Mattia Cecchinato

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.

West Nile virus circulation in Veneto region in 2008-2009

West Nile virus (WNV) was detected in Italy, in late summer 2008 in horses and birds in the Po valley. As a consequence, an intense WNV surveillance was implemented in that area involving Emilia-Romagna, Veneto and Lombardy. This paper presents the results of the September 2008-November 2009 surveillance on equines, mosquitoes, wild birds, dogs and cattle in Veneto. WNV was detected in equines and dogs, and, to a lesser extent in cattle and wild birds. Simultaneous circulation of Usutu virus was detected by testing wild birds found dead. Usutu virus but not WNV was also found in mosquitoes monitored during 2009. Equine practices monitoring allowed the definition of an area of WNV circulation and the 2008-2009 westward and northward spread of the infection. Although a relatively low number of human cases and a low virus circulation in vectors and birds detected in Veneto region could be considered favourable conditions for a limited risk of human exposure, it remains difficult to predict the possible evolution of the epidemiological situation.

Use of vaccination in avian influenza control and eradication.

Vaccination against avian influenza (AI) infections caused by viruses of the H5 and H7 subtypes has been used in several occasions in recent years with the general objective of controlling and in some cases eradicating the disease. To contain AI infections effectively, vaccination should only be used as part of a comprehensive control strategy that also includes biosecurity, quarantine, surveillance, education, and elimination of infected and at‐risk poultry. Although properly used, potent AI vaccines can prevent disease and death, increase resistance to infection, reduce virus replication and shedding, and reduce viral transmission, they cannot completely prevent AI virus replication. A wide variety of vaccines against AI has been developed and tested in experimental conditions, but only inactivated whole AI virus vaccines and recombinant H5‐AI vaccines have been licensed and widely used in various countries. AI vaccination programmes should be adapted to local conditions to guarantee efficacy and sustainability. In particular, vaccination programmes should be modulated in diverse situations according to the virus strain involved, the characteristics of the poultry producing sector, the capacity of the veterinary infrastructure, and the availability of adequate resources. Based on the eco‐epidemiological situation in the affected region/area/compartment and the assessment of the risk of AI introduction, different vaccination strategies could be implemented to control AI: (i) routine vaccination performed in endemic areas; (ii) emergency vaccination in the face of an epidemic; and (iii) preventative vaccination carried out whenever a high risk of virus incursion is identified.

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus.

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC(50)) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC(50) (concentration required to inhibit 50% of viral cytopathic effect). CC(50)s of tested compounds were > 200 microg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC(50) values ranging from 25 to 66 microg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC(50) 24 microg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.

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.

Porcine circovirus type 2 (PCV2) evolution before and after the vaccination introduction: A large scale epidemiological study

Since their commercialization, vaccines against Porcine circovirus type 2 (PCV2) have been the cornerstone control strategy. Nevertheless, the periodic emergence of new genotype waves and the recent reports of vaccine failure outbreaks have raised the question if widespread vaccination strategies could have driven viral evolution and affected different genotype fitness. To investigate this issue an in-deep analysis, based on a bioinformatics and biostatistics approach, has been implemented. ORF2 sequences from vaccinated and non-vaccinated populations (i.e. domestic pigs before and after vaccine introduction and wild boars) were considered. The action of selective forces on PCV2 strains has been analyzed and compared among groups. Remarkable differences were found in the selective forces acting on viral populations circulating in different “immune environments”. Particularly for PCV2a, a directional selection promoting a change in the viral capsid away from the vaccine specific antigenic determinants has been detected after vaccine introduction. Involved amino acids were previously reported to be part of viral epitopes whose variability is responsible of immune escape. Our findings support a change in PCV2 evolutionary pattern after widespread vaccination introduction and stress once more the compulsoriness of a continuous monitoring of PCV2 epidemiology to promptly act in response to the emergence of possible vaccine-escaping mutants.

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.

The effects of control measures on the economic burden associated with epidemics of avian influenza in Italy

In 1999, Italy experienced a devastating epidemic of high-pathogenicity avian influenza (HPAI) caused by an H7N1 virus subtype. After this epidemic, a ministerial decree was passed to implement control measures for low-pathogenicity avian influenza (LPAI) due to H5 and H7 subtypes. We investigated whether these control measures have decreased the public expenditure associated with epidemics of LPAI and HPAI by comparing the direct and consequential losses of the 1999 epidemic to the losses associated with successive epidemics. The estimated total economic burden of the epidemics was about euro650 million (euro217 million in direct losses and euro433 million in consequential losses). The 1999 epidemic accounted for most of these losses (euro507 million: euro112 million in direct losses and euro395 million in consequential losses), whereas the total economic burden for the 5 successive LPAI was euro143 million (euro105 million in direct losses and euro38 million in consequential losses). These results demonstrate that the implementation of a coordinated set of disease-control measures, which included both emergency and prophylactic vaccination, was able to reduce the overall costs associated with avian influenza epidemics. The results also show that the application of adequate LPAI control measures may limit the risk of emergence of an HPAI virus in an area with a high poultry density, allowing the complete disruption of the poultry market and its huge associated costs to be avoided.

Low Pathogenicity Avian Influenza in Italy During 2007 and 2008: Epidemiology and Control

Abstract Since 1999, the Italian poultry production system has experienced several outbreaks of avian influenza (AI), mainly located in northeastern Italy. This paper describes the low pathogenicity (LP) AI outbreaks detected during the surveillance activities implemented in 2007–08. From May to October 2007, ten rural and hobby poultry farms were infected by an LPAI virus of the H7N3 subtype. In August–October 2007, the H7N3 LPAI virus was introduced into the industrial poultry sector with the involvement of six meat turkey farms. Phylogenetic analysis of the hemagglutinin gene indicated that all but one of the H7N3 virus strains had a high level of homology (98.7%–99.8%). Furthermore, in August 2007, an LPAI H5N2 virus was identified in a free-range geese and duck breeder flock. The hemagglutinin and neuraminidase genes showed a high level of homology (99.8% and 99.9%, respectively) with H5N2 LPAI viruses isolated from mallards in July 2007 in the same area, suggesting a possible introduction from the wild reservoir. All the birds (in total 129,386) on the infected poultry farms were culled. The prompt implementation of AI control measures, including the enforcement of a targeted emergency vaccination plan, allowed the rapid eradication of infection. In 2008, three LPAI viruses (two H7N1 and one H5N1) were identified in dealer/rural farms. The surveillance activity implemented in this area allowed the prompt detection of LPAI viruses of the H5 and H7 subtypes in the rural sector, which, as observed in the 2007 epidemic, might be the source of infection for industrial poultry.