Abstract
Background: The genomes of several infectious pancreatic necrosis virus (IPNV) isolated in Chile were sequenced, by using a single amplification approach for both segments A and B. The obtained sequences were used to investigate how conserved were the primer binding regions used in PCR-based diagnosis methods described in literature. The analysis allowed us to study the robustness of each technique, which could be affected in the eventual case of further mutations within the primer binding sites. Results: Analysis showed that most of the methods to detect Chilean IPNV varieties are currently adequate. However, the primers themselves were designed in function of specific genogroups, implying that most detection methods present some level of risk for detecting all strains present in the country, since genogroups 1 and 5 coexist. Conclusions: Considering IPNV high genomic variability, negative results must be taken with caution. The use of detection techniques (qRT-PCR) based on degenerate primers should be considered to minimize possibilities of false negative detections. Normal 0 21 false false false ES-CL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:ES-CL; mso-fareast-language:EN-US;}
Highlights
Infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family and the Aquabirnavirus genus. It is composed of an unenveloped icosahedral capsid and a bisegmented genome with double-stranded RNA
The calculated Tm for these pairs was much lower, approximately 30°C for both forward and reverse primers, which indicated a considerably weaker binding and in turn the experimental genogroup specificity stated in the original publication
It is worth noting that the use of probes are included in the complete protocol [8], which further increase specificity
Summary
Infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family and the Aquabirnavirus genus It is composed of an unenveloped icosahedral capsid and a bisegmented genome with double-stranded RNA (dsRNA). The longer segment, segment A (3097 bp), contains two ORFs. The larger ORF encodes for a 106-kDa polyprotein that is cleaved cotranslationally by the nonstructural protease VP4, generating mature pre-VP2 and VP3 (the major and minor capsid proteins, respectively), and VP4. The larger ORF encodes for a 106-kDa polyprotein that is cleaved cotranslationally by the nonstructural protease VP4, generating mature pre-VP2 and VP3 (the major and minor capsid proteins, respectively), and VP4 The sequence of this ORF has been used to classify Aquabirnaviruses into 6 distinct genogroups by their geographical origin. Detection techniques (quantitative reverse transcription (qRT)-PCR) based on degenerate primers can be used to minimize the possibilities of false-negative detections
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