Mauro Delogu

Characterization of Low-Pathogenic H5 Subtype Influenza Viruses from Eurasia: Implications for the Origin of Highly Pathogenic H5N1 Viruses

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BackgroundAvian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.MethodsRRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.ResultsThe RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.ConclusionThe high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.

Influenza virus circulation in wild aquatic birds in Italy during H5N2 and H7N1 poultry epidemic periods (1998 to 2000)

Two epidemics of avian influenza due to H5 and H7 highly pathogenic viruses occurred in poultry in Italy in 1997/98 and 1999/2000, respectively. The circulation of these serotypes in wild aquatic birds was investigated examining 638 cloacal swabs and 621 sera collected from 150 gulls, 162 coots, and 326 ducks trapped in Italian wetlands from 1998 to 2000. Seroprevalences against influenza A viruses, detected by a double-antibody sandwich-blocking enzyme-linked immunosorbent assay (ELISA), were 11% in gulls, 16% in coots, and 45% in ducks. Among the Anatidae group, duck species wintering in Mediterranean areas showed significantly higher values than ducks wintering in South-Saharan areas of Africa. In order to detect H5 and H7 antibodies, the haemagglutination-inhibition assay and two competitive ELISA tests (H5-ELISA and H7-ELISA) using monoclonal antibodies specific for H5 and H7 subtypes were performed. None of the aquatic bird species were found seropositive for H7 subtype, whereas H5-positive sera were found by both the haemagglutination-inhibition and ELISA assays in ducks only. The highest H5 seroprevalences were detected by H5-ELISA; overall, 5% (10/201) of duck species wintering in Mediterranean areas tested positive by this assay, with annual seroprevalences ranging from 2% (2/123) to 12% (6/51). In the present study, only five viruses belonging to H1N1, H11N6, and H2N3 subtypes were isolated from ducks. However, the H5 seroconversion observed in one mallard duck at the beginning of 1998 indicates that H5 virus circulation also occurred in the study area. Circulation de virus influenza chez les oiseaux aquatiques sauvages, en Italie au cours des épisodes épidémiologiques de H5N2 et H7N1 observés chez les volailles entre 1998 et 2000 Deux épidémies dinfluenza aviaire dues à des virus H5 et H7 hautement pathogènes sont respectivement apparues chez les volailles en Italie en 1997/98 et en 1999/2000. la circulation de ces sous types chez les oiseaux aquatiques sauvages a été étudiée en examinant 638 écouvillons cloacaux et 621 sérums récoltés à partir de 150 mouettes, 162 foulques, et 326 canards piégés dans les marécages entre 1998 et 2000. Les séroprévalences contre les virus influenza, détectées par un test ELISA bloquant, système sandwich avec deux anticorps, ont été de 11% chez les mouettes, 16% chez les foulques, et 45% chez les canards. Parmi le groupe des Anatidae, les espèces de canards hivernant dans les zones méditerranéennes (DWMA) ont montré des valeurs significativement supérieures à celles des canards hivernant dans les zones Sud Sahariennes de lAfrique (DWSSA). Dans le but de détecter des anticorps H5 et H7, le test dinhibition de lhémagglutination (HI) et les deux tests ELISA de compétition (ELISA-H5 et ELISA-H7) utilisant des anticorps monoclonaux (Mabs) spécifiques des sous types H5 et H7 ont été appliqués. Aucune de ces espèces doiseaux aquatiques, na été trouvée séropositive vis-à-vis du sous type H7, alors que seuls des sérums de canards ont été trouvés positifs vis-à-vis de H5 par les tests HI et ELISA. Les séroprévalences les plus élevées vis-à-vis de H5 ont été détectées par lELISA H5. Au total, 5% (10/201) des DWMA se sont révélés positifs par ce test, avec des séroprévalences annuelles allant de 2% (2/123) à 12% (6/51). Dans cette étude, seuls 5 virus appartenant aux sous types H1N1, H11N6, et H2N3 