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SCIENCE CHINA Life Sciences, Volume 63 , Issue 5 : 623-634(2020) https://doi.org/10.1007/s11427-020-1657-9

A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

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  • ReceivedFeb 20, 2020
  • AcceptedFeb 27, 2020
  • PublishedMar 1, 2020

Abstract

African swine fever (ASF) is a devastating infectious disease in swine that is severely threatening the global pig industry. An efficacious vaccine is urgently required. Here, we used the Chinese ASFV HLJ/18 as a backbone and generated a series of gene-deleted viruses. The virulence, immunogenicity, safety, and protective efficacy evaluation in specific-pathogen-free pigs, commercial pigs, and pregnant sows indicated that one virus, namely HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, cannot convert to the virulent strain, and provides complete protection of pigs against lethal ASFV challenge. Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV, and as such is expected to play an important role in controlling the spread of ASFV.


Funded by

the National Key R&D Program of China(2018YFC1200601)

Applied Technology Research and Development Project of Heilongjiang Province(GA19B301)

Key-Area Research and Development Program of Guangdong Province(2019B020211004)

and the grant from the State Key Laboratory of Veterinary Biotechnology Program(SKLVBP201801)


Acknowledgment

We thank Susan Watson for editing the manuscript. This work was supported by the National Key R&D Program of China (2018YFC1200601), Applied Technology Research and Development Project of Heilongjiang Province (GA19B301), Key-Area Research and Development Program of Guangdong Province (2019B020211004), and the grant from the State Key Laboratory of Veterinary Biotechnology Program (SKLVBP201801).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Protective efficacy of long-lasting immunity induced by HLJ/18-7GD in commercial pigs.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


References

[1] Alejo A., Matamoros T., Guerra M., Andrés G.. A proteomic atlas of the African swine fever virus particle. J Virol, 2018, 92: pii: e01293-18 CrossRef PubMed Google Scholar

[2] Arias M., de la Torre A., Dixon L., Gallardo C., Jori F., Laddomada A., Martins C., Parkhouse R.M., Revilla Y., Rodriguez F.J.M., et al. Approaches and perspectives for development of African swine fever virus vaccines. Vaccines, 2017, 5: 35 CrossRef Google Scholar

[3] Borca M.V., Ramirez-Medina E., Silva E., Vuono E., Rai A., Pruitt S., Holinka L.G., Velazquez-Salinas L., Zhu J., Gladue D.P.. Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic Eurasia strain. J Virol, 2020a, : doi: 10.1128/JVI.02017-19 CrossRef Google Scholar

[4] Borca M.V., O’Donnell V., Holinka L.G., Risatti G.R., Ramirez-Medina E., Vuono E.A., Shi J., Pruitt S., Rai A., Silva E., et al. Deletion of CD2-like gene from the genome of African swine fever virus strain Georgia does not attenuate virulence in swine. Sci Rep, 2020b, 10: 494 CrossRef Google Scholar

[5] Coggins, L., Moulton, J.E., and Colgrove, G.S. (1968). Studies with HINDE attenuated African swine fever virus. Cornell Vet 4, 525–540. Google Scholar

[6] Gallardo C., Sánchez E.G., Pérez-Núñez D., Nogal M., de León P., Carrascosa Á.L., Nieto R., Soler A., Arias M.L., Revilla Y.. African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses. Vaccine, 2018, 36: 2694-2704 CrossRef Google Scholar

[7] Ge S., Li J., Fan X., Liu F., Li L., Wang Q., Ren W., Bao J., Liu C., Wang H., et al. Molecular characterization of African swine fever virus, China, 2018. Emerg Infect Dis, 2018, 24: 2131-2133 CrossRef Google Scholar

[8] Iglesias I., Rodríguez A., Feliziani F., Rolesu S., de la Torre A.. Spatio-temporal analysis of African swine fever in Sardinia (2012–2014): Trends in domestic pigs and wild boar. Transbound Emerg Dis, 2017, 64: 656-662 CrossRef Google Scholar

[9] Jancovich J.K., Chapman D., Hansen D.T., Robida M.D., Loskutov A., Craciunescu F., Borovkov A., Kibler K., Goatley L., King K., et al. Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins. J Virol, 2018, 92 CrossRef PubMed Google Scholar

[10] Kim H.J., Cho K.H., Lee S.K., Kim D.Y., Nah J.J., Kim H.J., Kim H.J., Hwang J.Y., Sohn H.J., Choi J.G., et al. Outbreak of African swine fever in South Korea, 2019. Transbound Emerg Dis, 2020, : doi: 10.1111/tbed.13483 CrossRef Google Scholar

