detection-tilapia-lake-virus-using-conventional-rt-pcr-sybr-green-rt

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

reverse-transcription-loop-mediated-isothermal-amplification-rt-lamp

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Kasetsart University

Fruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.

Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

During 2015-2016, several outbreaks of tilapia lake virus infection occurred among tilapia in Thailand. Phylogenetic analysis showed that the virus from Thailand grouped with a tilapia virus (family Orthomyxoviridae) from Israel. This emerging virus is a threat to tilapia aquaculture in Asia and worldwide.

Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015-2016.

Since 2015, a novel orthomyxo-like virus, tilapia lake virus (TiLV) has been associated with outbreaks of disease and massive mortality of cultured Nile and red tilapia (Oreochromis niloticus and Oreochromis spp., respectively) in Thailand. In this study, TiLV was isolated from field samples and propagated in the permissive E-11 cell line, with cytopathic effect (CPE) development within 3-5days post-inoculation. Electron micrographs of infected E-11 cells and fish tissues confirmed the rounded, enveloped virions of 60 to 80nm with characteristics very similar to those of Orthomyxoviridae. In vivo challenge studies showed that high mortality in Nile (86%) and red tilapia (66%) occurred within 4-12days post-infection. The virus was re-isolated from challenged fish tissues in the permissive cell line, and PCR analysis confirmed TiLV as a causative pathogen. The distinct histopathology of challenged fish included massive degeneration and inflammatory cell infiltration in the liver and brain as well as the presence of eosinophilic intracytoplasmic inclusions in hepatocytes and splenic cells. Our results fulfilled Koch's postulates and confirmed that TiLV is an etiologic agent of mass mortality of tilapia in Thailand. The emergence of this virus in many countries has helped increase awareness that it is a potential threat to tilapia aquacultured in Thailand, Asia, and worldwide.

Experimental infection of Tilapia Lake Virus (TiLV) in Nile tilapia (Oreochromis niloticus) and red tilapia (Oreochromis spp.).

Recent outbreaks of a novel tilapia lake virus (TiLV) have raised concerns regarding the international spread of TiLV in frozen tilapia products. This study investigated the potential risks of frozen tilapia fillet as a source of TiLV transmission. It revealed that TiLV genomic RNA could be detected in tilapia fillet and the virus isolated from non-frozen and frozen fillets with clinical TiLV infection stored up to 28 days caused a cytopathic effect (CPE) formation in the susceptible cell line in vitro. However, frozen fillets from clinical TiLV infection stored for 90 and 120 days did not cause CPE in the susceptible cell line. Similarly, CPE was not observed in TiLV isolated from subclinically TiLV-infected fish fillets. In addition, in vivo bioassay revealed that despite the presence of TiLV isolated from subclinically TiLV-infected fillet stored at -20°C for 14 days, there was no evidence of TiLV disease in naïve red hybrid tilapia based on the absence of clinical signs and mortality and without the detection of TiLV genomic RNA using reverse transcription-quantitative polymerase chain reaction assay. Collectively, these findings suggested minimal risk of transmission of TiLV via frozen tilapia fillets.

Minimal risk of tilapia lake virus transmission via frozen tilapia fillets.

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2-day-old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV-free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication.

Evidence of potential vertical transmission of tilapia lake virus.

To gain a better understanding of the pathogenesis of tilapia lake virus (TiLV) infections in Nile tilapia (Oreochromis niloticus), fingerlings were challenged with a single dose of 1 × 10  TCID /fish of TiLV utilizing intracoelomic/intraperitoneal (IC ) or intragastric (IG ) routes. Acute mortalities were present in both groups, reaching 70 and 40% in IC and IG after 10 days, respectively. Challenged fish presented erratic swimming, lethargy, anorexia, exophthalmia and cutaneous petechiae and ecchymoses. Histological changes in challenged groups included syncytial formation, intracytoplasmic inclusion bodies and multifocal hepatocellular degeneration and necrosis. In addition, multifocal areas of mild proliferation of glial cells and lymphocytic perivascular cuffing were observed in the brain of exposed challenged groups. TiLV RNA was detected in gills and faeces of challenged fish using quantitative reverse transcriptase-PCR, as well as in the tank water holding challenged fish. Moreover, TiLV RNA was detected in scrolls obtained from formalin-fixed paraffin-embedded tissue blocks from challenged fish. Results from this study suggest that IG methods represent an additional method to study the pathogenesis of the disease in this species, as it results in infection and diseases as in naturally occurring cases and does not bypass important mucosal immune responses as injectable routes do.

Intragastric and intracoelomic injection challenge models of tilapia lake virus infection in Nile tilapia (Oreochromis niloticus L.) fingerlings.

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H O ), 80 ppm formalin and 5,000 ppm (0.5%) Virkon . Factors that affect the disinfectants' efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log TCID  ml was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H O when tested at 10 min and 28°C; however, Virkon still achieved more than 5 log inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented.

Virucidal effects of common disinfectants against tilapia lake virus.

Here, we present the complete coding sequences of two tilapia lake virus (TiLV) isolates recovered during an investigation of a mortality event in farmed Nile tilapia in the United States. Phylogenetic analysis supported the isolates as each other's closest relatives and members of a clade of Thai TiLV strains.

Genomic Characterization of Tilapia Lake Virus Isolates Recovered from Moribund Nile Tilapia () on a Farm in the United States.

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1β, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses.

ArticleTotal : 2	Year
Publication title	2018
Publication title	2020

Article	Year
Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection. Parasites & vectors\| PubMed ID: 26283222	2015
Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015-2016. Emerging infectious diseases\| PubMed ID: 28518020	2017
Experimental infection of Tilapia Lake Virus (TiLV) in Nile tilapia (Oreochromis niloticus) and red tilapia (Oreochromis spp.). Veterinary microbiology\| PubMed ID: 28757020	2017
Minimal risk of tilapia lake virus transmission via frozen tilapia fillets. Journal of fish diseases\| PubMed ID: 30468255	2019
Evidence of potential vertical transmission of tilapia lake virus. Journal of fish diseases\| PubMed ID: 31243783	2019
Intragastric and intracoelomic injection challenge models of tilapia lake virus infection in Nile tilapia (Oreochromis niloticus L.) fingerlings. Journal of fish diseases\| PubMed ID: 31270828	2019
Virucidal effects of common disinfectants against tilapia lake virus. Journal of fish diseases\| PubMed ID: 31322740	2019
Genomic Characterization of Tilapia Lake Virus Isolates Recovered from Moribund Nile Tilapia () on a Farm in the United States. Microbiology resource announcements\| PubMed ID: 31974153	2020
Antiviral response of adult zebrafish (Danio rerio) during tilapia lake virus (TiLV) infection. Fish & shellfish immunology\| PubMed ID: 32201348	2020

Win Surachetpong

Biography

JoVE Journal Publications

Other Publications