ont été isolés à partir de canards. Toutefois, la séroconversion H5 observée chez un canard col vert au début de lannée 1998 indique que la circulation de virus H5 était également présente dans la zone où a été réalisée létude. Influenzaviruszirkulation in Wild-Wasservögeln während der H5N2- und H7N1-Epidemien (1998-2000) beim Geflügel in Italien In Italien traten beim Geflügel in den Jahren 1997/98 und 1999/2000 zwei durch die hochpathogenen H5- bzw. H7-Viren verursachten Influenzaepidemien auf. Die Zirkulation dieser Subtypen im wildlebendem Wassergeflügel wurde durch die Untersuchung von 638 Kloakenabstrichen und 621 Serumproben von 150 Möwen, 162 Wasserhühnern und 326 Enten, die in italienischen Feuchtgebieten zwischen 1998 und 2000 eingefangen wurden, überprüft. Das Vorkommen spezifischer Antikörper gegen Influenza A-Viren wurde mittels eines Doppel-Antikörper-Sandwich-Blocking-ELISA bei 11 % der Möwen, 16 % der Wasserhühner und 45 % der Enten nachgewiesen. Innerhalb der Familie der Anatidae wiesen die Entenspezies, die im Mittelmeerraum (DWMA) überwintern, signifikant höhere Werte auf als die Enten, die den Winter im Bereich der südlichen Sahara (DWSSA) in Afrika verbringen. Zum Nachweis von H5- und H7-Antikörpern wurden der Hämagglutinationshemmungs (HAH)- Test und zwei kompetitive ELISA-Tests (H5-ELISA und H7-ELISA) mit H5- und H7-Subtyp spezifischen monoklonalen Antikörpern (Mabs) durchgeführt. Keine der Wasservogelspezies war seropositiv gegen den H7-Subtyp, während H5-positive Seren sowohl mit dem HAH als auch den ELISAs nur bei Enten festgestellt wurden. Die höchsten H5-Seroprävalenzen wurden mittels des H5-ELISA ermittelt., insgesamt waren 5 % (10/201) der DWMA positiv in diesem Test mit jährlichen Seroprävalenzen zwischen 2 % (2/123) und 12 % (6/51). In der vorliegenden Studie wurden nur 5 Influenza A-Viren isoliert, die als H1N1, H11N6 und H2N3-Viren typisiert worden sind. Die H5-Serokonversion bei einer Stockente Anfang 1998 zeigt jedoch, dass das H5-Virus auch in dem untersuchten Areal zirkuliert. Circulación de virus de influenza en aves salvajes acuáticas en Italia durante los periodos de epidemia con H5N2 y H7N2 en aves de producción (1998-2000) Dos epidemias de influenza aviar debidas a virus altamente patogénicos H5 y H7 ocurrieron en aves de producción en Italia en 1997/98 y 1999/2000, respectivamente. La circulación de estos serotipos en aves salvajes acuáticas fue investigado mediante el examen de 638 hisopos cloacales y 621 sueros recogidos de 150 gaviotas, 162 fochas, y 326 patos atrapados en humedales italianos desde 1998 al 2000. Las seroprevalencias frente a virus de influenza A, detectados mediante un ELISA de sandwich de bloqueo con doble anticuerpo fueron del 11.3% en gaviotas, 16% en fochas y 45% en patos. Entre el grupo de Anatidae, las especies de patos invernando en las áreas del Mediterráneo (DWMA) mostraron valores significativamente mayores que los patos invernando en las áreas Subsaharianas (DWSSA) de África. Con el objetivo de detectar anticuerpos frente a H5 y H7, se utilizaron la técnica de inhibición de la hemaglutinación (HI) y dos técnicas de ELISA competitivas (H5-ELISA y H7-ELISA) mediante el uso de anticuerpos monoclonales (MAbs) específicos para los subtipos H5 y H7. Ninguna de las especies de aves acuáticas fueron seropositivas al subtipo H7, mientras que sí que se encontraron sueros positivos H5 tanto mediante HI como de ELISA en patos únicamente. Las seroprevalencias más altas frente a H5 fueron detectadas mediante H5-ELISA; en general, 5% (10/201) de los DWMA fueron positivas mediante esta técnica, con seroprevalencias anuales del 1.6% (2/123) hasta 11.8% (6/51), en 1998 y 1999 respectivamente. En el presente estudio, únicamente 5 virus pertenecientes a los subtipos H1N1, H11N6, y H2N3 fueron aislados de patos. Aún así, la seroconversión a H5 observada en un ánade real a inicios del 1998 indica que la circulación de virus H5 también ocurrió en el área de estudio.

