First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

An increasing incidence of enteric disorders clinically suggestive of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time reverse transcriptase-polymerase chain reaction assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37% of the intestinal samples collected from diseased turkey flocks. The full-length spike (S) gene of these viruses was amplified, cloned and sequenced from three samples. The French S sequences shared 98% identity at both the nucleotide and amino acid levels, whereas they were at most 65% and 60% identical with North American (NA) TCoV and at most 50% and 37% identical with IBV at the nucleotide and amino acid levels, respectively. Higher divergence with NA TCoV was observed in the S1-encoding domain. Phylogenetic analysis based on the S gene revealed that the newly detected viruses form a sublineage genetically related with, but significantly different from, NA TCoV. Additionally, the RNA-dependent RNA polymerase gene and the N gene, located on the 5' and 3' sides of the S gene in the coronavirus genome, were partially sequenced. Phylogenetic analysis revealed that both the NA TCoV and French TCoV (Fr TCoV) lineages included some IBV relatives, which were however different in the two lineages. This suggested that different recombination events could have played a role in the evolution of the NA and Fr TCoV. The present results provide the first S sequence for a European TCoV. They reveal extensive genetic variation in TCoV and suggest different evolutionary pathways in North America and Europe.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

Lack of antigenic relationship between French and recent North American non-A/non-B turkey rhinotracheitis viruses.

Twelve turkey rhinotracheitis viruses (TRTVs) including the Colorado isolate and two French non-A/non-B viruses were serologically compared. Six enzyme-linked immunosorbent assay (ELISA) antigens derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado TRTVs were used. Virus neutralization (VN) tests were performed with four Ma-104-adapted viruses derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado viruses. French strains isolated since 1995 were assigned to subgroup B in both ELISA and VN, whereas those isolated in 1985 and 1986 appeared more diverse: two strains belonged to subgroup B, one to subgroup A, and two others appeared antigenically different from both the A and B subgroups and are classified as non-A/non-B. The Colorado strain appeared different from these three groups of TRTVs. Assignment to subgroup A or B was confirmed by reverse transcription-polymerase chain reaction, but neither the French non-A/non-B strains nor the Colorado virus could be classified with the subgroup-specific G-based primers. These results suggest that at least three antigenically different viruses were present in France in 1985-86 and that the Colorado strain is different from all European TRTVs. Further serologic and phylogenic studies will be necessary to evaluate their actual prevalences and relationships.

Respiratory disease (rhinotracheitis) of turkeys in Brittany, France. III. Interaction of multiple infecting agents.

A candidate breeder flock of turkeys was studied during and after an outbreak of rhinotracheitis. Laboratory studies revealed the presence of three pathogens during the acute phase of the disease. These agents were hemorrhagic enteritis virus (HEV), paramyxovirus type 2 (PMV2), and chlamydia. Twenty-five turkeys in the flock were individually identified, and blood samples from these birds were collected for serological studies each week for 21 weeks. The serological results revealed high titers for HEV and chlamydia but very low titers for PMV2.

Respiratory disease (rhinotracheitis) in turkeys in Brittany, France, 1981-1982. II. Laboratory findings.

After discovering that numerous turkey flocks experiencing rhinotracheitis in Brittany, France, had antibodies against chlamydia, laboratory studies were conducted to determine whether chlamydia and/or viruses would explain the respiratory disease observed. Although both lentogenic paramyxoviruses of type 1 (Newcastle disease virus) and Chlamydia psittaci were isolated, it was concluded, based on epidemiologic and other laboratory findings, that C. psittaci was the primary cause of the disease.

Infectious bursal disease (Gumboro disease).

Infectious bursal disease (IBD) (Gumboro disease) has been described throughout the world, and the socio-economic significance of the disease is considerable world-wide. Various forms of the disease have been described, but typing remains unclear, since antigenic and pathotypic criteria are used indiscriminately, and the true incidence of different types is difficult to determine. Moreover, the infection, when not fatal, leads to a degree of immunosuppression which is often difficult to measure. Finally, the control measures used are subject to variations, and seldom follow a specific or standardised plan. In the context of expanding international trade, the authors provide an overview of existing knowledge on the subject to enhance available information on the epidemiology of IBD, the identification of reliable viral markers for diagnosis, and the implementation of specific control measures to ensure a global and co-ordinated approach to the disease.

European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages.

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus.

A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 x 10(7) clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

Significance of the genetic relationships deduced from partial nucleotide sequencing of infectious bursal disease virus genome segments A or B.

The rapid genomic characterization of infectious bursal disease virus (IBDV) requires determining which partial nucleotide (nt) sequences derived from IBDV segments A or B would produce phylogenetic information as significant as sequencing the whole corresponding segments. Long nt coding sequences of 27 IBDV segments A (aa 20-991) and 21 segments B (aa 7-stop codon) were retrieved from databanks and used to compute reference phylogenetic trees using Neighbor Joining (NJ) and Parsimony (P): clusters appearing in the NJ and P reference trees with a bootstrap value greater than 80% were considered as significant (Whole Segment Clusters, WSC). The sequences were then cut into overlapping regions. These were used to compute phylogenetic trees which were compared with reference ones. Of the partial sequences, the VP2 gene best represented IBDV segment A (10 out of 13 WSC were conserved), and the 5' two thirds of segment B best represented segment B (5 to 6 conserved WSC out of 6). Implementation of the Plato programme finally demonstrated that the region encoding VP2 variable domain (vVP2, segment A) is the only region of IBDV genome with a significantly different evolution rate, which result is consistent with vVP2 being subjected to a high selection pressure.

[Observations and isolation of pseudopicornavirus from sick turkeys]. / Observations et isolements de pseudopicornavirus a partir de dindonneaux malades.

Forty-nine sick turkeys (from 31 flocks), 2- to 6-week-old, were examined. They had enteritic and respiratory signs. Direct electron microscopy, inoculation of embryonated eggs by the yolk sac route and inoculation of young SPF turkeys with materials from affected turkeys showed in several cases presence of picornalike virus. Two strains of virus were isolated.

[Electron microscopical observations on materials from diseased turkeys]. / Observations au microscope electronique a partir de prelevements de dindes presentant des troubles pathologiques.

Many types of infectious agent have been observed during the past 2 years in studies on diseased turkeys in Brittany. Electron microscopy was found to be a very useful tool. This paper presents illustrations of a variety of infectious agents seen in the material examined by negative staining or in ultrathin sections.

[Comparison of sensitivity of four turkey genetic types infected with virulent or attenuated strains of haemorrhagic enteritis virus]. / Sensibilite comparee de quatre varietes genetiques de dinde a des souches virulentes ou attenuees du virus de l'enterite hemorragique.

Four turkey genetic strains reacted differently to an experimental infection with different strains, virulent or attenuated, of haemorrhagic enteritis virus. Criteria used were: clinical signs (diarrhoea, mortality), daily weight gain, histopathology and virus recovery in the spleen. Turkeys of one genetic type developed more quickly the disease; the serological response to vaccine using ELISA and agar gel precipitation techniques was also faster. Turkeys of two other genetic types responded later both to the virus and the vaccine. Turkeys of the fourth genetic type responded last. The origin of this discrepancy is discussed.