Aquaculture 565 2028) 730166 Contents lists available nt ScienceDirect Aquaculture ELSEVIER journal homepage: www elsevier comlocate/aquaculture ® Identification and pathogenetic study of tilapia lake virus (TiLV) isolated |S from naturally diseased tilapia Tao He", Yu-Zhou Zhang", Li-Hong Gao", Bo Miao", Ji-Shu Zheng‘, De-Cheng Pu’, Qing-Qing Zhang", Wei-Wei Zeng“, De-Shou Wang", Sheng-Qi Su , Song Zhu" 2 coli of Fahey Soh Unb, Conn 40715, Cha = ey Laan of rae ah Rar and Dent (ay of lac), ay Lara of gue soe of Chong, chal of ie Sees ‘Sur Unt, hong 40715, On Chena Acadony of Agate enc, Cong 40071, Cina Guo Prost Key Loreto Amel Molar Desen ad Prec Bet Scho of fe Scene and Enger, Fshn Ue Fehon S223, ARTICLE INFO ABSTRACT ower [Asan eneiglng pathogen, tilapia lke vnus (TLV) has eaused severe soclo-econonie Impacts and vemalns & ip Ike vie evastatng facto in wil ane farmed apn, Ealy diagnosis and ely reporting of TLV ae very important Tsp tnd necessary. Inthe study, a TILV infection event ina tapa farm is studied and reported. Naurlly diseased ‘lapa wih incl signs sch as anorexia, exopiuhalnia, skin abrasion and hemouage ae collected. Multiple tse lesions of liver, pen aed kde ae observed by histopathological analysis, sc as synetal fexmatin, loniaeytopasnieinelsion body and neeoss No betel pathogens ate identified fom Hive, spleen and kidney ‘ofthe dieased tilapia, while lof then show TV: positive. B11 ells ate employed for TILY bolaion, ane ‘ytopathle effects ate developed following ieubadon with supernatants ofthe homogenized tues collected ‘om the diseased tapi. Eleton micrographs ofthe infeted cells and pusifiedTILV show that neal round shaped, enveloped sill payieles (60-80 nin) can be dented. Segment 1 ofthe iolted TLV has high Sequence siniaity to ether isolates, but phylogenetic analysis show tha ii placed in «nique else Experimental infection with dhe solatedTILV can cause high movalty (> OM) la Healthy Ne apa, wih the slay cline sigas and sue lesions to those found in nautlly infected tape. Moveover,vacolation and neers in brai,inlison bod and neross in heat, megalocytes and neross in teste are observed in experimentally infected ait. Msc, spleen iver, intestine, bin, heat, kidoey and gil of the ve nfesed ‘apa show TILV pose, and liver, spleen and busin ae che main wget sues, The data ao far condita a TUN associated diese ouveak in Chins, eatiching the relevant information of TIL. 1. Introduetion, threatening dlapia farming, but this is no longer tue over the past de cades. A varity of devastating infectious diseases have emerged with {As the second most farmed freshwater fish worldwide, elapia has become a primary source of dietary protein and economic income in many developing countries (FAO, 2020; Honnmienow et sl 2018) “Tapia is Farmed in >100 counties with an estimated global production ‘of 6.8 million tons. Chine isthe largest producer of tilapia, followed by Indonesia, Egypt, Thailand, Philippines, ete. Tilapia is comprised of 100 ciel species, and Nile apa (Oreochromis niloeus) i the pre dominant cultured species (Bacharach et al, 2016; Hounmanon et al., 2018), Previously, was believed thar there were few serious diseases the improperly expanded, intensive and diversified farming (Elle cal, 2016 Surachetpong ct al, 2020). Most disease reports in tilapia fre related to bacterial pathogens, notably Aeromonas hydrophila and Streptococcus spp. With few report of vital diseases until the emergence of tilapia lake virus (TILV) (Aich et a, 2022; Hounmanou etal, 20185 Surachetpong et al, 2020). mn 2009, there was a sharp decline of tilapia production in Lake kKinnezet(srae)ffom an average level of 257 tons to 8 tons per year (Suivehetpong ef al, 2020), Subsequently, episodes of massive tilapia * Conesponding authors at: College of Fisheries, Southwest University, Choaggg 400715, Chine mal addres: sshengqiscsuen (6-9. Su), 2320200801 gsteden (Zh), ps /door/10.1016/Jaquacule.2022.739165 Received & July 2022; Received in revised fom 21 Noverber 2022; Acepted 14 December 2022 Available online 15 December 2022 ‘nea 4/6 2022 Eee BY, Al rights reserved,mortalities were quickly spread in wild and farmed tilapia all over