Long-term Monitoring for Avian Influenza Viruses in Wild Bird Species in Italy

M.A. De Marco1*, E. Foni2, L. Campitelli3, E. Raffini4, M. Delogu5 and I. Donatelli3 1Istituto Nazionale per la Fauna Selvatica, Ozzano Emilia (BO); 2Istituto Zooprofilattico Sperimentale della L ombardia e dell’Emilia Romagna, Parma; 3Department of V irology, Istituto Superiore di Sanita, Rome; 4Istituto Zooprofilattico Sperimentale della L ombardia e dell’Emilia Romagna, L ugo (RA); 5Department of Public Health and Animal Pathology, Faculty of Veterinary Medicine, University of Bologna, Italy *Correspondence: Istituto Nazionale per la Fauna Selvatica ‘ A. Ghigi ’, 9 V ia Ca’ Fornacetta, 40064 Ozzano Emilia (BO), Italy E-mail: infs.adem@iperbole.bologna.it

Ecological Aspects of Influenza A Virus Circulation in Wild Birds of the Western Palearctic

M. Delogu1*, M.A. De Marco2, I. Donatelli3, L. Campitelli3 and E. Catelli1 1Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Facolta di Medicina Veterinaria, Universita degli Studi di Bologna, Ozzano Emilia (BO); 2Istituto Nazionale per la Fauna Selvatica, Ozzano Emilia (BO); 3L aboratory of V irology, Istituto Superiore di Sanita, Rome, Italy. *Correspondence: Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Facolta di Medicina Veterinaria, Universita degli Studi di Bologna, 50 via T olara di Sopra, 40064 Ozzano Emilia (BO), Italy E-mail: delogu@vet.unibo.it

Serological evidence of avian pneumovirus infection in reared and free-living pheasants

The study was funded by the French Ministry of Agriculture and Fisheries, and followed by a scientific steering committee (Marc Girard, CERVI, Lyon, France, Jeanne Brugere-Picoux, ENVA, Maisons-Alfort, France, Dominique Costagliola, INSERM SC4, Paris, France, Jean-Claude Desenclos, INVS, SaintMaurice, France, Marc Eloit, ENVA, Maisons-Alfort, France, Dagmar Heim, Office veterinaire federal, Liebefeld, Switzerland, Francois Moutou, AFSSA, Maisons-Alfort, France, Annick Tibi, Pharmacie centrale des h6pitaux, Paris, France). The authors would especially like to thank J-N. Arsac, N. Besson, E. Charmette, S. Dechavanne and L. Maitrias (AFSSA Lyon) for their excellent technical assistance, and F. Gauchard and S. La Vieille (AFSSA Derns) for their efficient and valuable assistance in scientific coordination. The study was made feasible by the involvement of the veterinary practitioners, the CVLS, the county veterinary services and the Direction generale de lalimentation of the MAF in charge of the overall logistics of the study.

Avian Pneumovirus infection in turkey and broiler farms in Italy: a virological, molecular and serological field survey

Abstract Avian Pneumovirus (APV) is the casual agent of Turkey Rhinotracheitis (TRT), and also causes a respiratory infection in chickens, which can result in Swollen Head Syndrome. A survey of APV infection in Italian turkey and broiler farms, in a highly populated area of Northern Italy (Verona Province) is reported. Nine turkey farms and 6 broiler farms were sampled. Sixteen birds from each group were doubly swabbed from the choanal cleft for virus isolation on tracheal organ cultures (TOC) and RT nested PCR (A and B type specific) using extracted RNA. At the same time blood samples were collected for a blocking ELISA serological assay. The broiler samples for virological assays were treated with infectious bronchitis virus (IBV) antiserum raised against serotypes prevalent in the areas sampled, thus avoiding competitive growth of IBV on TOC. Ciliostasis on TOC was taken as the indicator of the presence of the virus, and confirmation was by indirect immunofluorescence. APV was isolated and detected by RT-PCR in 19 day-old turkeys, and in 34, 42 and 48 day-old broilers. All APV strains were found to be type B. All turkeys of more than 4 weeks old were APV positive by ELISA. APV infection was found to be widely spread in the area sampled and the protocol used for virus isolation was shown to be effective. Turkey rhinotracheitis first appeared in Italy in the late 1980s, and the APV isolates involved were subsequently characterised as B types. Our results confirm APV B type to be present in Italy, but the limited findings to date need to be extended by further surveys of other Italian regions.

Bayesian skyline plot inference of the Toscana virus epidemic: a decline in the effective number of infections over the last 30 years.

Toscana virus (TosV), a sandfly fever virus, is one of the main causes of the aseptic meningitis that occurs during the summer in some Mediterranean regions, and whose epidemiology is largely unknown. We used a Bayesian Markov Chain Monte Carlo approach and a relaxed molecular clock to estimate the demographic history of the TosV infection in a series of isolates sampled between 1980 and 2003. The estimated mean evolutionary rate was 2.5 x 10(-4) substitutions per site per year (95% HPD: 0.31-5.44 x 10(-4)subs/site/year). Bayesian skyline plot revealed a sharp decline in the effective number of infections over the last 30 years. In conclusion, our data suggest that continuous and prolonged perturbations of vector/phlebovirus interactions due to the relatively recent climate changes may have contributed to gradually reducing the viral population in endemic areas.