The attenuated African swine fever vaccine HLJ/18-7GD provides protection against emerging prevalent genotype II variants in China

ABSTRACT

Genetic changes have occurred in the genomes of prevalent African swine fever viruses (ASFVs) in the field in China, which may change their antigenic properties and result in immune escape. There is usually poor cross-protection between heterogonous isolates, and, therefore, it is important to test the cross-protection of the live attenuated ASFV vaccines against current prevalent heterogonous isolates. In this study, we evaluated the protective efficacy of the ASFV vaccine candidate HLJ/18-7GD against emerging isolates. HLJ/18-7GD provided protection against a highly virulent variant and a lower lethal isolate, both derived from genotype II Georgia07-like ASFV and isolated in 2020. HLJ/18-7GD vaccination prevented pigs from developing ASF-specific clinical signs and death, decreased viral shedding via the oral and rectal routes, and suppressed viral replication after challenges. However, HLJ/18-7GD vaccination did not provide solid cross-protection against genotype I NH/P68-like ASFV challenge in pigs. HLJ/18-7GD vaccination thus shows great promise as an alternative strategy for preventing and controlling genotype II ASFVs, but vaccines providing cross-protection against different ASFV genotypes may be needed in China.

KEYWORDS:

• African swine fever virus
• live attenuated vaccine
• cross-protection
• field prevalent isolate

Introduction

African swine fever (ASF) is a highly contagious disease caused by ASF virus (ASFV), which infects both wild boar and domestic swine with high mortality and morbidity [1]. ASF is listed as a notifiable disease by the World Organization for Animal Health (WOAH), and is a threat to global food security. ASF was first detected in Kenya in 1921, then spread into Europe and America. In 2018, genotype II Georgia07-like ASFVs invaded China, the world’s biggest pig producer and pork consumer, and then spread to 16 other Asian countries, causing considerable socioeconomic losses (www.woah.org) [2, 3]. ASF has continued to spread across five continents and poses a great threat to swine production worldwide (https://wahis.oie.int).

ASFV has been naturally evolving in China since its introduction in 2018, and its biological properties have also changed [4, 5]. During ASFV surveillance in 2020, we found that nucleotide mutations, deletions, insertions, or short-fragment replacement had occurred in isolates compared to the HLJ/18 virus, the earliest genotype II Georgia07-like ASFV in China [4]. Moreover, 11 isolates had four different types of natural mutations or deletions in the EP402R gene, resulting in the abortive expression of CD2v protein and the non-HAD phenotype [4]. Further ASF surveillance showed that genotype I non-HAD ASFVs emerged in domestic pigs in China [5]. Both non-HAD genotype I and II isolates have lower lethality and high transmissibility in pigs, which hampers early detection and poses challenges for ASF control in China [4, 5].

Despite extensive research on ASF, its control and eradication currently depend on rapid diagnosis, strict biosecurity measures, and the slaughter and disposal of all infected animals. Vaccination has been proven to be the most economical and powerful tool to control viral diseases. Different strategies for ASF vaccine development have been attempted [6–8]; however, inactivated, subunit, DNA, and engineered vectored vaccines could not provide solid protection against virulent ASFV challenge [9–12]. Some gene-deleted attenuated viruses have shown promise as ASF vaccines [13–17], and ASFV-G-ΔI177L has been approved for use in the field as a commercial product in Vietnam since July 24, 2023 [18, 19].

In previous studies, we developed a seven-gene-deleted live attenuated vaccine candidate (HLJ/18-7GD) based on the earliest virulent isolate in China HLJ/18. Animal studies demonstrated that HLJ/18-7GD is safe in pigs and effective against challenges with homologous lethal ASFV HLJ/18 in the laboratory and in the field [19, 20]. It remains unclear whether HLJ/18-7GD can provide efficient protection against heterogonous ASFVs. In this study, we examined the protective efficacy of HLJ/18-7GD against highly virulent and lower lethal isolates in China.