[11] King D.P., Reid S.M., Hutchings G.H., Grierson S.S., Wilkinson P.J., Dixon L.K., Bastos A.D.S., Drew T.W.. Development of a TaqMan® PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods, 2003, 107: 53-61 CrossRef Google Scholar

[12] Krug P.W., Holinka L.G., O'Donnell V., Reese B., Sanford B., Fernandez-Sainz I., Gladue D.P., Arzt J., Rodriguez L., Risatti G.R., et al. The progressive adaptation of a Georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome. J Virol, 2015, 89: 2324-2332 CrossRef PubMed Google Scholar

[13] Le V.P., Jeong D.G., Yoon S.W., Kwon H.M., Trinh T.B.N., Nguyen T.L., Bui T.T.N., Oh J., Kim J.B., Cheong K.M., et al. Outbreak of African swine fever, Vietnam, 2019. Emerg Infect Dis, 2019, 25: 1433-1435 CrossRef Google Scholar

[14] Malmquist, W.A., and Hay, D. (1960). Hemadsorption and cytopathic effect produced by African swine fever virus in swine bone marrow and buffy coat cultures. Am J Vet Res 21, 104–108. Google Scholar

[15] Monteagudo P.L., Lacasta A., López E., Bosch L., Collado J., Pina-Pedrero S., Correa-Fiz F., Accensi F., Navas M.J., Vidal E., et al. BA71ΔCD2: a new recombinant live attenuated African swine fever virus with cross-protective capabilities. J Virol, 2017, 91: pii: e01058-17 CrossRef PubMed Google Scholar

[16] Murgia M.V., Mogler M., Certoma A., Green D., Monaghan P., Williams D.T., Rowland R.R.R., Gaudreault N.N.. Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus. Arch Virol, 2019, 164: 359-370 CrossRef Google Scholar

[17] O’Donnell V., Holinka L.G., Gladue D.P., Sanford B., Krug P.W., Lu X., Arzt J., Reese B., Carrillo C., Risatti G.R., et al. African swine fever virus Georgia isolate harboring deletions of MGF360 and MGF505 genes is attenuated in swine and confers protection against challenge with virulent parental virus. J Virol, 2015a, 89: 6048-6056 CrossRef PubMed Google Scholar

[18] O’Donnell V., Holinka L.G., Krug P.W., Gladue D.P., Carlson J., Sanford B., Alfano M., Kramer E., Lu Z., Arzt J., et al. African swine fever virus Georgia 2007 with a deletion of virulence-associated gene 9GL (B119L), when administered at low doses, leads to virus attenuation in swine and induces an effective protection against homologous challenge. J Virol, 2015b, 89: 8556-8566 CrossRef PubMed Google Scholar

[19] O’Donnell V., Risatti G.R., Holinka L.G., Krug P.W., Carlson J., Velazquez-Salinas L., Azzinaro P.A., Gladue D.P., Borca M.V.. Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge. J Virol, 2017, 91: pii: e01760-16 CrossRef PubMed Google Scholar

[20] Pejsak Z., Truszczyński M., Niemczuk K., Kozak E., Markowska-Daniel I.. Epidemiology of African swine fever in Poland since the detection of the first case. Polish J Vet Sci, 2014, 17: 665-672 CrossRef Google Scholar

[21] Quembo C.J., Jori F., Vosloo W., Heath L.. Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype. Transbound Emerg Dis, 2018, 65: 420-431 CrossRef Google Scholar

[22] Reed, L.J., and Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497. Google Scholar

[23] Reis A.L., Abrams C.C., Goatley L.C., Netherton C., Chapman D.G., Sanchez-Cordon P., Dixon L.K.. Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response. Vaccine, 2016, 34: 4698-4705 CrossRef Google Scholar

[24] Reis A.L., Goatley L.C., Jabbar T., Sanchez-Cordon P.J., Netherton C.L., Chapman D.A.G., Dixon L.K.. Deletion of the African swine fever virus gene DP148R does not reduce virus replication in culture but reduces virus virulence in pigs and induces high levels of protection against challenge. J Virol, 2017, 91: pii: e01428-17 CrossRef PubMed Google Scholar

[25] Revilla, Y., Perez-Nunez, D., and Richt, J. A. (2018). African swine fever virus biology and vaccine approaches. Adv Virus Res 100, 41–74. Google Scholar

[26] Ribeiro, M., Nunes Petisca, J.L., Lopez Frazao, F., and Sobral, M. (1963). Vaccination contre la pest porcine africaine. Bul Off Internatl Epizoot 60, 921. Google Scholar

