail and pheasant avian coronaviruses share spike genes identical to chicken IBV spike genes and quail might host Deltacoronavirus.

Molecular characterization of whole genome sequence of infectious bronchitis virus 624I genotype confirms the close relationship with Q1 genotype

Abstract Infectious Bronchitis virus (IBV) genotype Q1 was detected for the first time in China in 1996, and then spread worldwide. The first report of Q1 genotype in Italy occurred in 2011 and a deep molecular investigation of a Q1 isolated in Italy in 2013 has led to speculation regarding the origin of this genotype. Phylogenetic analysis of the S1 sequence of a Q1 Italian strain revealed a close relationship with sequences of the 624I strains circulating in Italy in the early 1990s and this led to the idea that 624I was an ancestor of the Q1 genotype. Despite the fact that most heterogeneity of IBVs occurs in the S1 gene, the sequence analysis of this gene alone was not sufficient to confirm or deny this hypothesis. In the present study, an Italian 624I (gammaCoV/AvCov/Ck/Italy/IP14425/96) was fully sequenced for the first time and compared to all available complete Q1 genome sequences. This analysis confirmed the genetic correlation between GammaCoV/AvCov/Ck/Italy/IP14425/96 and Q1 strains, suggesting a common origin between 624I and Q1 genotypes.

Avian Metapneumovirus circulation in Italian broiler farms

With increasing frequency, avian Metapneumovirus (aMPV) is reported to induce respiratory signs in chickens. An adequate knowledge of current aMPV prevalence among Italian broilers is lacking, with little information available on its economical and health impact on the poultry industry. In order to collect preliminary data on the epidemiological context of aMPV in broiler flocks, a survey was performed in areas of Northern Italy with high poultry density from 2014 to 2016. Upper respiratory tract swabs were collected and processed by A and B subtype-specific multiplex real-time reverse transcription PCR (RT-PCR). Samples were also screened for infectious bronchitis virus (IBV) by generic RT-PCR and sequencing. Productive data and respiratory signs were detailed where possible. The high prevalence of aMPV was confirmed in broilers older than 26 d and also attested in IBV-negative farms. All aMPV detections belonged to subtype B. Italian strain genetic variability was evaluated by the partial attachment (G) gene sequencing of selected strains and compared with contemporary turkey strains and previously published aMPV references, revealing no host specificity and the progressive evolution of this virus in Italy.