Israel and Ecuador (Eyngor el 2014; Ferguson eal, 201). By April 2020, 16 countries across four continents have reported TILY (Surscetpony 150,000 rons of dispia and the income souree of a least 6 million people will be a risk (Hoan ‘now et al, 2018). Currently, there is no practieable measures ro control the disease (Aichi eal, 20225 Surachetpong etal, 2020). Therefore, ‘early diagnosis and timely reporting of TIL are urgently needed. TILV Is an enveloped, single stranded RNA virus consisting of 10, ‘genomic segments. Only the segment 1 shares weak sequence similarity to influenza € virus while the other segments show no homology t0 known viruses (Abu Ras et al, 2022; Bacharach et a, 2016), Based on sequence analysis, TLV has been assigned as a new species, Tapia tiapinevirs, under gens THapinevrs and family Ammoonvirdae (Adan Cal, 2017). THLV is roughly eircular with a diameter of 55-100 nm. (Del Pozo eral, 2017; Eyngor et al, 2014). Presently, several proced- lures have been developed for diagnosis of TLV, including observation of clinical symptoms and tissue lesions (Belira et al, 20185 Eyagor eta 2014, serological dlagnosts (Fu et aly 2020; Prewbaug etal, 2020, molecular techniques (Sakon etal, 2022 Waiyanitra etal, 2018), ‘and virus isolation in cell entre (Li etal, 2022; Tsofack etal, 2017) In the study, a case of TILV associated disease outbreak in & dapia farm in Yangiiang is studied and reported. The diseased tilapia was diagnosed by clinical sign, histopathology, pathogen Tsolaion and Identification, molecular and ultrastructural techniques. Healy Nile tilapia was experimentally infected with the isolated TILV to verify TLV fulfils Kochs postulates. Moreover, tissue tropism of the isolated TILV was checked, Our data wil eatich the relevant information of TIL, and ‘promote the diagnosis, prevention and control ofthe disease 2. Materials and methods 2.1. Gall, fish and ethical saxement E-11 cells, a clone of SSN-1 cells (derived from the whole fy tissue of ‘Channa strats; Iwan0t0 etal, 2000), were kindly provided by Prof. Wei Wei Zeng (Foshan University, Foshan, China) and maintained at 26 °C in Lebovitz 15 medium (1-15; Gibeo, USA) supplemented with 101% fetal bovine serum (FBS; Gibco, USA), Nawurally diseased capi at the moribund stage were colleced from a tilapia farm in Yangiiang (Guangdong, China). Healthy Nile tilapia wsed for experimental chal lenge were kindly provided by Prof. De-shou Wang (Southueest Unt versity, Chongaing, China) and reared in recirculating aerated freshwater canks. at 28 °C. Animal experiments were performed following the Guide for Care and Use of Laboratory Animals approved by the Committee of Laboratory Animal Experimentation of Southwest University 22, Diagioss ofthe diseased tipia 2.2.1. Clinical sigs anu rssue pathology ‘nthe tata farm, lpia were farmed in 12 ponds (about 6600 0° pond) supplied with river water. Ar the time ofthe disease outbreak, Ulapia were approximately 25,0-85.0 em in length and 1.0-1.3 kin weight, Clinical signs of the diseased tilapia included lethargy, loss of appatte slow movement, exophthalmia, skin abrasion and hemorrhage, ‘nd gil congestion. The diseased tilapia atthe moribund stage were Agua 56S (202) 720166 ‘Table 1 Primets used inthe suey rer ane ‘Primer sapere om 519) Se) muvee suTaTOACAGTCOACoGAAT 18 muvok ‘TeaaccearraaTencaae peso. ‘tacTacocercaceacagca ssi pasta THOR atearecaccaTeacteae Feces E ‘ecrocarecrecaccucsas 0 pactneTeDR feccactaaTaaacccTaces Peametor ‘ccoscenccTeAcAGACTAG mm Pasta ELL OR ‘ectetaccanaccenacere carefully collected in plastic bags filled with pond water and oxygen, and then rapidly transported to our laboratory, Tissue samples (liver, spleen aud kidney) fromthe healthy and diseased tilapia were collected, fd then fixed in 109% formalin for 24 h. Following dehydrated and eubeded in paraffin, the samples were sectioned ac5 ym thickness and stained with Hematoxylin and Eosin (H&E) for microscopic analysis. 