[27] Sánchez-Vizcaíno J.M., Mur L., Martínez-López B.. African swine fever (ASF): five years around Europe. Vet Microbiol, 2013, 165: 45-50 CrossRef Google Scholar

[28] Sánchez E.G., Pérez-Núñez D., Revilla Y.. Development of vaccines against African swine fever virus. Virus Res, 2019, 265: 150-155 CrossRef Google Scholar

[29] Sunwoo S.Y., Pérez-Núñez D., Morozov I., Sánchez E., Gaudreault N., Trujillo J., Mur L., Nogal M., Madden D., Urbaniak K., et al. DNA-protein vaccination strategy does not protect from challenge with African swine fever virus Armenia 2007 strain. Vaccines, 2019, 7: 12 CrossRef Google Scholar

[30] Wade A., Achenbach J.E., Gallardo C., Settypalli T.B.K., Souley A., Djonwe G., Loitsch A., Dauphin G., Ngang J.J.E., Boyomo O., et al. Genetic characterization of African swine fever virus in Cameroon, 2010–2018. J Microbiol, 2019, 57: 316-324 CrossRef Google Scholar

[31] Wang N., Zhao D., Wang J., Zhang Y., Wang M., Gao Y., Li F., Wang J., Bu Z., Rao Z., et al. Architecture of African swine fever virus and implications for viral assembly. Science, 2019, 366: 640-644 CrossRef Google Scholar

[32] Wen X., He X., Zhang X., Zhang X., Liu L., Guan Y., Zhang Y., Bu Z.. Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018. Emerg Microb Infect, 2019, 8: 303-306 CrossRef Google Scholar

[33] Zhao D., Liu R., Zhang X., Li F., Wang J., Zhang J., Liu X., Wang L., Zhang J., Wu X., et al. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microb Infect, 2019, 8: 438-447 CrossRef Google Scholar

[34] Zsak L., Caler E., Lu Z., Kutish G.F., Neilan J.G., Rock D.L.. A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine. J Virol, 1998, 72: 1028-1035 CrossRef Google Scholar

  • Figure 1

    Generation and virulence evaluation of different gene-deleted African swine fever viruses (ASFVs). A, Schematic representation of the gene(s) and region(s) deleted in each gene-deleted ASFV. The deleted gene segments were replaced with the p72eGFP, eGFP, or p72mCherry reporter gene cassette as indicated. The virus-infected primary porcine alveolar macrophages expressing different fluorescence are shown on the right of the panel. Nucleotide positions indicating the boundaries of the deletion relative to the ASFV HLJ/18 genome are indicated. B, Survival rates of pigs inoculated with the wild-type ASFV HLJ/18 and different gene-deleted ASFVs.

  • Figure 2

    Protective efficacy induced by different gene-deleted ASFVs in pigs. Groups of specific-pathogen-free pigs inoculated with 103 or 105 TCID50 of the indicated gene-deleted ASFVs were challenged intramuscularly (i.m.) with lethal ASFV HLJ/18. The indicated samples were collected from dead pigs or surviving pigs that were euthanized on day 21 post-challenge for virus DNA detection. A, Survival rates of pigs. B–E, Viral DNA detection of pigs in the control group, the HLJ/18-9GL&UK-del-inoculated groups, the HLJ/18-6GD-inoculated groups, and the HLJ/18-7GD-inoculated groups. The dashed lines indicate the lower limit of detection. The red asterisk indicates that some blood samples were not collected from pigs that died during the night.

  • Figure 3

    Safety evaluation of HLJ/18-6GD and HLJ/18-7GD in pigs. HLJ/18-6GD and HLJ/18-7GD were serially passed in pigs, and the indicated samples from the dead pigs or surviving pigs that were euthanized on day 21 post-inoculation (p.i.) were collected for viral DNA detection. The viral HLJ/18-6GD DNA copies from the 2nd to 6th passage are shown in panels A to E, respectively. The survival rate of pigs inoculated with different passages of HLJ/18-6GD is shown in panel F. Viral DNA was not detected in any samples collected from the HLJ/18-7GD initially inoculated pigs or the blindly passed pigs (Table 3). Therefore, 14 pigs were inoculated with the 107.7 TCID50 dose of HLJ/18-7GD and two pigs at each timepoint were euthanized on days 2, 5, 8, 10, 12, 16, and 21 p.i.. Viral DNA was detected in a few lymph nodes of some pigs (G), and in two lymph nodes of one pig after the second passage (H), but was not detected in the blood, heart, liver, spleen, lung, kidney, tonsil, thymus, or other lymph nodes of any pigs (data not shown). The dashed lines indicate the lower limit of detection. LN 1: intestinal lymph node; LN 2: inguinal lymph node; LN 3: submaxillary lymph node; LN 4: bronchial lymph node; LN 5: gastrohepatic lymph node; LN 6: mediastinal lymph node.

