Viral haemorrhagic septicaemia virus 11287 Viral Hemorrhagic Septicemia (VHS) is a deadly fish virus and aquatic invasive species that attacks and weakens the blood vessels of fish. Broken blood vessels and severe blood loss ultimately causes death. It afflicts more than 50 species of freshwater and marine fish in several parts of the northern hemisphere. Scientists are not sure how the virus arrived. It may have come in with migrating fish, or may have hitchhiked in ballast water from ships. VHS was first found in European freshwater trout in the late 1930s and continues to cause epidemics in European trout farms. It appeared on the U.S. West Coast in 1988 in marine trout and salmon (WISC - VHSV). Baboon Papio cynocephalus 9556 Bank vole Clethrionomys glareolus 447135 Bear Ursus americanus 9643 Birds Passeroidea 175121 Brown Trout Salmo trutta 8032 Buffalo Bison bison 9901 Carnivores Vulpes 9625 Cat Felis catus 9685 Catfishes Siluriformes 7995 Cattle Bos taurus 9913 Chicken Gallus gallus 9031 Chimpanzee Pan troglodytes 9598 chinchillas Chinchillidae 10150 Copper Pheasant Syrmaticus soemmerringii 9067 Deer Cervus elaphus 9860 Deer mouse Peromyscus maniculatus 10042 Dog Canis familiaris 9615 Ducks Anas 8835 Ferret Mustela putorius furo 9669 Fish Hyperotreti 117565 Gerbil Gerbillina 10045 Goat Capra hircus 9925 Gray wolf Canis lupus 9612 Guinea pig Cavia porcellus 10141 Hamster Mesocricetus auratus 10036 Horse Equus caballus 9796 Human Homo sapiens 9606 Macaque Macaca fascicularis 9541 Mongolian Gerbil Meriones unguiculatus 10047 Monkey Platyrrhini 9479 Mouse Mus musculus 10090 None None Parrot Psittacidae 9224 Pig Sus scrofa 9823 Rabbit Oryctolagus cuniculus 9986 Rainbow trout Oncorhynchus mykiss 8022 Rat Rattus 10114 Raven Corvus corax 56781 sei whale Balaenoptera borealis 9768 Sheep Ovis aries 9940 Squirrel Spermophilus richardsonii 37591 Tree shrew Tupaiidae 9393 Trouts, salmons & chars Salmoninae 504568 Turkey Meleagris gallopavo 9103 Vole Microtus ochrogaster 79684 Water buffalo Bubalus bubalis 391902 17D YF Vaccine plus Ig Sanofi Pasteur Live, attenuated vaccine Clinical trial Intramuscular injection (i.m.) Yellow fever (YF) virus vaccine with human immunoglobulin (Ig) that contained YF virus-neutralizing antibodies. (Edupuganti et al., 2013) The first 64 participants received the YF vaccine from one lot. Immediately after the administration of the YF vaccine subcutaneously into the deltoid, immunoglobulin (Ig) or saline injections were administered intramuscularly into the upper outer quadrants of the buttocks. The placebo for Ig was sterile saline (0.9% sodium chloride injection, USP; Hospira Inc., Lake Forest, IL) administered similarly. Participants returned on post-vaccination days 1, 2, 3, 5, 7, 9, 11, 14, 30, and 91 for determination of viremia and immunological and safety assessment. (Edupuganti et al., 2013) Intramuscular injection (i.m.) Human Leukocyte Antigen VHSV DNA vaccine pCMV-vhsG encoding glycoprotein G VO_0004338 DNA vaccine Research pcDNA3 [Ref2400:Heppel et al., 1997] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pcDNA3 expressed glycoprotein G gene of the virus (Heppel et al., 1997). VO_0000286 Young rainbow trout injected with the G construct, alone or together with the N construct, were strongly protected against challenge with live virus (Heppel et al., 1997). VHSV DNA vaccine pCMV-vhsN encoding nucleocapsid protein N and glycoprotein G VO_0004339 DNA vaccine Research pcDNA 3 [Ref2400:Heppel