Materials and methods

Ethics statements

All animal experiments were conducted in compliance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) and the Animal Ethics Committee of Heilongjiang Province, China.

Biosafety statement and facility

All experiments with live ASFVs were carried out within the enhanced biosafety level 3 (P3+) facilities in the HVRI of the CAAS approved by the Ministry of Agriculture and Rural Affairs and China National Accreditation Service for Conformity Assessment.

Cell culture and viruses

Primary porcine alveolar macrophages (PAMs) were prepared from 1 to 2-month-old specific-pathogen-free (SPF) piglets, and maintained in RPMI 1640 medium (Thermo Scientific, USA) supplemented with 5% porcine serum (HyClone, USA), 100 U/ml penicillin and streptomycin, and 2 mmol/ml glutamine at 37°C with 5% CO₂.

HLJ/18-7GD was previously constructed by deleting the following seven genes: MGF505-1R, MGF505-2R, MGF505-3R, MGF360-12L, MGF360-13L, MGF360-14L, and EP402R [20]. During ASF surveillance, clinical samples of blood, lung, or lymph nodes were first tested by using quantitative PCR (qPCR), and ASFV positive samples were used for virus isolation as described previously [5].

ASFV stocks were amplified and titrated in PAMs as described previously [3–5]. Live ASFV titers were determined in PAMs. Briefly, ASFV samples were serially diluted and inoculated into PAMs in 96-well plates. Haemadsorption (HAD) was observed for 7 days, or indirect immunofluorescence was performed using an anti-P72 antibody (in-house) on day 7 post-inoculation (p.i.). The 50% HAD values (HAD₅₀) or 50% Tissue Culture Infectious Doses (TCID₅₀) were calculated by using the method of Reed and Muench [21].

Viral gene sequencing and genetic analysis

Viral genomic DNA was amplified by PCR and sequenced using segment-specific primers and first-generation sequencing (Applied Biosystems: ABI3500xL). Genetic analysis was carried out by using the software DNAStar and MEGA 4.0. Primer sequences for PCR amplification and sequencing were published in a previous study [22].

Pig studies

Large White and Landrace-crossed SPF pigs were obtained from the Laboratory Animal Center of the HVRI. To evaluate the protective efficacy of the seven-gene-deleted ASFV vaccine candidate against challenge with newly emerging ASFVs, groups of pigs were intramuscularly (i.m.) vaccinated with 10⁶ TCID₅₀ of HLJ/18-7GD, and then challenged with 10^1.5 HAD₅₀ of HuB/628/20, 10⁶ TCID₅₀ of HLJ/HRB1/20, and 10⁶ TCID₅₀ of SD/DY-I/21 on 28 days post-immunization (p.im.), respectively. Unvaccinated SPF pigs were challenged with the corresponding viruses as controls. Detailed information about the challenge viruses and doses is provided in Table S1. The pigs were monitored daily for 28 days post-challenge (p.c.) for rectal temperature, mortality, and clinical signs including anorexia, recumbency, rash, and swelling of joints. Fever was recorded when the rectal temperature rose above 40^oC. Clinical scores for each pig were recorded daily and summed based on a scoring system described previously with minor modifications [23]; rectal temperature was not included in this scoring system. Oral and rectal swabs, and blood from the pigs were collected at the indicated timepoints. Tissues including heart, liver, spleen, lung, kidney, and four lymph nodes (intestinal lymph node, inguinal lymph node, submaxillary lymph node, and bronchial lymph node) were collected from the dead pigs, as well as from the surviving pigs, which were euthanized at the end of the observation period, for viral DNA detection by using qPCR, or for histopathological analyses.

qPCR

ASFV genomic DNA was extracted from oral and rectal swabs, EDTA-treated whole peripheral blood samples or tissue homogenates. WOAH-recommended qPCR was carried out using a QuantStudio 5 system (Applied Biosystems, USA) as described previously [24].

