A total of 12 wild boars in the immunized groups II and III were tested, and after 24 days of direct contact with the challenged wild boars carrying the ASF virus, 11 wild boars showed good immune effect against the ASF virus and did not react, and the survival rate of the 12 wild boars in the immunized groups II and III was 92%.
Professor José Manuel Sanchez-Vizcaino
Director of the African Swine Fever Virus Research Laboratory of the National and World Organisation for Animal Health (OIE) in Spain, and Professor of the Veterinary Health Surveillance Center of the Complutense University of Madrid, Spain
Since the 60s of the last century, the research and development of African swine fever virus (ASFv) vaccine has been close to 60 years, and due to the lack of clear research and understanding of many aspects of protective immunity against ASF virus infection, there has been no significant progress in the research and development of traditional inactivated vaccines, subunit vaccines, nucleic acid vaccines, live virus vector vaccines, live attenuated vaccines, and gene mutation vaccines (including gene deletion vaccines).
At present, at least 15 research institutions in 8 countries around the world are working on the research and development of ASFV vaccines.
In January 2019, China launched a research and development project for a live vaccine with deletion of African swine fever virus (ASFV).
In October 2018, Zoetis was granted the "MGF Gene Deletion-based Attenuated African Swine Fever Vaccine" published by the University of Connecticut in the United States.
In March 2018, scientists in Spain published the results of research on the prevention and control of ASF virus infection with a live attenuated vaccine that lacks certain genes.
At the end of 2017, the European Union launched an innovative campaign against the ASF virus.
Because ASF virus gene mutation vaccines (including gene deletion vaccines) can provide complete protection, they are currently promising vaccines.
Although the threat posed by African swine fever virus to domestic pigs is more serious, the development of ASF virus vaccine for wild boars is of great significance for global ASF control and eradication, especially in Africa and the European Union, followed by China, because wild boars are densely distributed in many areas of China, and the situation of wild boars infected with ASF virus will be more serious in China in the future, and the management of forest ASF virus transmission cannot be ignored.
At the 2019 International Swine Science Forum held in Beijing in March, José Manuel Sanchez-Vizcaino, a world-renowned African swine fever expert and director of the African Swine Fever Virus Research Laboratory of the World Organisation for Animal Health (OIE) in Spain and a professor at the Veterinary Health Surveillance Center of the Complutense University of Madrid, Spain, presented a speech in the R&D and testing of wild boar oral vaccine against ASF virus genotype II strain" theme report said that the current immune challenge test shows that this ASF virus gene mutation vaccine has a good immune effect on wild boar, and has a good safety, and the next step will be to carry out wild boar actual immunization research and domestic pig immunization experimental research.
"ASF virus is transmitted both in forests and between livestock. We want to study why African soft ticks, African wild boars/African warthogs are resistant to ASF virus, and although they carry ASF virus for a long time, they do not develop disease. We hope to have a vaccine against these ASF virus hosts and
"In 2017, there was an outbreak of African swine fever in Latvia, which had a very high infection rate, mainly due to the ASF genotype II strain. At that time, we found a wild boar infected with the genotype II strain of the ASF virus in Latvia, and after taking samples, we tested and studied them in our laboratory. "We sequenced the ASF virus genes in this diseased wild boar sample and found that there was a mutation in the genes, and we studied the mutated genes to develop the world's ASF virus genotype II wild boar oral vaccine (Lv17/WB/RIE1, a vaccine developed through the study of mutated genes in the diseased wild boars infected with the ASF virus in Latvia in 2017)," said Professor Sánchez Skino. Here, we would like to introduce two experiments for this vaccine: one is the wild boar immunization and strong challenge experiment after immunization of this ASF virus genotype II oral vaccine, and the other is the experiment of observing the cross-infection and protective effect of strong virus challenge after immunization with wild boars with this ASF virus genotype II oral vaccine. In these two experiments, the strong strain of ASF virus Armenia07 genotype II was used for challenge (the African swine fever epidemic in Armenia in 2007 was mainly ASF virus genotype II, and the infection rate was also very high). ”
Professor Sánchez Skaeno also introduced that the two experiments lasted for nearly two months, using TCID50 (half of the tissue infection, that is, the amount of virus required to cause half of the cells to be damaged or killed in the culture plate well or test tube), erythrocyte adsorption test (HAD), real-time fluorescence PCR (polymerase chain reaction), immunoperoxygenase test (IPT), ELISA (enzyme-linked immunosorbent assay) and other methods. The temperature of excrement and rectum was measured twice, and comprehensive analysis and research were carried out in combination with viremia, antigen, clinical symptoms and autopsy results.
"In these two experiments, we selected a total of 18 baby wild boars with a month age of 3~4 months. Before the experiment, their individual weight was 10~15 kg, and after the experiment, their individual weight increased to 20~25 kg. "In Spain, wild boars are hunted for breeding. The baby wild boars we bought for the experiment came from a local pig farm. ”
"At the beginning, the main purpose of the experiment was to test the immune response, clinical symptoms, challenged infection and death of the experimental group (wild boars vaccinated with the oral vaccine of this ASF virus genotype II strain). Professor Sánchez Skaeno said that in the experiment, there were 6 wild boars in the control group and 12 wild boars in the experimental group (see figure above):
Group √ I (BOX1): Control group with 6 wild boars not infected with ASF virus.
