Didier Toquin

Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup

Sequence analysis was performed of all or part of the genes encoding the fusion (F), polymerase (L) and attachment (G) proteins of two French non-A/non-B avian pneumovirus (APV) isolates (Fr/85/1 and Fr/85/2). The two isolates shared at least 99.7% nt and 99.0% aa sequence identity. Comparison with the F genes from subgroup A, subgroup B or Colorado APVs revealed nt and aa identities of 70.0-80. 5% and 77.6-97.2%, respectively, with the L gene sharing 76.1% nt and 85.3% aa identity with that of a subgroup A isolate. The Fr/85/1 and Fr/85/2 G genes comprised 1185 nt, encoding a protein of 389 aa. Common features with subgroup A and subgroup B G proteins included an amino-terminal membrane anchor, a high serine and threonine content, conservation of cysteine residues and a single extracellular region of highly conserved sequence proposed to be the functional domain involved in virus attachment to cellular receptors. However, the Fr/85/1 and Fr/85/2 G sequences shared at best 56.6% nt and 31.2% aa identity with subgroup A and B APVs, whereas these isolates share 38% aa identity. Phylogenetic analysis of the F, G and L genes of pneumoviruses suggested that isolates Fr/85/1 and Fr/85/2 belong to a previously unrecognized APV subgroup, tentatively named D. G-based oligonucleotide primers were defined for the specific molecular identification of subgroup D. These are the first G protein sequences of non-A/non-B APVs to be determined.

Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses

SummaryClassical serotype 1 infectious bursal disease viruses (IBDV), but not very virulent (vv) isolates, react with neutralizing monoclonal antibody (NMab) 3 in virus neutralization tests or antigen-capture ELISA. Two other NMabs, 6 and 8, bind to both classical and most vv strains, but not to the atypical 94 432 and 91 168 vv strains, respectively. The basis for such reactivities was investigated by sequencing the genome region encoding the VP2 major immunogenic domain. In classical, variant, vaccine or vv IBDV strains, negative reactions with NMab3 were associated with changes in the Proline-Glycine pair at amino-acid (aa) positions 222–223 (hydrophilic peak A), and negative reactions with NMabs 6 and 8 with aa changes from positions 318 to 324 (hydrophilic peak B). The 91 168 and 94 432 viruses are the first vvIBDVs to present aa changes in peak B.

Antigenic and genetic relationships between European very virulent infectious bursal disease viruses and an early West African isolate

The antigenic and genetic relationships between very virulent (vv) infectious bursal disease viruses (IBDV) from different countries were investigated. Antigenic characterization was performed using an antigencapture ELISA based on a panel of seven neutralizing monoclonal antibodies (Mabs), which probe at least three VP2-located antigenic domains. All these domains are reactive in the Faragher 52/70 (F52/70) reference strain for European classical serotype 1 IBDV. Genomic characterization was achieved by reverse transcription, amplification and direct sequencing of a genome fragment encoding the VP2 variable domain. Eleven vv isolates from France were compared to the British, Dutch and Belgian UK661, DV86 and 849VB viruses, and to an early vv isolate obtained from the Ivory Coast in 1988. All viruses exhibited antigenic profiles characterized by no binding of Mabs 3 and 4. Lack of binding of Mabs 3 and 4 might thus be helpful for differentiating classical and vvIBDVs. None of the non-French strains resemb...

Comparison of F-, G- and N-based RT-PCR protocols with conventional virological procedures for the detection and typing of turkey rhinotracheitis virus.

SummaryFifty-six reverse transcriptions followed by a polymerase chain reaction (RT-PCR) were developed and/or assessed to detect and to type turkey rhinotracheitis virus (TRTV). Twenty-seven primers corresponding to sequences either common to both A and B viruses, or type-specific were respectively defined in the fusion (F), attachment (G) and nucleocapsid (N) proteins genes. Only one N-based RT-PCR detected 21/21 TRTVs isolated in four countries since 1985. Molecular typing (RT-PCR) and antigenic typing (ELISA) showed that TRTV strains antigenically related either to the 3BOC18 (UK/85/1) or to the 86004 (Fr/86/1) viruses belonged to the A or B genomic type respectively. Neither typing approach allowed assignment of two 1985 French isolates (Fr/85/1 and Fr/85/2) to either type A or B, these strains might thus belong to a third type. RT-PCR assays on tracheal and nasal swabs sampled during experimental and field infections significantly outperformed concurrent virus isolation in tissue culture and ELISA: G- and N-based RT-PCRs detected more positive samples than conventional methods. Molecular and serological results were concordant and demonstrated that all the recent French field viruses belonged to type B. Thus, N- and G- based RT-PCR are respectively specific and sensitive tools for rapid diagnosis and typing of TRTV in field samples.