Results

Emergence of different variants and genotypes of ASFVs in the field in China

ASFV has continuously spread and evolved in nature in China since 2018, and genetic variations have been identified in different ASFVs including virulent genotype II virus, moderately virulent genotype II variant, low virulent genotype I virus, and lethal genotype I and II recombinant virus [4, 5, 22].

Genotype II virus HuB/628/20 was isolated in the Hubei province of China in 2020 and showed high lethality in pigs similar as the earliest isolate in China, HLJ/18 [4]. To characterize the genetic changes in HuB/628/20, we sequenced its whole genome by using a segmentation PCR strategy as previously described [2]. The sequence data have been deposited in GenBank and the accession number is OR126359. Compared to the whole genome of virulent HLJ/18, HuB/628/20 has 10 nucleotide mutations that result in six amino acid changes in six viral genes (MGF_110-1L, MGF_110-3L, MGF_110-4L, MGF_110-5L-6L, MGF_360-12L, and QP383R), a C insertion in a noncoding region, and 20 nucleotide deletions in eight sites involving four viral genes (MGF_360-2L, MGF_360-7L, MGF_110-14L, and MGF_110-13L) (Figure 1 and Table 1). Our genetic analysis showed that many genetic alterations have occurred in the genome of the prevalent virulent genotype II virus HuB/628/20 compared with the early isolate HLJ/18.

Figure 1. Genetic changes of new field prevalent ASFVs compared with HLJ/18. The whole genome sequences of genotype II field isolates HuB/628/20 and HLJ/HRB1/20 were respectively compared with HLJ/18 for nucleotide mutations (A), nucleotide deletions, insertions, and replacements (B). The names of the ORFs are shown at the top of each panel.

Table 1. Genetic changes in the prevalent genotype II ASFVs compared with HLJ/18.

Virus	Position	ORF/Region	Nucleotide change	ORF/amino acid change
HuB/628/20	434	Noncoding Region	C insertion	/^a
	794	Noncoding Region	A deletion	/
	2386	MGF_360-2L	A deletion	18 AA truncation
	6260	MGF_110-1L	G → T	T → N
	6868	Noncoding Region	G → A	/
	7368	MGF_110-3L	A → T	H → Q
	8084	MGF_110-4L	T → C	Q → R
	8613	MGF_110-5L-6L	G → C	H → Q
	9733	MGF_110-7L	C deletion	112 AA truncation
	13275	MGF_110-14L	5 C deletion	5 AA extension
	14708	MGF_110-13L	7 C deletion	148 AA truncation
	16886	Noncoding Region	G deletion	/
	19470	Noncoding Region	T deletion	/
	20835	Noncoding Region	3 G deletion	/
	29426	MGF_360-12L	A → G	M → T
	46581	A224L	G → A	84 AA truncation
	97103	B438L	G → A	/
	160727	QP383R	G → A	S → N
	180649	Noncoding Region	T → C	/
HLJ/HRB1/20	1231	MGF_360-1L	A → G	/
	6043	MGF_110-1L	86 nt insertion	73 AA extension
	6260	MGF_110-1L	G → T	T → N
	6868	Noncoding Region	G → A	/
	7384	MGF_110-3L	C → T	G → D
	8084	MGF_110-4L	T → C	Q → R
	8613	MGF_110-5L-6L	G → C	H → Q
	10880	Noncoding Region	C → T	/
	13266	MGF_110-14L	5 C deletion	5 AA extension
	16886	Noncoding Region	G deletion	/
	19032	ASFV-G-ACD-00350	4 G deletion	29 AA truncation
	42533	Noncoding Region	C → A	/
	50632	A238L	14 nt deletion^b	4 AA extension
	63596	K205R	C → T	T → I
	65215	Noncoding Region	T deletion	/
	65610	K421R	C → T	/
	72908	EP153R	A deletion	138 AA truncation
	73428	EP402R	25 nt deletion^c	343 AA truncation
	144740	D205R	G → A	/
	160727	QP383R	G → A	S → N
	167740	E248R	G → A	S → N
	169629	E66L	G → A	H → Y

^a means no change.

