Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 from emus from the Ein Gedi oasis by the Dead Sea.

An avian influenza virus (AIV), A/Emu/Israel/552/2010/(H5N1), was isolated from a dead emu that was found in the Ein Gedi oasis near the Dead Sea. The virus molecular characterization was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR using AIV subtype-specific primers. The virus was of high pathogenicity, according to its intravenous pathogenicity index of 2.85 and the nucleotide sequencing at the cleavage site of the hemagglutinin gene, GERRRKKR, which is typical for highly pathogenic chicken influenza A viruses.

Fatal Infection in a Wild Sandbar Shark (Carcharhinus plumbeus), Caused by Streptococcus agalactiae, Type Ia-ST7.

Streptococcus agalactiae is one of the most important fish pathogenic bacteria as it is responsible for epizootic mortalities in both wild and farmed species. S. agalactiae is also known as a zoonotic agent. In July 2018, a stranded wild sandbar shark (Carcharhinus plumbeus), one of the most common shark species in the Mediterranean Sea, was found moribund on the seashore next to Netanya, Israel, and died a few hours later. A post-mortem examination, histopathology, classical bacteriology and advanced molecular techniques revealed a bacterial infection caused by S. agalactiae, type Ia-ST7. Available sequences publicly accessible databases and phylogenetic analysis suggest that the S. agalactiae isolated in this case is closely related to fish and human isolates. To the best of our knowledge, this is the first description of a fatal streptococcosis in sandbar sharks.

Optimized polypeptide for a subunit vaccine against avian reovirus.

Avian reovirus (ARV) is a disease-causing agent. The disease is prevented by vaccination with a genotype-specific vaccine while many variants of ARV exist in the field worldwide. Production of new attenuated vaccines is a long-term process and in the case of fast-mutating viruses, an impractical one. In the era of molecular biology, vaccines may be produced by using only the relevant protein for induction of neutralizing antibodies, enabling fast adjustment to the emergence of new genetic strains. Sigma C (SC) protein of ARV is a homotrimer that facilitates host-cell attachment and induce the production and secretion of neutralizing antibodies. The aim of this study was to identify the region of SC that will elicit a protective immune response. Full-length (residues 1-326) and two partial fragments of SC (residues 122-326 and 192-326) were produced in Escherichia coli. The SC fragment of residues 122-326 include the globular head, shaft and hinge domains, while eliminating intra-capsular region. This fragment induces significantly higher levels of anti-ARV antibodies than the shorter fragment or full length SC, which neutralized embryos infection by the virulent strain to a higher extent compared with the antibodies produced in response to the whole virus vaccine. Residues 122-326 fragment is assumed to be folded correctly, exposing linear as well as conformational epitopes that are identical to those of the native protein, while possibly excluding suppressor sequences. The results of this study may serve for the development of a recombinant subunit vaccine for ARV.

Wide-range protection against avian reovirus conferred by vaccination with representatives of four defined genotypes.

Many isolates of the contagious avian reovirus have been characterized, mainly based on the sequence of their sigma C protein. These isolates have been classified into four genotypes. Currently available vaccines are of limited effectiveness, likely due to the existence of many variants. The aim of this study was to test the efficacy of a vaccine consisting of a mixture of prototypes (representatives) of the four defined genotypic groups of avian reovirus. The prototypes were selected based on their distance from the isolates within each genotype. All prototypes were found to be virulent. Antibodies produced against each of the prototypes neutralized all members of its genotype. Birds were then vaccinated with a mixture of the four prototypes. Results suggest that the 4-valent vaccine can prevent disease and confer broad protection against field isolates of avian reovirus.

Differentiating infected from vaccinated animals, and among virulent prototypes of reovirus.

Birds are most susceptible to infection by avian reovirus, genus Orthoreovirus family Reoviridae, at a young age. Although chicks are protected by antibodies transferred from vaccinated maternal flocks, due to the many variants in the field, the efficiency of the vaccines is limited. The level of antibodies against viruses is generally determined by enzyme-linked immunosorbent assay (ELISA), using the whole virus as the antigen. This has some disadvantages: first, the test measures antibodies against all capsid proteins, most of which are irrelevant for neutralizing the virus, and as such does not reflect the real protection status; second, it is impossible to distinguish between vaccine- and infection-derived antibodies. In the case of a virus that changes frequently, a third disadvantage is the inability to distinguish among serotypes. The aim of this study was to develop a test that would address these concerns. Four prototypes of the avian reovirus protein sigma C were used as antigens on the ELISA plate. Sigma C is the main protein inducing neutralizing antibodies and the most variable among strains and isolates, and it is used for reovirus classification. This differentiating ELISA enabled distinguishing between vaccine and field strains of the virus, identifying the infection source, and in the case of vaccination, exclusively determining the level of protective antibodies. Whereas the whole virus detected antibodies against all strains, differentiating ELISA enabled differentiating between infected and vaccinated animals (DIVA) and in most cases, identifying the sigma C genotype. In a field study, a correlation was found between disease symptoms and antibodies identified against virulent strains in the flock. Thus virulent strains can be identified in the field, enabling adjustment of the relevant vaccines.