2.2.2. Pathogen detection For bacterial detection, liver, spleen and Kidney from the diseased Ulapia were collected in sterile environment. The collected tissues were separately homogenized and spread onto brain-heart extractive ‘medium (BHD) plates. The plates were chen incubated at 30°C for 48 h For TILV detection, total RNA ofthe collected tissues (approximately 30 rg/tissue) was extracted using 1 mil of RNAibo plus (Takara, Japan) according to the manufacturer's instructions. Total RNA concentration rand quality were detected using a NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, USA), and then use to synthesis eDNA using Prime script RT reagent Kit (Takara, Japan) according tothe manufac turer's protocol, PCR analysts using the specif primer pars (TILV-D-F and TILV-D-R (Nicholson etal, 2017), Pactin-TE-D-F and aeti-T-D-R; “Inble 1) was performed for TILV detection, The PCR eyeing conditions were denaturation at 95 °C for 5 mia, followed by 30 eyeles at 95 °C for 305, 60°C for 30s, and 72“ for 40s, with inal elongation at 72 °C for 10 min using a PCR thermoeyeler (MyCyeler, Bio-Rad, USA). The PCR products were electrophoresed in 1.59% agarose gel with ethidium bro ‘mide and visualized wnder a Gel Documentation System (Bio-Rad, USA) 23. Vins isolation -11 cells wore employed for vir isolation. refly, S00 mig of each collected sue 34s respectively homogenized with 5 mL of L15 me- dium (29 FBS), and then centrifuged at 12,000 xg for 10 min. The ematants were collected and filtered through 0.22 ym filers (Millipore, USA), The filtered samples were respectively inoculated into onfhient E11 eels in 25 em asks and mainte at 26°C for 7 days [cytopathic effects (CPE) were observed daly, and the supernatants were collected following 809% of the cells showed CPE for experimental challenge std. Besides, the infected cells were colleted with 1 ml of| RNAIso plus CFakara, Japan), and rota RNA was extracted according to the manufacturer's instrictons. Concentration and quality of the RNA Were detected using @ NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, USA), and then used to synthesis eDNA using Prime seript RT reagent Kic (Takara, Japan) according roche manufacturers provocol. PCR analysis using the specific primer pairs (TILV-D-F and ‘TLV-DR, Pactin-EL1-D-F and pactin-E1 LD; Table 1) was performed as described above to detect TILV.T tesa Agua 56S (202) 720166 Dom 2x Fig. 1. Clini signs an histopahologeal analysis ofthe diseased tpl Typical elinial signs of the diseased tape, including exophthalnia (A), skin abvasion (@), skin emonthage (and gil congestion (0); Histopathoioglal sections of liver, spleen and Kidney collected fom healthy and diseased lapis. (5) nial iver; (and) liver ofthe disease apts sueytn cells formation Cccles), incetopiasmni easion body (vows) an mifoca ten of meres anowhend CH) outa spleen; (and J) spleen ofthe diseased tilapia; MMCS (cvs) and intaeytoplasmieinluson body (arrows); (K) normal kidney CL ane M) Kidney of the ‘seas tape; vacuolation (artowheads), nesis (artows) and cell disintegration (ee). 24, Transmission electron microscope observation with 2.5% glutaraldehyde for 24 hy and then rinsed five cmes in PBS (pH 7.2), Following pos fixed in 1% 0:04, the pellet was dehydrated ‘Transmission electron microscope (TEND observation was performed with inereasing concentrations of ethanol, washed with 100% propylene to check the virions in E-11 cells according to Eyngor el. (2014). oxide, treated with propylene oxide-Epon (1, vv) for 30 min, followed Briefly, cell were scraped from the flask following infection for S days by propylene oxide-Epon (1:1, viv) for 15 min. The pellets were ‘and centrifuged at 2000 rpm for 5 mui, The sedimental cells were fixed emnbedded in 100% Epon, and then thin sections (70 co 90 nn) were eut‘and placed on Formvar-coated copper sri. Finally, the sections were ‘observed under a TEM (JEM-1200€X Il, JEOL, Japan) afer staining with uranyl acetate and lend citrate Besides, the collected euleure supernatants were ultacentrfuged through 25% (wt/vol) suerose cushions at 145,000 g for 1h using @ Beckman ultracentrifige (Optima XPN-100, USA). The preliminarily purified visions were examined under the TEM, 25. Phylogenetic analysis ‘The complete coding sequence (CDS) of TILV segment 1 was cloned, using the specific primer pair (S1—F and SLR; fable 1). Amplified DNA produets were