Denaturing Gradient Gel Electrophoresis (DGGE) as a Powerful Novel Alternative for Differentiation of Epizootic ISA Virus Variants

Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses

This work received support from the Biotechnology and Biological Sciences Research Council (grant BB/M010996/1) and Marine Scotland Science. The authors thank Dr Elena Fringuelli (Agri-Food and Biosciences Institute, Belfast) for providing some of the SAV material used.Peer reviewedPublisher PD

REPLICATION OF LOW PATHOGENIC AVIAN INFLUENZA VIRUS IN NATURALLY INFECTED MALLARD DUCKS (ANAS PLATYRHYNCHOS) CAUSES NO MORPHOLOGIC LESIONS

Although the Mallard (Anas platyrhynchos) is considered an important maintenance host for low pathogenic avian influenza (LPAI) viruses, viral cell tropism and pathology in naturally infected birds are largely unknown. In August 2006, we collected 19 free-living hatch-year Mallards that were positive for LPAI virus by real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) in combined oropharyngeal and cloacal swabs. We investigated virus infection and associated lesions in the digestive and respiratory tracts by RRT-PCR, virus culture, immunohistochemistry (IHC), and histology. By RRT-PCR, 15 birds were positive in cloacal bursa, colon/cloaca, or both, and three were positive in lungs. Virus was isolated from eight birds and typed as H2N3 (three birds), H3N3 (two birds), H3N8 (one bird), H4N6 (one bird), and H?N3 (one bird). By IHC, birds were positive in the cloacal bursa (eight birds), colon (three), cecum (two), or ileum (one). Cell types infected were superficial epithelial cells of the bursa and epithelial cells of the intestinal villi and, less commonly, mucosal glands. By histology, there was no evidence of lesions associated with LPAI virus infection. These results show that epithelia of the cloacal bursa and of the lower intestine are important sites of natural LPAI virus infection in free-living hatch-year Mallards. The lack of lesions associated with this infection suggests that there is a strong selection by LPAI virus to cause minimal virulence in this maintenance host species