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Fulltext Detection of Laem-Singh virus in cultured Penaeus monodon shrimp from several sites in the Indo-Pacific region

Sittidilokratna N, Dangtip S, Sritunyalucksana K, Babu R, Pradeep B, Mohan CV, et al.

Dis Aquat Organ, 2009 Apr 27;84(3):195-200.
PMID: 19565696 DOI: 10.3354/dao02059

Laem-Singh virus (LSNV) is a positive-sense single-stranded RNA (ssRNA) virus that was recently identified in Penaeus monodon shrimp in Thailand displaying signs of slow growth syndrome. A total of 326 shrimp collected between 1998 and 2007 from countries in the Indo-Pacific region were tested by RT-PCR for evidence of LSNV infection. The samples comprised batches of whole postlarvae, and lymphoid organ, gill, muscle or pleopod tissue of juvenile, subadult and adult shrimp. LSNV was not detected in 96 P. monodon, P. japonicus or P. merguiensis from Australia or 16 P. monodon from Fiji, Philippines, Sri Lanka and Mozambique. There was no evidence of LSNV infection in 73 healthy juvenile P. vannamei collected during 2006 from ponds at 9 locations in Thailand. However, LNSV was detected in each of 6 healthy P. monodon tested from Malaysia and Indonesia, 2 of 6 healthy P. monodon tested from Vietnam and 39 of 40 P. monodon collected from slow-growth ponds in Thailand. A survey of 81 P. monodon collected in 2007 from Andhra Pradesh, India, indicated 56.8% prevalence of LSNV infection but no clear association with disease or slow growth. Phylogenetic analysis of PCR amplicons obtained from samples from India, Vietnam, Malaysia and Thailand indicated that nucleotide sequence variation was very low (>98% identity) and there was no clustering of viruses according to site of isolation or the health status of the shrimp. The data suggests that LSNV exists as a single genetic lineage and occurs commonly in healthy P. monodon in parts of Asia.

Matched MeSH terms: RNA Viruses/classification; RNA Viruses/genetics*; RNA Viruses/isolation & purification*
Fulltext Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Taengphu S, Kayansamruaj P, Kawato Y, Delamare-Deboutteville J, Mohan CV, Dong HT, et al.

PeerJ, 2022;10:e13157.
PMID: 35462762 DOI: 10.7717/peerj.13157

BACKGROUND: Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems.
METHODS: Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV.
RESULTS: The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.

Matched MeSH terms: RNA Viruses*
Fulltext Tilapia Lake Virus was not detected in non-tilapine species within tilapia polyculture systems of Bangladesh

Debnath PP, Dinh-Hung N, Taengphu S, Nguyen VV, Delamare-Deboutteville J, Senapin S, et al.

J Fish Dis, 2022 Jan;45(1):77-87.
PMID: 34580880 DOI: 10.1111/jfd.13537

Sixteen countries, including Bangladesh, have reported the presence of tilapia lake virus (TiLV), an emerging tilapia pathogen. Fish polyculture is a common farming practice in Bangladesh. Some unusual mortalities reported in species co-cultivated with TiLV-infected tilapia led us to investigate whether any of the co-cultivated species would also test positive for TiLV and whether they were susceptible to TiLV infection under controlled laboratory experiments. Using 183 samples obtained from 15 farms in six districts across Bangladesh, we determined that 20% of the farms tested positive for TiLV in tilapia, while 15 co-cultivated fish species and seven other invertebrates (e.g. insects and crustaceans) considered potential carriers all tested negative. Of the six representative fish species experimentally infected with TiLV, only Nile tilapia showed the typical clinical signs of the disease, with 70% mortality within 12 days. By contrast, four carp species and one catfish species challenged with TiLV showed no signs of TiLV infection. Challenged tilapia were confirmed as TiLV-positive by RT-qPCR, while challenged carp and walking catfish all tested negative. Overall, our field and laboratory findings indicate that species used in polycultures are not susceptible to TiLV. Although current evidence suggests that TiLV is likely host-specific to tilapia, targeted surveillance for TiLV in other fish species in polyculture systems should continue, in order to prepare for a possible future scenario where TiLV mutates and/or adapts to new host(s).

Matched MeSH terms: RNA Viruses*
Fulltext A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV)

Delamare-Deboutteville J, Meemetta W, Pimsannil K, Sangpo P, Gan HM, Mohan CV, et al.

Sci Rep, 2023 Nov 20;13(1):20276.
PMID: 37985860 DOI: 10.1038/s41598-023-47425-w

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

Matched MeSH terms: RNA Viruses*
Sistem minigenom virus Penyakit Newcastle (NDV) AF2240 sebagai sistem pengekspresan gen sel mamalia

SHAHRUL HISHAM ZAINAL ARIFFIN, ROSLINA SHAMSUDIN, NURUL ATIKAH AHMAD, ZULKIFLIE ZAMROD

Sains Malaysiana, 2012;41:423-430.