purified using a GEL/PCR Purification Kit CTANGEN, (Ching), and then ligated into the pET-28a (+) vector using a One Step ‘Cloning Kit (Vazyme, China), The recombinant plasmid containing segaieat 1 (verified by colony PCR using vector primers) was sequenced, by Sangon Biological Engineering Technology Services Co.,Ltd. (Ghangha, Chins), The sequence was compared with the available se ‘quences in NCBI sing the MegeX sftwvnre, and phylogenetic analysis on ‘genome segment 1 sequences of 38 TILY isolates was performed using Neighbor-Joining method (Saitou and Nei, 1987), 2.6. Experimental challenge 26.41. TLV infection Four hundred healthy ie tilapia (engi 5.5 + 0.9 cx; weight: 9.9, 40.8 g) were separated into two groups (the contol group Ad the TILY challenge group 4 replieates/ group) in a toal of 8 tanks, with 50 ish tank. Tilapia in the control group were injected intraperitoneal with supernatant (60 il /fish) from uninfected E-11 cells, while fish in the ‘TLV challenge group were injected with the same volume supernatant from the infected E-11 cell, For each group, 3 tanks were nse to record tortality, and the fourth tank used for sample election, Clinical sig ‘and mortality after TILV challenge were monitored and recorded daily for 14 days, At the end of the infection, all surviving fish were eutha. zed using an overdose of eugenol solution. All samples were processed ‘8 high temperature (120 °C, 30 mun) to prevent virus leakage. 26.2. symptoms linia signs of the re infected tilapia were checked and recorded, ‘Tissue samples (Liver, spleen, kidney, rain, heart and intestine) fom. the healthy and infected tilapia were collected and fixed in 10% formalin. Histopathological analysis and virus isolation were performed ‘as described in 2.2.1 and 2.3, respectively. 26.3, Tse tropin To check the tissue tropism of TILV, tissue samples (amsele, ey, spleen, liver, intestine, brain, heat, kidney and gill) from the infected Ulapia were collected, Total RNA ofthe collected tssues (approximately 90 mig/tisse) was extracted, and then used to syuthesis cDNA, PCR alysis was performed as described in 2.2.2. & SYBR Premix Ex Taq I it (Vazyme, China) and a StepOnePlus!™ Real-Time PCR system. (Applied Biosystem, USA) were employe for real-time quantitative PCR (RT-QPCR) using the specie primer pairs (TILV-Q-F and TILV-O-R, Dractin-T-Q- and pactinyT-Q-R; Table 1). The RT-gPCR eyeing eon dlitions were denaturation at 95 °C for 5 min, followed by 40 cycles at 95°C for 10 sad 60 *Cfor 305, Ache end ofthe reaction, melting curve analysis was carried out from 65 “C t0 95 °C with 0.5 °C per s incre ment, Relative expression was caleulated using the 29 method (Gehmittgen and Livak, 2008) and normalized to the expression of Fractin in the same sample. Results were expressed as “old change” ‘compared with the normalized expression of TILY in intestine. 27. Statistical analysis She fish were collected for histopathologieal analysis each group. The Agua 56S (202) 720166 survival rte i presented asthe mean survival nt from three replicates (80 fish/replicate), Six fish were employed for tissue tropism analysis, and the results are presented as mean + sd. Statistical comparisons berween «wo groups were performed using the rwo-tlled unpsired Student’ est, and values of “P< 0,05 and **P < 0.01 were applied ta notate statistical significance. 3. Results and discussion, ‘Tilapia are worldwide farmed and served as an important protein sand income source for human beings, playing key roles in ensuring food security and promoting economic development (AO, 2020; How now ec al, 2018). in recent decades, many devastating infectious dis eases have emerged in wild and farmed tilapia (Abu Hla etal, 2016; Surache(pong etal, 2020), TIL isa movel Orthomyxo-like virus, whieh was firstly isolated and identified at 2014 (Eyngoret al, 2014). THLV has caused Jos of devastaing events in at least 16 countries, and >45 counties are thigh risk of TILV, leading to serious socio-economic consequences (Aich et al, 2022;’Hounmanou et al, 2018). As the largest producer and exporter of tilapia, China needs to strictly monitor the occurrence of TILV fo ensure the heathy development ofthe tlapia Industry. Here, we investigated a TILV-assocated disease outbreak in & tilapia farm in Yanglang, Chins. 