Amplification and cloning of a long RNA virus genome using immunocapture-long RT-PCR

A rapid and easy method was developed in order to amplify long fragments of the genome of potato virus Y (PVY), a virus possesing a 10-kb genomic RNA. The method of immunocapture-RT-PCR was adapted, by using thermostable DNA polymerases with proofreading activity and the proper buffers and cycles, to amplify almost the whole genome of PVY in two fragments (5.6 and 4.3 kb) without purifying virions nor viral RNA. Both fragments were cloned subsequently and their ends sequenced. The method is applicable to the rapid cloning and molecular characterization of the genomes of many other RNA viruses

A strain-type clustering of potato virus Y based on the genetic distance between isolates calculated by RFLP analysis of the amplified coat protein gene

Potato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I, Eco RV, Hinf I, Rsa I and Taq I). Genetic distances between the different PVY 'restrictotypes' were calculated and used to define the PVY genetic strains. Three main clusters were found PVY(O), PVY(N), and non-potato PVY (PVY(NP)) in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed

Characterization of typical pepper-isolates of PVY reveals multiple pathotypes within a single genetic strain

Potato virus Y (PVY) isolates originally coming from infected pepper plants, were biologically and genetically characterized, especially in comparison with PVY potato-isolates. Pepper PVY isolates could be differentiated from potato isolates in their host range, aphid transmission efficiencies, Mab serology, and genetic status. The genetic distances estimated for PVY pepper-isolates, based on their restrictotypes with five restriction enzymes and on their coat protein gene sequences, indicated that they form a single genetic strain with different pathotypic properties. This situation is essentially different to that of PVY potato-isolates.This work was supported by INIA grants 8562 and SC94-105.Peer reviewe

Potato virus Y group C isolates are a homogeneous pathotype but two different genetic strains

Potato virus Y group C isolates (PVY(C)) have been characterized according to biological, molecular and genetic criteria. Two genetic strains PVY(C1) and PVY(C2), were identified on the basis of genetic distances (among them and other PVY strains), host range (ability or inability to infect pepper), MAb response (ELISA recognition with MAb 10E3) and coat protein processing site. Some characteristics, such as aphid transmission and ELISA using other MAbs, did not correlate with classification into these two genetic strains. All isolates tested induced a hypersensitive response on potatoes bearing the Nc resistance gene, confirming the nature of PVY(C) isolates as a homogeneous pathotype

Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

International audienc

Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

A collection of 38 PVY isolates from seed potato batches, originating from several Western European countries, was characterized by using current biological, serological and molecular tools differentiating PVY strains and groups. The correlation between the three kinds of tests was good but not absolute. No single serological or PCR method was able to discriminate among the five isolate groups found. Twenty-nine isolates belonged to the PVY(N) strain and six to the PVY(O) strain. No PVY(C) was found. Two other isolates reacted serologically like PVY(O), but were unable to elicit a hypersensitive response from the Ny(tbr) gene and probably represent the PVY(Z) group. At the molecular level, these two isolates showed a combination of both PVY(O) and PVY(N) and could be recombinants of these strains. Another isolate reacted serologically like PVY(O), but induced vein necrosis in tobacco, like PVY(N)-Wilga. Some PVY(N) isolates caused tuber ring necrosis in glasshouse conditions. These might belong to the PVY(NTN) group. The PVY(NTN), PVY(N)-Wilga and PVY(Z) groups probably represent pathotypes within strains PVY(N) and PVY(O), respectively. The present study also confirms previous reports showing a high genetic variation at the 5' end within the PVY(N) strain

The woodchuck interferon-alpha system: Cloning, family description, and biologic activity

Interferon-alpha (IFN-alpha) is a key element in the defense against viral infection because, in addition to a direct antiviral effect, it exhibits potent immunostimulatory activity. To investigate the function of this cytokine in the woodchuck model of chronic hepatitis B, the woodchuck IFN-alpha gene (IFNA) family was cloned and examined. The data indicate that this is a multigenic family from which 12 IFNA functional sequences and four pseudogene sequences were isolated. The overall identity of the amino acid sequence among the members of the woodchuck IFN-alpha family is 85%, and the identity with the IFN-alpha family from other species such as mice and humans is 50%. The analysis of hepatic expression of IFNA genes showed that wIFNA5a was the subtype transcribed preferentially in the woodchuck liver. The wIFNA genes transcribed in the liver were tested in an eukaryotic expression system and were found to enhance 2-5-oligoadenylate synthetase (2-5-OAS) mRNA levels and to posses a potent antiviral activity. Cloning of woodchuck IFNA genes will allow testing diverse forms of IFN-alpha delivery as well as different combination therapies in woodchuck hepatitis virus infection, thus providing useful information for the design of new strategies for the treatment of patients with chronic hepatitis B