  • Figure 4

    Vaccine efficacy of the HLJ/18-7GD in commercial pigs. Groups of clean commercial pigs were inoculated once (A, D) or twice (B, C) with 105 TCID50 (A, B, C) or 106 TCID50 (D) of LJL/18-7GD vaccine, and then challenged at the indicated timepoints with lethal HLJ/18 virus intramuscularly (i.m.) (A, B, D) or orally (C); similar aged unvaccinated pigs were used as controls. Pigs were observed for 21 days post-challenge and then euthanized for detection of the viral DNA of the challenging virus. The survival rates and viral DNA copies in the organs and tissues of the pigs are shown in the panels. The dashed lines indicate the lower limit of detection. The red asterisk indicates that some blood samples were not collected from pigs that died during the night. LN 1: intestinal lymph node; LN 2: inguinal lymph node; LN 3: submaxillary lymph node; LN 4: bronchial lymph node; LN 5: gastrohepatic lymph node; LN 6: mediastinal lymph node.

  • Table 1   Virulence of gene-deleted ASFVs in SPF pigs

    Virus

    Dose

    Characteristics of fever in pigs after inoculation with each gene-deleted virus

    No. of survivingpigs/total

    No. of pigs with fever/total

    Duration (day)

    Highest body temperature (°C)

    HLJ/18-6GD

    103 TCID50

    0/4

    /

    /

    4/4

    105 TCID50

    0/4

    /

    /

    4/4

    HLJ/18-9GL&UK-del

    103 TCID50

    0/6

    /

    /

    6/6

    105 TCID50

    0/6

    /

    /

    6/6

    HLJ/18-7GD

    103 TCID50

    0/4

    /

    /

    4/4

    105 TCID50

    0/4

    /

    /

    4/4

    HLJ/18-DP148R-del

    103 TCID50

    3/3

    4

    41

    0/3

    105 TCID50

    3/3

    5

    41.4

    0/3

    HLJ/18-CD2v&UK-del

    103 TCID50

    2/4

    3–6

    41

    2/4

    105 TCID50

    4/4

    2–9

    42

    2/4

    HLJ/18-CD2v-del

    103 TCID50

    3/4

    4–5

    41.1

    1/4

    105 TCID50

    2/4

    5–6

    41.4

    2/4

  • Table 2   Protective efficacy of gene-deleted ASFVs against lethal ASFV HLJ/18 challenge in SPF pigs

    Virus

    Dose

    Characteristics of fever in pigs after challenge with lethal HLJ/18

    No. of survivingpigs/total

    No. of pigs with fever/total

    Duration (day)

    Highest body temperature (°C)

    Control

    Not applicable

    4/4

    3–5

    41.4

    0/4

    HLJ/18-9GL &UK-del

    103 TCID50

    6/6

    5–8

    41.7

    0/6

    105 TCID50

    6/6

    4–8

    41.6

    0/6

    HLJ/18-6GD

    103 TCID50

    1/4

    9

    41.4

    4/4

    105 TCID50

    1/4

    6

    41.9

    4/4

    HLJ/18-7GD

    103 TCID50

    4/4

    3–9

    42

    4/4

    105 TCID50

    1/4

    1

    40.7

    4/4

  • Table 3   Viral DNA detection from pigs that were used for the safety evaluation of the HLJ/18-6GD and HLJ/18-7GD viruses

    Virus

    No. of passagein pigs

    Number of viral DNA-positive pigs/total number of pigs

    No. of pigs with fever/total

    Blood on days post-inoculation (p.i.) (highest copy number, log10)

    Samples collected from pigs euthanized on day 21 p.i. or from pigs that died before day 21 p.i.