^b 14 nt denotes GTTTTGGTAGTCAT.

^c 25 nt denotes GTATTGATTATTGGGTTAGTTTTAA.

Genotype II variant HLJ/HRB1/20 was isolated in the Heilongjiang province of China in 2020, and showed less lethality than HLJ/18 in pigs due to a 25-nt deletion in its EP402R gene that resulted in the abortive expression of CD2v protein, and a 10⁶ TCID₅₀ dose led to the deaths of 75% of pigs [4]. Compared to the whole genome of virulent HLJ/18, HLJ/HRB1/20 (Accession number: MW656282) has 23 selected ORFs or regions with high-frequency mutations, a 25-nucleotide deletion in the EP402R gene, a 14-nucleotide deletion in the A238L gene [4], 14 nucleotide mutations that result in eight amino acid changes in eight viral genes (MGF_110-1L, MGF_110-3L, MGF_110-4L, MGF_110-5L-6L, K205R, QP383R, E248R, and E66L), a short 86 bp-replacement in MGF_110-1L, and 51 nucleotide deletions in seven sites involving five viral genes (MGF_110-14L, ASFV-G-ACD-00350, A238L, EP153R, and EP402R) (Figure 1 and Table 1).

Genotype I viruses of SD/DY-I/21 and HeN/ZZ-P1/21 were detected in the field in Shandong and Henan provinces in 2021, respectively [5]. Phylogenetic analysis using the whole genome sequences showed that SD/DY-I/21 (GenBank: MZ945537) and HeN/ZZ-P1/21 (GenBank: MZ945536) belong to the same clade with the genotype I Portuguese isolates NH/P68 and OURT88/3, and differ from the genotype II ASFVs in China [5, 25, 26]. Animal studies have shown that the genotype I NH/P68-like virus SD/DY-I/21 has low virulence but transmits efficiently in pigs, and causes chronic clinical signs including necrotic skin lesions and joint swelling [5].

Attenuated live vaccine candidate HLJ/18-7GD induced protection against challenge with a prevalent highly virulent genotype II HLJ/18-like variant

To evaluate whether the genetic changes of HuB/628/20 affect the protective capability of HLJ/18-7GD, five 7-week-old SPF pigs were i.m. inoculated with 10⁶ TCID₅₀ of HLJ/18-7GD, and then i.m. challenged with 10^1.5 HAD₅₀ of HuB/628/20 on day 28 post-immunization (p.im.). Four unvaccinated SPF pigs were i.m. challenged with the same dose of HuB/628/20 as a control. All four control pigs showed obvious ASF clinical signs and fever from day 6 post-challenge (p.c.), and died between day 9 and day 11 p.c. (Figure 2). In the immunized group, three pigs immunized with HLJ/18-7GD showed normal rectal temperature without any clinical signs, and two vaccinated pigs showed transient fever from day 4 p.c.. All five vaccinated pigs survived the 28-day observation period (Figure 2).

Figure 2. Protection efficacy of HLJ/18-7GD in pigs challenged with the prevalent highly virulent genotype II variant HuB/628/20. HLJ/18-7GD-vaccinated and control pigs were challenged with 10^1.5 HAD₅₀ of HuB/628/20, and were monitored daily for rectal temperature (A and B) and survival (C and D) for 28 days post-challenge. Viral DNA was detected by qPCR in the indicated tissue samples collected from the dead pigs in the control group and the surviving pigs in the vaccinated group, which were euthanized on day 28 post-challenge (E and F). The dashed black lines in panel A indicate the threshold of normal rectal temperature. LN1, intestinal lymph node; LN2, inguinal lymph node; LN3, submaxillary lymph node; LN4, bronchial lymph node.