Group √ II (BOX2): Experimental group, there were 5 wild boars, of which 4 were immunized by oral administration and 1 was immunized by contact into the body.
Group √ III (BOX3): Experimental group, there were 7 wild boars, of which 5 were immunized by oral administration and 2 were immunized by contact into the body.
After analyzing the experimental testing and monitoring data within 30 days, the results show (see the figure above):
√ compared with the control group, the different clinical symptoms were body temperature changes - among the 12 wild boars in the experimental group, 8 wild boars had an average body temperature of 40.1~40.8 °C, with a slight increase;
√ real-time PCR results showed that in terms of viremia, there were occasional (sporadic) weak positive expressions - 8 out of 12 wild boars had Ct values equal to 33.02±4.07 (Ctvalues, Cycle thresholdvalues, the number of cycles experienced when the fluorescence signal in each reaction tube reached the set threshold).
Data from ELISA antibody (Ab) detection shows (see figure above):
√ there was no significant change in antibody testing at 1 week (day 1 to day 7) after immunization;
√ one week after immunization (day 7 to day 30), the number of wild boars with positive antibody tests began to increase, and the antibody positivity rate began to increase;
√ to the 30th day of immunization (the day before challenge), the number of wild boars with positive antibody test among the 12 wild boars in the experimental group reached 8: 6 of the 9 wild boars who were orally immunized were positive, 2 of the 3 wild boars immunized by contact into the body were positive for antibody testing, and the positive rate of antibody in the experimental group reached 66.6%.
√ on the 31st day after immunization, the wild boars in the experimental group were challenged with the strong strain of ASF virus Armenia07 genotype II, and in the first 5 days after the challenge (the 31st to the 35th day after immunization), the positive rate of wild boar antibodies in the experimental group began to increase compared with the day before the challenge, although the increase was small, but it was more than 70% From the 6th day to the 0th day after challenge (the 35th to the 41st day after immunization), the positive rate of wild boar antibody in the experimental group increased significantly, and the increase showed a slight decrease on the 8th day after challenge, and the positive rate of wild boar antibody in the experimental group reached about 90% on the 9th day after challenge (the 40th day after immunization).
Professor Sánchez Scaino said that from the above antibody test results, the overall antibody positive rate of immunization and immune challenge in the experimental group remained at a high level, which indicates that the oral vaccine of ASF virus genotype II strain developed by us has a good immune protection effect on wild boars (the higher the antibody positive rate detected by ELISA, the better the immune effect/challenge protection effect of the pig herd against ASF virus).
In the second experiment, 4 of the 6 wild boars not infected with ASF virus were challenged with the strong strain of ASF virus Armenia07 genotype II, and 2 wild boars were used as the control for the challenge test, and then they were divided into 2 groups and mixed with wild boars in groups II and III who were vaccinated with the oral vaccine of this ASF virus genotype II strain for 30 days (see figure above).
Then, sampling tests were used to monitor whether wild boars that had been infected with ASF virus after challenge would transmit their ASF virus to other wild boars in the same group, and whether ASF virus cross-infection would occur between wild boars in the same group.
Group √ I (BOX1): 6 wild boars not infected with ASF virus were divided into 2 batches: the first batch of 3 wild boars, 1 wild boar as the challenge test control (not infected with ASF virus), 2 wild boars challenged by intramuscular injection (IM) (infected with the strong strain of ASF virus Armenia07 genotype II, the same below), and then mixed with group II wild boars, and the second batch of 3 wild boars: 1 wild boar as the challenge test control (not infected with ASF virus), entered by intramuscular injection
2 wild boars were attacked, and then mixed with wild boars from group III;
Group √ II (BOX2): From the day of immunization to day 30, there were 5 wild boars, of which 4 were immunized by oral administration and 1 was immunized by contact, and from the day of challenge (day 31 of immunization), group II was mixed with 8 wild boars.
Group √ III (BOX3): From the day of immunization to the 30th day, there were 7 wild boars, of which 5 were immunized orally and 2 were immunized by contact, and one of the 2 wild boars that had been immunized by contact entry was challenged by intramuscular injection, and since the day of the challenge (the 31st day of immunization), there were 10 wild boars in group III.
According to Professor Sánchez Skino's analysis, the results of 54 days of immunization and 24 days of immunization and 24 days of immunization and polyculture showed (see figure above):
√ 12 wild boars in groups II and III (including 1 immunized boar challenged by intramuscular injection) were tested, and 1 wild boar in oral immunization died due to ASF virus infection on the 5th day after direct contact with the challenged wild boars carrying ASF virus, and the mortality rate of 12 wild boars in groups II and III was 8%;
A total of 12 wild boars from immunized groups II and III were tested √, and after 24 days of direct contact with the challenged wild boars carrying the ASF virus, 11 of them showed good immunity to the ASF virus and did not react, and the survival rate of 12 wild boars in groups II and III was 92%.