    Day 5

    Day 10

    Day 15

    Blood

    Heart

    Liver

    Spleen

    Lung

    Kidney

    Tonsil

    Thymus

    LN 1

    LN 2

    LN 3

    LN 4

    LN 5

    HLJ/18-6GD

    1

    4/6 (6.0)

    4/6 (6.1)

    4/6 (6.1)

    ND

    ND

    ND

    1/6

    ND

    ND

    ND

    ND

    0/6

    0/6

    0/6

    0/6

    0/6

    0/6

    2

    3/3 (6.6)

    3/3 (7.2)

    3/3 (7.1)

    3/3

    1/3

    1/3

    2/3

    1/3

    0/3

    0/3

    0/3

    0/3

    0/3

    1/3

    1/3

    2/3

    0/3

    3

    2/3 (6.6)

    3/3 (7.9)

    ND

    3/3

    1/3

    1/3

    2/3

    2/3

    1/3

    0/3

    1/3

    1/3

    0/3

    2/3

    2/3

    2/3

    1/3

    4

    3/3 (8.3)

    3/3 (7.9)

    3/3 (5.9)

    3/3

    0/3

    0/3

    3/3

    0/3

    1/3

    1/3

    1/3

    0/3

    0/3

    2/3

    0/3

    0/3

    0/3

    5*

    3/3 (8.1)

    3/3 (8.0)

    3/3 (6.0)

    3/3

    3/3

    2/3

    3/3

    3/3

    2/3

    2/3

    3/3

    2/3

    1/3

    2/3

    1/3

    1/3

    2/3

    6**

    4/5 (8.7)

    5/5 (9.4)

    4/4 (8.2)

    4/4

    4/5

    5/5

    5/5

    5/5

    5/5

    3/5

    3/5

    4/5

    3/5

    3/5

    4/5

    4/5

    4/5

    HLJ/18-7GD

    1

    0/6

    0/6

    0/6

    ND

    ND

    ND

    0/6

    ND

    ND

    ND

    ND

    0/6

    0/6

    0/6

    0/6

    0/6

    0/6

    2 to 5

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    0/12

    Groups of six pigs were intramuscularly injected with 107 TCID50 of each test virus, and their blood, organs, and tissues were collected at the indicated timepoints for viral DNA detection. Viral DNA-positive blood samples collected from the HLJ/18-6GD-inoculated pigs were used to inoculate pigs in the subsequent passage (passages 2–6). Since the viral DNA was not detected from any pigs that were inoculated with the HLJ/18-7GD virus, the pooled blood samples collected from pigs on days 5 and 10 post-inoculation were used to inoculate pigs in the subsequent passage (passages 2–5). *, one pig in this group died on day 11 post-inoculation. **, one pig in this group died on day 13 post-inoculation, and its blood sample was not collected. LN 1: intestinal lymph node; LN 2: inguinal lymph node; LN 3: submaxillary lymph node; LN 4: bronchial lymph node; LN 5: gastrohepatic lymph node. ND, not done.

  • Table 4   Protective efficacy of HLJ/18-7GD in commercial pigs

    Administration

    Characteristics of fever in pigs after challenge with lethal HLJ/18

    No. of surviving pigs/total

    Challengeroute

    Challenge time (days after last vaccination)

    Vaccine dosage

    Group

    No. of pigs with fever/total

    Duration (day)

    Highest body temperature (°C)

    Intramuscular

    28

    105 TCID50, once

    Vaccinated

    1/5

    2

    40.6

    5/5

    Control

    5/5

    4–8

    42.0

    0/5

    Intramuscular

    14

    105 TCID50, twice

    Vaccinated

    1/5

    2

    41.2

    5/5

    Control

    4/4

    5–6

    42.0

    0/4

    Oral

    21

    105 TCID50, twice

    Vaccinated

    1/5

    2

    41.8

    5/5

    Control

    4/4

    4–6

    42.0

    0/4

    Intramuscular

    70

    106 TCID50, once

    Vaccinated

    2/6

    2

    41.3

    6/6

    Control

    4/4

    4–8

    42.0

    0/4

  • Table 5   The safety of HLJ/18-7GD in pregnant sows

    Sow number

    Time (days) of pregnancy of the sow

    Piglet information

    Transferred to the lab (stage of gestation)

    Vaccinated

    Delivered

    Total number

    Number of mummified fetuses

    Number of stillborn piglets

    Healthy rate(%)

    1

    89 (late)

    94

    112

    11

    0

    0

    100

    2

    89 (late)

    94

    114

    19

    1

    0

    94.7

    3

    58 (middle)

    63

    117

    15

    1

    1

    86.7

    4

    57 (middle)

    62

    114

    18

    2

    0

    88.9

    5

    57 (middle)

    62

    114

    18

    0

    1

    94.4

    6

    30 (early)

    35

    115

    18

    1

    0

    94.4

    7

    54 (middle)

    NA

    110

    19

    0

    2

    89.5

    8

    27 (early)

    NA

    115

    14

    1

    1

    85.7

    All pigs were primiparous sows.

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