et al., 1997] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pcDNA 3 expressed the gene encoding the nucleocapsid protein N (Heppel et al., 1997). DNA vaccine construction Vector pcDNA3 expressed glycoprotein G gene of the virus (Heppel et al., 1997). VO_0000286 Young rainbow trout injected with the G construct together with the N construct were strongly protected against challenge with live virus (Heppel et al., 1997). VHSV DNA vaccine pVHS-G encoding glycoproteins VO_0004494 DNA vaccine Research pcDNA3.1(+) vector [Ref2433:Lorenzen et al., 2002] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction The pcDNA3.1(+) vector expressed viral glycoproteins (G proteins) (Lorenzen et al., 2002). VO_0000286 Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the VHSV and IHNV whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high (Lorenzen et al., 2002). VHSV Inactivated Vaccine Chitosan Adjuvant Inactivated or "killed" vaccine Research oral Inactivated VHSV vaccine using Chitosan adjuvant protects olive flounder against VHSV challenge. (Jung et al., 2022) Chitosan (Jung et al., 2022) VHSV (10 TCID50/mL) was inactivated by adding 0.3% formalin with continuous stirring using a magnetic stirrer at 4 ◦C for 24 h. Complete inactivation of the inactivated VHSV (IV) was confirmed by re- inoculation into FHM cells and infection in olive flounder. The combination of saponin and chitosan was used for the evaluation of survival rate at two different concentrations (0.29 and 2.9 mg/g of fish body weight/day). Adjuvant mixtures were mixed using a magnetic stirrer. IV (10^8.8 TCID50/mL) (50% of the total volume) was added to each combination and homogenised on ice for 15 min using PolytronPT 1200E homogeniser (Kinematica, Switzerland) followed by gentle vortexing. (Jung et al., 2022) oral Inactivated VHSV (Jung et al., 2022) Trial III was conducted to study the duration of protection of saponin (0.29 or 2.9 mg) + IV (inactivated virus), chitosan (0.29 or 2.9 mg) + IV, and saponin (0.29 or 2.9 mg) + chitosan (0.29 or 2.9 mg) + IV vaccines. In this study, for primary immunisation, 120 fish (12.1 ± 0.2 g, 10.4 ± 0.6 cm) in each group were orally administered saponin + IV, chitosan + IV, and saponin + chitosan + IV. Each formulation was mixed with feed constituting 1% of total body weight and orally administered for 10 days. Additionally, 120 fish in the non-administered (virus-injected control) and naive groups were maintained at 21–23 ◦C in tanks (200 L) with continuous supply of seawater and aeration. (Jung et al., 2022) For the first challenge, the RPS values obtained after were 15%, 0%, 42.9%, and 25% (0.29 mg chitosan + IV), and 10%, 0%, 0%, and 12.5% (2.9 mg chitosan + IV) for the respective challenge time points. For re-challenge, the survival rate was 100% for chitosan (2.9 mg) + IV and 80% in the chitosan (0.29 mg) + IV group. Specific mortality was observed after six days in the virus-infected control group (not previously exposed to virus) with characteristic clinical signs of VHSV and 100% mortality rate at 12 dpi. (Jung et al., 2022) The group administered 2.9 mg of chitosan displayed severe red liver after 14 days of initial administration. Therefore, our results indicated that oral administration of chitosan at a dose of 2.9 mg (2916 mg/kg of fish) was toxic to olive flounder. (Jung et al., 2022) At 4, 9, 14, and 20 wpv (weeks post vaccination), each group of 15 fish was infected with VHSV (106.8 TCID50/100 