Modified activity of a VP2-located neutralizing epitope on various vaccine, pathogenic and hypervirulent strains of infectious bursal disease virus

SummaryNine monoclonal antibodies (Mabs) to a vaccine strain of infectious bursal disease virus (IBDV) of intermediate virulence were characterized in Western-blot, radioimmunoprecipitation, ELISA additivity, and neutralization assays. At least two distinct serotype 1-specific conformation-dependent overlapping neutralizing antigenic domains were shown to be present on IBDV-VP2, and were respectively probed by Mabs 3 and 4, and by Mabs 6 and 7. Ten serotype 1 vaccine or pathogenic IBDV strains were tested for neutralization. Most mild or intermediate vaccine strains were efficiently neutralized by all Mabs, whereas US variant A, European pathogenic strain Faragher 52/70 and French hypervirulent isolate 89 163 were not neutralized by Mabs 3 and 4. In addition, these two Mabs were shown to bind to the Faragher 52/70 strain, but not to the 89 163 isolate, in an antigen-capture ELISA. These results suggest that a neutralizing epitope is possibly modified in European pathogenic IBDV strains, and that Mabs 3 and 4 may prove useful for antigenic differentiation between European classical and hypervirulent isolates.

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.

Limited antigenic variation among recent infectious bursal disease virus isolates from France

SummarySeven neutralizing monoclonal antibodies (Mabs) to infectious bursal disease virus (IBDV) were used in an antigen-capture ELISA for the antigenic characterization of 58 IBDV isolates obtained in France since 1989. Fifty-six isolates exhibited an antigenic profile which was different from reference strain Faragher 52/70, and similar to French very virulent IBDV strain 89 163 (no binding of two Mabs). Two strains (3.4%), isolated in 1991 and 1994, showed additional antigenic modifications resulting in suppressed or reduced binding activity of three other Mabs. The two atypical viruses were not re-identified in field samples subsequently collected, hence showing that antigenic variants of IBDV do not tend to replace the antigenically dominant 89 163-like strains that have prevailed in France since 1989.

Both Genome Segments Contribute to the Pathogenicity of Very Virulent Infectious Bursal Disease Virus

ABSTRACT Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.

Deletion of the SH gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the G gene or M2-2 open reading frame.

Subgroup A avian metapneumoviruses lacking either the SH or G gene or the M2-2 open reading frame were generated by using a reverse-genetics approach. The growth properties of these viruses were studied in vitro and in vivo in their natural host. Deletion of the SH gene alone resulted in the generation of a syncytial-plaque phenotype and this was reversed by the introduction of the SH gene from a subgroup B, but not a subgroup C, virus. Infected turkeys were assessed for antibody production and the presence of viral genomic RNA in tracheal swabs. The virus with a deleted SH gene also showed the greatest impairment of replication both in cell culture and in infected turkeys. This contrasts with the situation with other pneumoviruses in culture and in model animals, where deletion of the SH gene results in little effect upon viral yield and a good antibody response. Replication of the G- and M2-2-deleted viruses was impaired more severely in turkeys than in cell culture, with only some animals showing evidence of virus growth and antibody production. There was no correlation between virus replication and antibody response, suggesting that replication sites other than the trachea may be important for induction of antibody responses.

Serological survey of the king penguin, Aptenodytes patagonicus, in Crozet archipelago for antibodies to infectious bursal disease, influenza A and Newcastle disease viruses

Abstract. Sera from adult and chick king penguins were examined for antibodies to infectious bursal disease, influenza A and Newcastle disease viruses. This study was completed by a 1-year survey of clinical signs of disease in the colony. About 4% and 1% of the birds were positive for serotypes 1 and 2, respectively, of infectious bursal disease virus. In 1996, two coughing peaks occurred in adults, during autumn and spring, and a peak of conjunctivitis occurred at the end of the winter. Although this showed the presence of infectious agents, it was not possible to be precise about whether the detected viruses were responsible for the clinical signs, or to assess if the infectious agents were native or introduced.