Sistem minigenom telah digunakan untuk mengkaji replikasi dan transkripsi virus RNA tidak bersegmen. Objektif kajian ini adalah untuk membina sistem minigenom bagi virus NDV strain tempatan, AF2240 serta bagi mengkaji mekanisme transkripsi dan replikasi virus ini. Bagi tujuan ini lima plasmid digunakan iaitu pMGNDV, pCITENP, pCITEP, pTriEX-T7, dan pGEML. Kesemua plasmid diekstrak secara berskala besar dan dimendakkan menggunakan polietilina glikol. Hasil ekstrak ini digunakan untuk transfeksi ke dalam sel. Translasi in vitro dilakukan dengan menggunakan pCITENP, pCITEP, dan pTriEX-T7 untuk memastikan kesemua konstruk ini berfungsi. Hasil pemblotan western menunjukkan protein bersaiz ~100 kDa (T7), ~53 kDa (NP), ~53 dan 55 kDa (P) berjaya diekspreskan. Protein CAT diperoleh apabila plasmid yang mengekodkan minigenom NDV ditransfeksi bersama plasmid yang mengekodkan protein nukleokapsid (NP), fosfoprotein (P) dan subunit besar polimerase (L) ke dalam sel BHK-21. Dianggarkan 55 pg protein CAT berjaya diperoleh menggunakan kit CAT ELISA. Hasil pemblotan western turut menunjukkan protein CAT bersaiz 25 kDa dihasilkan. Kesimpulannnya, system minigenom ini berupaya untuk berfungsi dan mampu mengekspreskan gen asing di dalam sel mamalia BHK-21.

Matched MeSH terms: RNA Viruses
Fulltext A Novel RNA Virus, Macrobrachium rosenbergii Golda Virus (MrGV), Linked to Mass Mortalities of the Larval Giant Freshwater Prawn in Bangladesh

Hooper C, Debnath PP, Biswas S, van Aerle R, Bateman KS, Basak SK, et al.

Viruses, 2020 10 02;12(10).
PMID: 33023199 DOI: 10.3390/v12101120

Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single‑stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

Matched MeSH terms: RNA Viruses/classification*; RNA Viruses/genetics; RNA Viruses/isolation & purification*
Immunological characterization of rice tungro spherical virus coat proteins and differentiation of isolates from the Philippines and India

Druka A, Burns T, Zhang S, Hull R

J Gen Virol, 1996 Aug;77 ( Pt 8):1975-83.
PMID: 8760450

Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.

Matched MeSH terms: RNA Viruses/immunology*; RNA Viruses/isolation & purification
Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter

Yee SY, Fong NY, Fong GT, Tak OJ, Hui GT, Su Ming Y

Int J Environ Health Res, 2006 Feb;16(1):59-68.
PMID: 16507481

Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6 x 10(2) pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.

Matched MeSH terms: RNA Viruses/genetics; RNA Viruses/isolation & purification
Fulltext Influenza A Viral Infection with Septic Shock in Pregnancy

Lwin S, San Yi M, Shi Leong M, Suharjono H, Moe Nwe T

Case Rep Obstet Gynecol, 2019;2019:2470352.
PMID: 31139480 DOI: 10.1155/2019/2470352

The influenza virus is RNA virus and is classified into four subtypes, influenza A, influenza B, influenza C, and influenza D. One of the subtypes of influenza A, the H1N1 strain, also known as swine flu, is especially of high risk for development of complications in pregnant women. The influenza A virus infection is difficult to diagnose clinically because its presenting symptoms are similar to those of the common cold but are more severe, last longer, and can be potentially life-threatening. This case also presented with common cold symptoms but her condition worsened later. Fortunately, obstetric health providers were vigilant enough to address the developing infection and its related complications. It was the cooperative effort of multidisciplinary team care which resulted in a favourable outcome in both mother and baby.

Matched MeSH terms: RNA Viruses
Fulltext Novel RNA viruses associated with Plasmodium vivax in human malaria and Leucocytozoon parasites in avian disease

Charon J, Grigg MJ, Eden JS, Piera KA, Rana H, William T, et al.

PLoS Pathog, 2019 12;15(12):e1008216.
PMID: 31887217 DOI: 10.1371/journal.ppat.1008216

Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.

Matched MeSH terms: RNA Viruses
Transcriptome analysis of liver elucidates key immune-related pathways in Nile tilapia Oreochromis niloticus following infection with tilapia lake virus

Sood N, Verma DK, Paria A, Yadav SC, Yadav MK, Bedekar MK, et al.

Fish Shellfish Immunol, 2021 Apr;111:208-219.
PMID: 33577877 DOI: 10.1016/j.fsi.2021.02.005

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.

Matched MeSH terms: RNA Viruses/physiology
Antibodies in human sera reacting with an insect pathogenic virus

MacCallum F, Brown G, Tinsley T

Intervirology, 1979;11(4):234-7.
PMID: 107144

Precipitating antibodies to an insect pathogenic RNA virus of Darna trima from East Malaysia have been found in a small percentage of human sera from several different groups of persons in West Malaysia and the United Kingdom. No associated illness was identified. The results suggest that an antigenically related virus or viruses are present in the environment that may be associated with symptomless or inapparent infections in man.

Matched MeSH terms: RNA Viruses

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