μL/fish) via i. p. route and maintained for 21, 21, 35, and 91 days, respectively, in an aquarium containing 30 L of UV-treated seawater at 15 ◦C. All survivors from 20 wpv groups (Trial III) at 70 dpi were re-challenged with VHSV. The survivors were administered vaccines containing saponin + IV (10 and 9 fish), chitosan + IV (9 and 8 fish) and saponin + chitosan + IV (10 and 10 fish), respectively, for the administration doses of 0.29 and 2.9 mg. (Jung et al., 2022) VHSV Inactivated Vaccine Saponin + Chitosan Adjuvants Inactivated or "killed" vaccine Research oral Inactivated VHSV vaccine using mixture of Saponin and Chitosan adjuvants protects olive flounder against VHSV challenge. (Jung et al., 2022) Chitosan + Saponin (Jung et al., 2022) VHSV (10 TCID50/mL) was inactivated by adding 0.3% formalin with continuous stirring using a magnetic stirrer at 4 ◦C for 24 h. Complete inactivation of the inactivated VHSV (IV) was confirmed by re- inoculation into FHM cells and infection in olive flounder. The combination of saponin and chitosan was used for the evaluation of survival rate at two different concentrations (0.29 and 2.9 mg/g of fish body weight/day). Adjuvant mixtures were mixed using a magnetic stirrer. IV (108.8 TCID50/mL) (50% of the total volume) was added to each combination and homogenised on ice for 15 min using PolytronPT 1200E homogeniser (Kinematica, Switzerland) followed by gentle vortexing. (Jung et al., 2022) oral Inactivated VHSV Trial III was conducted to study the duration of protection of saponin (0.29 or 2.9 mg) + IV (inactivated virus), chitosan (0.29 or 2.9 mg) + IV, and saponin (0.29 or 2.9 mg) + chitosan (0.29 or 2.9 mg) + IV vaccines. In this study, for primary immunisation, 120 fish (12.1 ± 0.2 g, 10.4 ± 0.6 cm) in each group were orally administered saponin + IV, chitosan + IV, and saponin + chitosan + IV. Each formulation was mixed with feed constituting 1% of total body weight and orally administered for 10 days. Additionally, 120 fish in the non-administered (virus-injected control) and naive groups were maintained at 21–23 ◦C in tanks (200 L) with continuous supply of seawater and aeration. (Jung et al., 2022) For first challenge, at 4, 9, 14, and 20 wpv, the RPS values obtained after administration were 15%, 26.7%, 42.9%, and 37.5% (0.29 mg saponin +0.29 mg chi- tosan + IV), and 10%, 26.7%, 42.9%, and 37.5% (2.9 mg saponin +2.9 mg chitosan + IV) for the respective challenge time points. For re-challenge, survival rate was 100% for saponin (0.29 and 2.9 mg) + chitosan (0.29 and 2.9 mg) + IV. Specific mortality was observed after six days in the virus-infected control group (not previously exposed to virus) with characteristic clinical signs of VHSV and 100% mortality rate at 12 dpi. (Jung et al., 2022) Results indicated toxic effects of saponin at a concentration of 2.9 mg/fish. The group administered 2.9 mg of chitosan displayed severe red liver after 14 days of initial administration. These observations suggested that a combination containing saponin and chitosan, each at a concentration of 0.29 mg (291.6 mg/kg of fish), was less toxic and acceptable for vaccination studies. (Jung et al., 2022) At 4, 9, 14, and 20 wpv (weeks post vaccination), each group of 15 fish was infected with VHSV (106.8 TCID50/100 μL/fish) via i. p. route and maintained for 21, 21, 35, and 91 days, respectively, in an aquarium containing 30 L of UV-treated seawater at 15 ◦C. All survivors from 20 wpv groups (Trial