51, Diagiosts ofthe diseased pia The routine diagnostic methods of fish diseases minty included clinical symproms observation, histopathotogical analysis, antibody- based immunological test, molecular biology detection, pathogen isolation and identifiation. In May 2021, lage numberof tilapia in & farm in Yangliang (Guangdong province, China) were died. Clinical signs ofthe diseased tilapia atthe moribund stage are lethargy, 1oss of appetite, slow movement, exophthalmia (ig. 1A), skin_ abrasion (Gg. 1B), skin hemorrhage (Ps. 1C), and gill congestion (Fig. 1D). Liver, spleen and kidney ofthe diseased tilapia were collected for histopat ological analysis. Liver (Pig. 1B), spleen (Fig. 1H) and kidney (Fig. 1K) of| heslehy lapis show normel strictures. As shown in Fig. 1Fand G, liver ofthe diseased tilapia shows syncytial cells Formation, intracytoplasmic inchison body and multifocal area of necrosis. For spleen of the diseased tilapia, depletion of red blood cells, increase of mielanomacrophage centers (MMs) and intracytoplasmic inclusion body can be observed (ig. 1 and J). As compared with the Kidney of healthy tilapia, vaewo: lization, necrosis and cell disintegration are present especialy in renal ‘tubular epithelium in the kidney of diseased tapia (Fig. 1L and MD, Similar clinical signs and histopathological phenomena have been reported in TLV Infection, Including. anotexia and exophthalmis (Ramirez Paredes et al, 2021; Waiyamitra ec a, 2021), skin abrasion and hemorsuage (Tattiyapong etl, 20175 Waiyamitre et al, 2021), syncytial cells and inclusion body formation in liver (Plerezan et al. 2020), red blood cells depletion and MACs increase in spleen (Way Inlet al, 2021), neerosis and cell disintegration of renal eubulae epithelium in kidney (Ras el. 2020). However, these elinieal sigs td histopathological phenomena are not unique, which are generalized aniong other viral and bacterial diseases (Ail al, 2022). Therefore, based on the clinical signs and histopathological results, it ean be only fered that the diseased tilapia may be infected by TILV. More ding nostie methods need to be pefornied 10 confi the infection 8.2, Pathogen detection Most disease reports in tilapia are related 0 bacterial pathogens, notably Aeramanas hydrophila and Streptococcus sp. (Ach etal, 2022; Nicholson etal, 2020), Sinee 2009, disease caused by TILV has been outbreaking worldwide, leading to serious socio-economic couse ‘quences (Eyngor etal, 2014; Hounmanou et al., 2018; Surachetpong 1, 2020). Moreover, co-infection of TILV with different bacterialT tesa Agua S65 (2023) 729166 (A) Liver Spleen Kidney TiLv 358 bp) Peactin (590 bp) LY (358 bp) actin (590 bp) riLy (358 bp) actin 590 bp) Fig. 2. Pathogen dtetion of de sensed tap. (A) Barta pthogens dtetin;(B) LY deteton. Fig. 3. oaton and idenifeation of THY. (A) E11 cells inoculated wi supernatants of liver collected from felt apn for 7 das; (8) EL cle ine Iated wich supernatants of liver allcted from diseased tilapia for 7 days show obvious CPE inciuing ytopannic acwolton, page forntion, cel aggregation and detachment; (C) PCR analss of the Infected E11 cells to confirm the presence of TILY; (D) Representative TEM micogtaphs of the infected E11 cells and the puifed virion fom st peratants ine,pathogens have been reported (Nicholson et al, 20205 Surachetpong ‘til, 2017), Diferent species of bacterial pathogens have been detected from TILV infected fishes (Abdulla etal, 20185 Amal etal, 20185 Nietiolson etl, 2020). For example, Nicholson et sl. (2020) invest ‘gated the TILV-bacteril co-infection in farmed tilapia, and demon: trated thet TILV- bacterial concurrent infections are occurred in 31% of the fis, especially TILV.Aeromonas spp. co-infection (Nicholson et 9 2020), Therefore, the posible involventent of bacterial pathogens needs to be detected during TILV infection. For bacterial pathogens detection, tssues (liver, spleen and Kidney) from the diseased tapia were separately homogenized and spread onto BHM plates. As shown in Fig, 28, no bacterial coloules can be observed from