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.

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.

A novel endopeptidase with a strict specificity for threonine residues at the P1' position.

An endopeptidase was purified from Archachatina ventricosa by chromatography on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose. The preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular weights of 90,000 and 121,000. The protease exhibited maximum proteolytic activity at 55 degrees C and at pH 8.0, but it retained more than 85% of its activity in the pH range 7.5 to 8.5. It was completely inactivated by the chelating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibitors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydrolyzed by the purified protease. This endopeptidase constitutes a novel tool for the study of proteins in view of its narrow and unique substrate specificity.