III) at 70 dpi were re-challenged with VHSV. The survivors were administered vaccines containing saponin + IV (10 and 9 fish), chitosan + IV (9 and 8 fish) and saponin + chitosan + IV (10 and 10 fish), respectively, for the administration doses of 0.29 and 2.9 mg. (Jung et al., 2022) VHSV Inactivated Vaccine Saponin Adjuvant Inactivated or "killed" vaccine Research oral Inactivated VHSV vaccine using Saponin adjuvant protects olive flounder against VHSV challenge. (Jung et al., 2022) Saponin VHSV (10 TCID50/mL) was inactivated by adding 0.3% formalin with continuous stirring using a magnetic stirrer at 4 ◦C for 24 h. Complete inactivation of the inactivated VHSV (IV) was confirmed by re- inoculation into FHM cells and infection in olive flounder. The combination of saponin and chitosan was used for the evaluation of survival rate at two different concentrations (0.29 and 2.9 mg/g of fish body weight/day). Adjuvant mixtures were mixed using a magnetic stirrer. IV (10^8.8 TCID50/mL) (50% of the total volume) was added to each combination and homogenised on ice for 15 min using PolytronPT 1200E homogeniser (Kinematica, Switzerland) followed by gentle vortexing. (Jung et al., 2022) oral Inactivated VHSV (Jung et al., 2022) Trial III was conducted to study the duration of protection of saponin (0.29 or 2.9 mg) + IV (inactivated virus), chitosan (0.29 or 2.9 mg) + IV, and saponin (0.29 or 2.9 mg) + chitosan (0.29 or 2.9 mg) + IV vaccines. In this study, for primary immunisation, 120 fish (12.1 ± 0.2 g, 10.4 ± 0.6 cm) in each group were orally administered saponin + IV, chitosan + IV, and saponin + chitosan + IV. Each formulation was mixed with feed constituting 1% of total body weight and orally administered for 10 days. Additionally, 120 fish in the non-administered (virus-injected control) and naive groups were maintained at 21–23 ◦C in tanks (200 L) with continuous supply of seawater and aeration. (Jung et al., 2022) After first challenge, the RPS values obtained after vaccine administration were 20%, 13.4%, 14.3%, and 37.5% (0.29 mg saponin + IV), and 10%, 26.7%, 28.6%, and 25% (2.9 mg saponin + IV) for the respective challenge time points. After re-challenge. survival rate was 100% in saponin (0.29 and 2.9 mg) + IV. Specific mortality was observed after six days in the virus-infected control group (not previously exposed to virus) with characteristic clinical signs of VHSV and 100% mortality rate at 12 dpi. (Jung et al., 2022) Results indicated toxic effects of saponin at a concentration of 2.9 mg/fish (2916 mg/kg of fish); therefore, a concentration below 1.45 mg/fish is recommended as safe for administration to olive flounder. (Jung et al., 2022) At 4, 9, 14, and 20 wpv (weeks post vaccination), each group of 15 fish was infected with VHSV (10^6.8 TCID50/100 μL/fish) via i. p. route and maintained for 21, 21, 35, and 91 days, respectively, in an aquarium containing 30 L of UV-treated seawater at 15 ◦C. All survivors from 20 wpv groups (Trial III) at 70 dpi were re-challenged with VHSV. The survivors were administered vaccines containing saponin + IV (10 and 9 fish), chitosan + IV (9 and 8 fish) and saponin + chitosan + IV (10 and 10 fish), respectively, for the administration doses of 0.29 and 2.9 mg. (Jung et al., 2022) Glycoprotein Viral hemorrhagic septicemia virus Danish isolate DK-3592B 299444 Y18263 NP_049548 CDD:279337 11287 2958 4532 ? envelope glycoprotein 7.14 54010.72 647 G protein >gi|9632550:2958-4532 Viral