Through the analysis of the above two experimental studies, we have found the answers to three questions. Professor Sánchez Skaeno concluded:
√ First, the immune efficacy and safety of vaccines. The oral vaccine of ASF virus genotype II strain developed by us can produce a good immune response to wild boars, and there are no adverse reactions in wild boars and no death of wild boars, indicating that the immune effect and safety of this ASF virus vaccine against wild boars are high.
√2. Will I be infected with ASF through direct contact after vaccination, and will the infection rate be high or low? The answer is yes, but the infection rate is low (8%). Because the monitoring shows that after direct contact between 12 wild boars and wild boars infected with ASF virus after direct contact, after 3~4 days, the phenomenon of ASF virus infection will occur in the immunized wild boars, but only 1 wild boar that was orally immunized was infected, and then died on the 5th day after direct contact with polyculture.
√3. Can this ASF virus vaccine play a good protective role against cross-infection?Through surveillance, the ASF virus vaccine we developed has a good protective effect against cross-infection of the strong strain of ASF virus Armenia07 genotype II, and among the 12 wild boars in groups II and III who have been immunized, 11 have no onset of disease after testing, and the survival rate is 92% (11/12).
Dissections need to be performed in a biosafety-level laboratory and cannot be dissected in the wild. Dissect the waste for harmless treatment.
"For the oral vaccine of the wild boar ASF virus genotype II strain developed in our laboratory, we conducted an autopsy on the diseased wild boar after vaccination with this immunization vaccine and the diseased wild boar after being infected with the strong strain of the ASF virus Armenia07 genotype II (Armenia/07), and conducted a pathological comparison study on their respective organ tissues and lymphoid organs, and found that there were obvious differences between the former and the latter in multiple organ tissues and multiple lymphoid organs (see the figure above). Professor Sánchez Skaeno analyzed.
"We analyzed the changes in 18 groups of organ tissues and lymphatic organs, including spleen, kidney, liver, lungs, heart, brain, bone marrow, bladder, intestines, tonsils, and mandibular lymph nodes, inguinal lymph nodes, retropharyngeal lymph nodes, mesenteric lymph nodes, lymph nodes between the stomach and liver, lymph nodes in front of the scapula, lymph nodes in the kidneys, and mediastinal lymph nodes (see figure above). Professor Sánchez Skaeno said that compared with the non-diseased wild boars after immunization, the spleen of the diseased wild boars infected with the ASF virus was particularly obvious, as well as the lesions of the Limba system, the inflammation of the lung infection, and the lesions of the intestinal system.
"In order to further test the immune effect of the oral vaccine of this ASF virus genotype II strain, we also performed autopsy on 16 tissues of 18 wild boars in the control group and the experimental group by real-time fluorescence PCR, as shown in the figure from left to right, arranged into 4 groups in each group, including bone marrow, brain, lymph nodes between the stomach and liver, heart, inguinal lymph nodes, kidneys, liver, lungs, mediastinal lymph nodes, mesenteric lymph nodes, lymph nodes in front of the scapula, renal lymph nodes, retropharyngeal lymph nodes, Spleen, mandibular lymph nodes, inguinal lymph nodes, retropharyngeal lymph nodes, mesenteric lymph nodes, lymph nodes between the stomach and liver, lymph nodes in front of the scapula, renal lymph nodes, Bladder system, and the measured Ct values (Cycle thresholdvalues, the number of cycles that the fluorescent signal in each reaction tube goes through when it reaches a set threshold) are compared to the number of days after viremia was detected at the end of the autopsy to determine the level of ASF virus (see figure above). "The blue color in the graph indicates that the virus has been immunized, the gray color indicates that it has been immunized but has no protective effect, the yellow color indicates that there has been contact at a later stage, and the red color indicates that the virus has been challenged by intramuscular injection." Judging from the Ct values of each of these 16 tissues, the current immune protection effect is good, and the dose used for immunization of this wild boar oral vaccine we developed is not particularly high, and the experimental results are relatively satisfactory. ”
"At present, the effect of this ASF virus vaccine on immunization of wild boars is relatively stable, and our research and development of this vaccine is still ongoing, and we will compare what is the difference between taking more doses and fewer doses per wild boar, and whether the former has a better immune effect and is safer than the latter. "As a next step, we hope to be able to conduct in vivo and in vitro immunization studies against this vaccine and determine the dose to be immunized against each wild boar in the herd during actual use, as well as the exact timing and appropriate interval for immunization." ”
"So far, we have conducted 50 days of experimental studies on the immunization and immune challenge of this ASF virus genotype II strain in wild boars, and we believe that this time is not long enough. "In the future, we will invest a longer period of experimental research on the immunization of wild boars against this vaccine and the practical application of immunization in wild boar herds to analyze how long the immunization effect in the laboratory and practical application can last, and at the same time, we also hope to do further immunization and immune challenge experiments on domestic pigs to study the immune response and immune effect of this vaccine in domestic pigs, as well as the safety and protection of this vaccine in the process of immunization in domestic pigs." ”
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