Detection and characterization of a novel marine birnavirus isolated from Asian seabass in Singapore.

BACKGROUND: Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods. METHODS: In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2'-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses. RESULTS: The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days' post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family. CONCLUSIONS: These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence.

Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤ 2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.

Development of Diagnostic Quality Metrics for Prosthetic Joint Infection.

ABSTRACT: Although well-accepted clinical practice guidelines exist for the diagnosis of prosthetic joint infection (PJI), little is known about the quality of diagnosis for PJI. The identification of quality gaps in the diagnosis of PJI would facilitate the development of care structures and processes to shorten time to diagnosis and reduce the significant morbidity, mortality, and economic burden associated with this condition. Hence, we sought to develop valid clinical quality measures to improve the timeliness and accuracy of PJI diagnosis. We convened a nine-member multidisciplinary national panel of PJI experts including orthopedic surgeons, infectious disease specialists, an emergency medicine physician, and a patient previously treated for PJI to review, discuss, and rate the validity of proposed measures using a modification of the RAND-UCLA appropriateness method. In total, 57 permutations of six proposed measures were rated. Populations considered to be at high enough risk for PJI that certain care processes should always be performed were identified by the panel. Among the proposed quality measures, the panel rated five as valid. These novel clinical quality measures could provide insight into care gaps in the diagnosis of PJI.