hemorrhagic septicemia virus Fil3, complete genome CATGGAATGGAACACTTTTTTCTTGGTCATCTTGATCATCATCATAAAGAGCACCACACCACAGATCACT CAACGACCTCCGGTCGAAAACATCTCGACGTACCATGCAGATTGGGACACTCCGCTATACACTCATCCCT CCAACTGCAGGGACGATTCCTTTGTCCCGATTCGACCAGCTCAACTCAGGTGTCCTCATGAATTTGAAGA CATAAACAAGGGACTGGTCTCCGTCCCAACCAAGATCATCCATCTCCCGCTATCAGTCACCAGCGTCTCC GCAGTCGCGAGTGGCCACTACCTGCACAGAGTGACTTATCGAGTCACCTGTTCGACCAGCTTCTTTGGAG GGCAAACCATTGAAAAGACCATCTTAGAGGCGAAACTGTCTCGTCAGGAGGCCACAGACGAGGCAAGCAA GGATCACGAGTACCCGTTCTTCCCTGAACCCTCCTGCATCTGGATGAAAAACAATGTCCATAAGGACATA ACTCACTATTACAAGACCCCAAAAACAGTATCGGTGGATCTCTACAGCAGGAAATTTCTCAACCCTGATT TCATAGAGGGGGTTTGCACAACCTCGCCCTGTCAAACTCATTGGCAGGGAGTCTATTGGGTCGGTGCCAC ACCCAAAGCCCATTGCCCCACGTCGGAAACACTAGAAGGACACCTGTTCACCAGGACCCATGATCACAGG GTGGTCAAGGCAATTGTGGCAGGCCATCATCCCTGGGGACTCACAATGGCATGCACAGTGACATTCTGCG GGGCAGAATGGATCAAGACTGACCTGGGAGACCTGATCCAGGTAACAGGACCGGGGGGCACGGGGAAACT GACTCCAAAGAAGTGTGTCAATGCTGATGTCCAGATGAGGGGGGCAACAGATGACTTTTCTTATCTCAAC CATCTCATCACCAACATGGCTCAAAGAACCGAGTGCCTAGATGCCCATAGTGATATCACCGCTTCTGGGA AAATATCCTCATTTCTCCTCTCAAAGTTTCGTCCCAGCCACCCTGGACCCGGCAAGGCACACTATCTTCT CAACGGTCAAATCATGCGAGGTGACTGTGACTATGAGGCAGTAGTCAGCATCAACTACAACAGCGCTCAA TACAAGACAGTGAACAACACATGGAAATCATGGAAACGGGTAGACAACAACACAGACGGGTACGATGGGA TGATATTTGGGGACAAATTGATCATCCCGGACATCGAGAAGTATCAGAGTGTCTATGACAGTGGAATGCT CGTTCAAAGAAACCTTGTGGAAGTCCCTCATCTGAGCATTGTGTTTGTCTCCAACACATCTGATCTTTCC ACTAATCACATCCACACCAACCTAATCCCTTCGGATTGGTCATTCCACTGGAGTATTTGGCCCTCATTAT CTGGGATGGGGGTTGTGGGAGGGGCCTTCCTTCTACTGGTACTCTGCTGTTGCTGCAAGGCGTCCCCTCC CATTCCAAATTACGGGATTCCGATGCAGCAGTTCTCCAGAAGTCAGACGGTCTGAGCACACCTGTCCGAA TGACCACAATTCCTCTATTAGGTAGATAGAAAAAA >AAB26115.1 envelope glycoprotein, G protein [viral haemorrhagic septicaemia virus VHSV, Danish isolate DK-3592B, Peptide, 507 aa] MEWNTFFLVILIIIIKSTTPQITQRPPVENISTYHADWDTPLYTHPSNCRDDSFVPIRPAQLRCPHEFED INKGLVSVPTKIIHLPLSVTSVSAVASGHYLHRVTYRVTCSTSFFGGQTIEKTILEAKLSRQEATDEASK DHEYPFFPEPSCIWMKNNVHKDITHYYKTPKTVSVDLYSRKFLNPDFIEGVCTTSPCQTHWQGVYWVGAT PKAHCPTSETLEGHLFTRTHDHRVVKAIVAGHHPWGLTMACTVTFCGAEWIKTDLGDLIQVTGPGGTGKL TPKKCVNADVQMRGATDDFSYLNHLITNMAQRTECLDAHSDITASGKISSFLLSKFRPSHPGPGKAHYLL NGQIMRGDCDYEAVVSINYNSAQYKTVNNTWKSWKRVDNNTDGYDGMIFGDKLIIPDIEKYQSVYDSGML VQRNLVEVPHLSTVFVSNTSDLSTNHIHTNLIPSDWSFHWSIWPSLSGMGVVGGAFLLLVLCCCCKASPP IPNYGIPMQQFSRSQTV Protective antigen nucleocapsid Viral hemorrhagic septicemia virus DK-3592B 3282333 CDD:281242 11287 ? nucleocapsid protein 4.89 41709.47 490 N protein >AAC24962.1 nucleocapsid protein, partial [Viral hemorrhagic septicemia virus] MEGGIRAAFSGLNDVRIDPTGGEGRVLVPGDVELIVYVGGFGEEDRKVIVDALSALGGPQTVQALSVLLS YVLQGNTQEDLETKCKVLTDMGFKVTQAVRATSIEAGIMMPMRELALTVNDDNLMEIVKGTLMTCSLLTK YSVDKMIKYITKKLGELADTQGVGELQHFTADKAAIRKLAGCVRPGQKITKALYAFILTEIADPTTQSRA RAMGALRLNGTGMTMIGLFTQAANNLGIAPAKLLEDLCMESLVESARRIIQLMRQVSEAKSIQERYAIMM SRMLGESYYKSYGLNDNSKISYILSQISGKYAVDSLEGLEGIKVTEKFREFAELVAEVLVDKYERIGEDS TEVSDVIKEAARQHARRTSAKPEPKARNFRSSTGRGKEQETGESDDDDYPEDSD Protective antigen Heppel et al., 1997 journal JOËL HEPPELL, NIELS LORENZEN, NEIL K. ARMSTRONG, TONG WU, ELLEN LORENZEN, KATJA EINER-JENSEN, JOACHIM SCHORR, HEATHER L. DAVIS 1997 8 4 271-286 Fish & Shellfish Immunology Lorenzen et al., 2002 journal Lorenzen N, Lorenzen E, Einer-Jensen K, LaPatra SE 2002 26 2 173-179 Developmental and comparative immunology Standish et al., 2016 journal Standish IF, Millard EV, Brenden TO, Faisal M 2016 13 1 203 Virology journal WISC - VHSV website http://www.invasivespecies.wa.gov/priorities/viral_hemorrhagic.shtml