Despite being known for almost half a century, Vibrio harveyi was considered only a pretty, biolu... more Despite being known for almost half a century, Vibrio harveyi was considered only a pretty, bioluminescent, environmental bacterium (Color Plate 13) that made balmy nights more magical when it contributed to water bioluminescence as bathers splashed around tropicallagool1s. With the advent of large scale prawn aquaculture, V. harveyi started to get attention as a pathogen, particularly in Australia and the Philippines. From the mid-1980s, more and more animals, particularly invertebrates in the tropics and fish in the more temperate areas, were recorded as being infected by V. harveyi. Recently, literature on V. harveyi has gained a wider audience as control of its bioluminescent pathway is elucidated and shown to be substantially different .from and more complicated than that of Vibrio fischeri. Similarly, the bacteriophage- mediated virulence in V. harveyi has also made scientists more interested in this bacterium.
[Extract] The ecology of viruses predominately involves the interaction of the virus at the anima... more [Extract] The ecology of viruses predominately involves the interaction of the virus at the animal and cellular level. When the virions are in the extracellular environment, they are quiescent waiting to infect a living cell. The virions bind to the cell receptors, undergo decapsidation in the phagolysosome, pass the nucleic acid to the cytoplasm or nucleus and begin replication. Evidently, the ecology of viroses starts with entry of the virus into the crustacean, evading the immune system, invasion of the cell, replication, and release of new virions. The crustacean immune system is critical to the functional ecology of their viruses, so we must start with an understanding of this system to have any hope of understanding the ecology of the viruses in crustaceans.
Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merg... more Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merguiensis leading to at least 28% production loss due to reduced growth rates and mortality of juveniles. In the present study, we reduced PmergDNV titres and subsequent mortality by feeding Acheta domesticus (previously determined as an appropriate animal model for P. merguiensis) with dsRNA specific to the capsid protein by mixing it into their food. Feeding A. domesticus with PmergDNV-specific dsRNA in advance of viral challenge increased their longevity, decreased mortality by 84.4% and reduced viral loads 24-fold below the threshold level required for mortality. Mortalities and viral loads were significantly (both P < 0.001) lower in treatments challenged with PmergDNV following exposure to bacterially expressed PmergDNV-dsRNA. This is the first study to demonstrate gene silencing via RNAi against PmergDNV in vivo through oral administration of live bacteria expressing dsRNA in a model system.
Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging di... more Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi-square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10(5) cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10(5) cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10(4) and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.
Despite being known for almost half a century, Vibrio harveyi was considered only a pretty, biolu... more Despite being known for almost half a century, Vibrio harveyi was considered only a pretty, bioluminescent, environmental bacterium (Color Plate 13) that made balmy nights more magical when it contributed to water bioluminescence as bathers splashed around tropicallagool1s. With the advent of large scale prawn aquaculture, V. harveyi started to get attention as a pathogen, particularly in Australia and the Philippines. From the mid-1980s, more and more animals, particularly invertebrates in the tropics and fish in the more temperate areas, were recorded as being infected by V. harveyi. Recently, literature on V. harveyi has gained a wider audience as control of its bioluminescent pathway is elucidated and shown to be substantially different .from and more complicated than that of Vibrio fischeri. Similarly, the bacteriophage- mediated virulence in V. harveyi has also made scientists more interested in this bacterium.
[Extract] The ecology of viruses predominately involves the interaction of the virus at the anima... more [Extract] The ecology of viruses predominately involves the interaction of the virus at the animal and cellular level. When the virions are in the extracellular environment, they are quiescent waiting to infect a living cell. The virions bind to the cell receptors, undergo decapsidation in the phagolysosome, pass the nucleic acid to the cytoplasm or nucleus and begin replication. Evidently, the ecology of viroses starts with entry of the virus into the crustacean, evading the immune system, invasion of the cell, replication, and release of new virions. The crustacean immune system is critical to the functional ecology of their viruses, so we must start with an understanding of this system to have any hope of understanding the ecology of the viruses in crustaceans.
Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merg... more Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merguiensis leading to at least 28% production loss due to reduced growth rates and mortality of juveniles. In the present study, we reduced PmergDNV titres and subsequent mortality by feeding Acheta domesticus (previously determined as an appropriate animal model for P. merguiensis) with dsRNA specific to the capsid protein by mixing it into their food. Feeding A. domesticus with PmergDNV-specific dsRNA in advance of viral challenge increased their longevity, decreased mortality by 84.4% and reduced viral loads 24-fold below the threshold level required for mortality. Mortalities and viral loads were significantly (both P < 0.001) lower in treatments challenged with PmergDNV following exposure to bacterially expressed PmergDNV-dsRNA. This is the first study to demonstrate gene silencing via RNAi against PmergDNV in vivo through oral administration of live bacteria expressing dsRNA in a model system.
Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging di... more Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi-square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10(5) cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10(5) cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10(4) and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.
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