the plates after incubation for 48 h at 30 °C indicating that the liseased tilapia fs non-bactoral infection, Subseqvently, PCR analysis ‘wos performed for TILV detection. As shosin in Fig. 28, all of liver, spleen and kidney collected from 12 diseased tlapia are TILV- postive ‘Therefore, the diseased tilapia may be only attributed to TILV infection 4.9, Isolation of TLV Virus isolation using sensitive cell Lines is also an effective way t0 directly detect vital Infetion. Different cell Ines have been used for isolation and propagation of TILV, seh as E-11 cells (Eyagor ot 2014), THB cell (Wang et al, 2018), and OnH cells (Yad etal. 2021). E-11 calls, clone of SSN-1 eels derived from the whole fry tssve of ‘Channa sri) (Ivanoto et al, 2000), are the first cll line used to sucessfully isolate TILV CEyngor ets, 201). To further eonfimy TL fa the primary cause of the diseased tilapia, supemnatants of the ho rogenized tissues (liver, spleen and kidney) were respectively inoc lated into confluent E11 cells to isolate TLV. E11 cells inoculated with supernatants of liver collected from healthy tilapia show normal morphological features (Fis. 9A). After Incubation with supernatants of liver collected from disewsed tilapia for 7 days, obvious CPE were observed in E11 eels, including cytoplasmic ‘vacuolation, plague formation, cell aggregation and detachment (ig. 9B), Similar CPE have been reported by previous studies (Eyngor ct al, 2014 Tattiyapong et al, 2017). £-11 cells inoculated with su pemnatants of spleen and kidney collected from disensed tilapia also ‘showed the same CPE (not shown), The infected cells were collected and subjected to PCR atnlsisto confirm the presence of TILV. As shown in Fig. SC, all stmples show a postive PCR product on 1.58% agarose ge, ‘confirming the presence of TLV. To further support for TILV infection, the infected cells were observed under a TEM As showin in ig. 2D, lots ‘of electvon-dense particles ean be obviously observed in the eytoplasm, jperuatants ofthe infected cell cultures were purified by wltracent gation through #259 sucrose cushion, and then examined using TEM. ‘Nearly round shaped, enveloped viral particles (60-80 um in diameter) ‘with a central variable electron-dense care cat be identified frm the electron micrographs. Combining the abave diagnostic results, ean be concluded that the diseased tilapia i attributed to TILV infection, 5.4. Phylogenetic analysis using genome segment 1 TILV is an RNA virus consisting of 10 genomic segments, and every, ‘segment contains open reading frames (ORES) flanked by non-coding regions at the 3 and Stends (Acharya el, 2019). Among the 10 seg rents, only segment 1 shows weak sequence homology (37% segment coverage, —17% amino acid identity) (0 the influenza C virus PBL subunit, and is presuaied to encode forthe RNA polymerase of TILV. The ‘other segments have no recognizable homology to known viruses achwraeh et al, 2016). AS the lrgest segment in TILV genome, ‘segment 1 (1641 bp) has been used for phylogenetic analysis of TIL in previous studies (Chaput etal, 2020; Debnath etal, 2020; Taengpha fetal, 2020) For phylogenetic analysis, dhe complete CDS of TILV segment 1 was sequenced snd submitted to GenBank (accession muniber: ON53686). Agua 56S (202) 720166 rt rere Fig 4. Phylogenetic tee ofthe slated TIL segment 1 (ON863686) and 32 silable TILY segment 1 sequences in NRL wing Neighbor Joining method. Tufenza G viv PB sequence saved aa fotigh lence gene. The boo staps <50 ate not show. Nucleotide BLAST shows that the nucleotide sequence sinllarity of (0N863686 (0 other available sequences in NCBI of TILV segment 1 is bermreen 95.0686 and 96.92%, Phylogenetic tee (Fi 4) was constructed based on the 38 available sequences of TILV segment 1, and inuenza © virus PBI sequence was used asa foreign reference gene. Result shows that the Isolated TILY in present study Ts placed In stall and unique cluster 13, Experimenta ingetion of TLV ‘The pathogen confirmation should full Koch's postulates. 