Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA

In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6–10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Background Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. Methods The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. Results There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. Conclusions In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Background: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. Methods: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.Results: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P>0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P<0.05) of viral mRNA and DNA copies (dpb+dpc: 79%, 68%; dpb+ie2b: 68%, 60%; dpb+dpc+ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. Conclusions: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

Molecular Characterization and Pathogenicity of an Indian Isolate of Duck Enteritis Virus Recovered From a Natural Outbreak

Background: Duck plague is a highly contagious viral disease reported in our country very often with significant economic loss. There are some bottlenecks with the currently used ‘Holland strain’ vaccine that involves cumbersome process of vaccine production in embryonated chicken eggs. With the future goal of development of an indigenous cell culture vaccine for duck plague, the present study is aimed at isolation of an Indian strain of DEV from a natural outbreak and its characterization for the seed virus purpose. Methods: Liver samples were collected from the suspected ducks died during a natural outbreak in Kerala and subjected to polymerase chain reaction (PCR) to confirm presence of viral DNA. The duck enteritis virus (DEV) was isolated by inoculation of PCR positive samples in embryonated duck eggs/ducklings and its pathogenicity was studied. Further, the DEV recovered from the infected duck embryo and duckling liver was confirmed by PCR amplification of the viral DNA polymerase gene and its sequence analysis. Result: Out of 12 liver samples tested eight (8) were found to be positive for duck plague by PCR. The DEV infected duck embryos and ducklings died showing typical signs and characteristic gross and microscopic lesions. PCR amplification of viral DNA targeting the DNA polymerase gene yielded amplicon of expected size of 446bp. The amplicon sequence showed 99-100% homology with other DEV isolates, thus confirming the new isolate as DEV, named as DEV/India/IVRI-2016 and the gene sequence has NCBI acc. no. KX511893.

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Background Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. Methods The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by TCID50, cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. Results There was no cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and no any mutation was identified, thereby avoiding the formation of mutant with potential resistant viruses. Conclusions In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream – in the city of Caxias do Sul, Rio Grande do Sul, Brazil – was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.