1 Is rnocossary (0 verify that a pathogen isolated from natural outbreaks ean ‘sesame infection when used ro challenge healthy animals Besides, the pathogen can be resolated from the experimentally infected ant tals (attiyapong etal, 2017). To confirm de isolated TILV in present study fulfils the Koeh’s postulates, healthy Nile tilapia were exper mentally infeeted wit the isolated TILV. Clinical syn resolation of TILV and histopathological analysis were performed. {s shown in ig, 9A, siniarelinieal signs ofthe reinected tapia to naturally diseased tilapia are obviously appeared, including exopk thai (ig. 9B), skin abrasion (ig. $C) and skin hemorrhage Pig. 9D). ‘The cumulative mortality of the re-infected apia is higher than 90% at 14th day post-infeetion (pi), while that is <5% for the control group (ig, 9B). Different mortalities caused by TILY have beea reported in diferent parts of the world (Belicra et al, 2018; Fathi et al, 20175 Ferguson etal, 2014). For examples, high mortality ranging from 10% to 80% was described in Beusdor(Fergison eta, 2014, higher thanT tesa Agua 56S (202) 720166 Day pocon| 5. xpeimenal infection of lpia with the isolated THLV. (A) The re nfecte lpia a 7 dp; exopalma (8), skin abrasion (C) ana skinhemantage (D) of the ve inete ap; E) Suis curves for tapi injected incapesitonelly with superna 50 pl sh) fom niafeted the “conte” gp) and inetd (he “TW challenge” soup) E-1 cells; E11 cel inoculated with supernatants of iver collected ron healthy (F) and experinentaly infected (G) api for 7 das eS Ami! Sdn S88 6, Histopathological analysis ofthe experimentally TLV infected tilapia. (A normal iver; (8) liver of te einfected tap; syncytial cells formation (cies), Inclusion body (arom) and necrosis (aroweeds);(C) noma spleen; (D) spleen ofthe reinfeted tapi: melanomacrophage ceaters (MOC), inlusion body Gavions) and MC inition (arrssheads; CE) normal kidnes (F idey of the re infected lapis vavolizacion (atowheads), nesonis(xt0%s) and ell slsintegration ctl) (6) nora bran; (H) brain of dhe se infected lpi vacuolation and multifocal area of necrosis (sows) (0) noua ett (3) hea ofthe Infected lapis red Hood cells depletion, inlison body (arrows) and necrosis arrowheads); (K normal intestine; (intestine ofthe re infected apis megaocytes (orros) and necrosis (rosea). (Fr inerpretation ofthe references to coon inthis fige legen, he render ie refered to the web version ofthis rile.)We £9 FSS Ho 7 ma BY, — EEE @) 15: * 12: “- | 2 i : é 9 Ee : A i 2 Ss os wee see wees Fig. 7. Tissue sopism of TLV checked by PCR (A) and RT-PCR (B) analysis For RT-gPCR analysis, results are express as Told change” eompated withthe socalled expression of TILV in neste, Data ate presented as etn SD. "P< 005 and "P< 0.01, a compated with the nowmalied expression of TLV {80% in Israel (Eyngor et al, 2014), 20%-909% in Thalland (Sur ‘acherpong, eal, 2017), and 80%-90% in India (Behera et sl, 2018), ‘The potential reasons ofthe wide variation in mortality are unknown, may be actributed to the species (Bsrvn etl. 20205 Ferguson et al 2014), life stages (Dong et al., 20174; Huamanela Pulido et a., 2019) ‘and weight (Roy etal, 2021) of tilpin. For example, previons studies reported that TILV can affect ish aa stages bur mortalities ace higher rainly in early developmental stages (larvae, fy and fingerings) due to the low immunity (Del Pozo et sl, 2017; Surschetpong et sl, 20175 ‘Tatiyapong et al, 2017). Subsequently, TILV was 1e-isolated using E-11 cells from the tissues collected from there infected tilapia E11 cells inoculated with super natants of liver collected from healthy tapia show normal morpho: logical festures (Pig 5). Similar GPE ig. 5G) were observed following incubation with supernatants of liver collected from the reinfected ‘lapia,indieating chat THLV ean be reisolnted from the experineatally infected tilapia, For histopathologic analysis, tlssue samples (liver, spleen, Kidney, brain, Heart und intestine) from the reinfected Glapia were collected ‘and subjected to section observation. Liver (Fg. 6A), spleen (Fig. 6C), kidney (Pig. 6E), brain (ig, 6), hear (Pg. 6D and intestine (Pg. 6K) oF healthy tilapia show normal structures, Following infection with TILV, the same histological cianges ean be identified from the liver (ig. 6B), spleen (Fg. 6D) and kidney (Pig. 6) compared tha collected from the naturally diseased tilapia. Moreover, vacuolation and multifocal area of neerosi in brain (i. OF), xed blood cells depletion, inclusion body and necrosis in hear (Fis. ), large numbers of megalocytes and necrosis in Inestine are observed from the sections ofthe experimentally infected Ulapia. Similar histopathological phenomena have been reported in previons studies (ierezan et al, 2020; Ramirez Paredes et al, 20213 ‘Yanlesen eta, 2021), indicating that experimental infection of TILV ‘an cause disease in tilapia, Agua 56S (202) 720166 8.6, Tissue ropism of TLV According to the above results, tissue lesions can be identified in diferent tissues of TILV-infected rilpin, suggesting that many’ organs ray be the target tse for vial transeripion and replication. To check the target tissues and viral tropism, different tissues of the infected Ulapia were collected and subjected to PCR and RT-PCR analysis, respectively. As shown in Fig. 7A, positive TILV bands with diferent brightness ae identified for muscle, spleen, liver, intestine, brain, heart, kidney and gil of the infected tilapia, RT-qPCR analysis was performed To qunntfy TLV in different disses, and results were expressed ns “fold clang” compared with the normalized expression of TILV in intestine (ig. 7B). Data show that liver i the primary target tissue of TILV, and followed by spleen, brain, hear. TILV fans been detected in miulkple tissues, such as liver, spleen, kidney, ails, heart and nusscle, while liver and brain are considered to be the main target tissues (Sacharach etal, 2016; Saranya and Sud eran, 2020; Waiyamitr et al, 2021). Nevertheles, ffeent rests have also been reported in previons studies (iiginba etal, 2028; Pierezan etal, 2020). For example, Nhgima etal (2018) investigated the TILV infection in Nile tilapia fom Lake Victoria. They demonstrated that the main prevalence of TLV isin kidney, spleen and heart, while the prevalence is low in liver and absent in brain (gina el, 2018). Potential reasons for the different target tissues of TILV are unknown, ray be attribted to the different TILV isolates and fish species. The rain target tissues of LV isolated in present study’ are liver spleen and brain, which can be used for LV detection. 4. Conel Inthe study, a natural TLV infection eventis reported. The infection is confirmed by clinical signs, histopathological analysis, pathogen ‘eteetion and virus isolation. Alchough segment 1 ofthe isolated TLV has a high sequence similarity to other isolates, phylogenetic analysis shows that it is placed in a unique cluster. Experimental infection with the isolated THLV can cause disease in Nile tilapia, with the similae clinical signs and tissue lesions to dhose found in the naturally infected tilapia. Besides, ILV can bere isolated from the experimentally infected Ulapia, verifying that TILY infection fulfils Kochs postulates, Liver, spleen and brain are che main target tissues ofthe isolated TILV, and ean be used for TILV detection. Author statement He Tao: Investigation, Data curation, Writing original draft; Zhang, Yu-Zhou: Investigation, Validation, Data curation; Gao Li:Hong: Re- sources, Supervision, Waiting reviewing & editing; Mino Bor Investign tion, Validation; Zheng Ji-shu: Validation, Date curation; Pu De Chen: Resources, Supervision; Zhang Qing-Qing: Validation, Daca curation; Zeng Wei-Wei: Resources, Supervision; Wang De Shou: Conceptuaiza tion, Resouces, Supervision; Su Sheng-Ql: Methodology, Supervision, Wiriting reviewing & editing: Zhw Song: Conceptualiztion, Resonees, Supervision, Writing reviewing & editing Declaration of Competing Interest ‘The authors declare that they have no competing interests Data availability Dats wil be msde availabe on request. ‘This work was supported by the Agricultural Technology Innovation Project of Chongqing in 2022, Natural Sclence Foundation of Chongaing{ste2021jeyjmsemX04S0), National Key Research and Development Program of China (2019¥FD0900305-03), Fundamental Research Funds {for the Cental Universities (SWU120034), and China Postdoctoral Sci ‘ence Foundation (2020683222 and 20217140567). 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