Oral and rectal swabs and blood were collected at different timepoints after challenge. In the control pigs, viral DNA was detected in oral swabs from day 5 p.c. and in rectal swabs and blood from day 3 p.c., and viral DNA levels gradually increased until the pigs died (Figure S1). In the vaccinated pigs, viral DNA was detected in 4 oral swabs and 5 rectal swabs between days 5 and 10 p.c., and in 7 blood samples between days 5 and 15 p.c. (Figure S1). High viral loads were detected in all tissues and lymph nodes of the dead control pigs (Figure 2), whereas when the vaccinated pigs were euthanized on day 28 p.c. and their tissues excised, no viral DNA was detected in the examined tissues and lymph nodes of two vaccinated pigs, and low levels of viral DNA was only detectable in the spleen and kidney from one pig and in several lymph nodes from two pigs (Figure 2).

Histopathological analysis of the lung, spleen, kidney, tonsil, inguinal lymph node, and submaxillary lymph node showed that vaccination with HLJ/18-7GD ameliorated histopathological changes (Figure S2). Tissues from non-immunized piglets exhibited remarkable pathological alterations including interstitial pneumonia with dense infiltration of inflammatory cells within the pulmonary alveolar walls, red blood cell accumulation and white pulp atrophy in the spleen parenchyma, hemorrhaging and infiltration of inflammatory cells in the kidney, and lymphocyte necrosis in the tonsil (Figure S2). The structure of lymph nodules was blurred, and the lymphocytes was obviously reduced and necrotic in inguinal lymph nodes and submaxillary lymph nodes from non-immunized piglets (Figure S2). However, such pathological changes were not clearly observed in the immunized pigs except for mild to moderate interstitial pneumonia observed in the lungs (Figure S2). These results indicate that HLJ/18-7GD induced protection against challenge with a prevalent highly virulent HLJ/18-like variant.

HLJ/18-7GD provided efficient protection against challenge with a lower lethal genotype II HLJ/18-like variant

To investigate whether HLJ/18-7GD could induce protection against the emerging lower lethal genotype II ASFV variants, five 7-week-old SPF pigs were i.m. immunized with 10⁶ TCID₅₀ of HLJ/18-7GD, and then i.m. challenged with 10⁶ TCID₅₀ of HLJ/HRB1/20 on day 28 p.im.. Four control pigs were i.m. challenged with the same dose of HLJ/HRB1/20. Rectal temperature, survival, and clinical signs including anorexia, recumbency, rash, and swelling of joints were recorded daily for 28 days. The daily clinical scores for each pig were then summed using a scoring system described as previous description with minor modifications [23]. In the control group, one pig had a fever from day 9 p.c., had high clinical scores and depression from day 8 p.c., and died on day 11 p.c.. Another control pig began experiencing fever on day 4 p.c., had high clinical scores and depression from day 13 p.c., developed phyma, cutaneous necrosis, and arthroncus on days 19, 20, and 21 p.c., respectively, and died on day 22 p.c. (Figure 3 and Table 2). The remaining two control pigs survived the duration of the observation period, although one of them showed fever from day 7 p.c., had high clinical scores and depression from day 11 p.c., and developed arthroncus from day 22 p.c. (Figure 3 and Table 2). In the vaccinated group, no pig showed a fever or any chronic clinical sign, and all pigs survived the 28-day observation period (Figure 3 and Table 2). Oral and rectal swabs and blood were collected at different timepoints after challenge. Viral DNA was randomly detected in oral and rectal swabs and blood from the control pigs even on day 28 p.c. (Figure 4). Low levels of viral DNA were detected in three swabs from two vaccinated pigs before day 6 p.c., and no viral DNA was detected in the blood of the vaccinated pigs (Figure 4).

Figure 3. Clinical signs and survival of HLJ/18-7GD-vaccinated pigs challenged with the genotype II lower lethal variant HLJ/HRB1/20. HLJ/18-7GD-vaccinated and control pigs were challenged with 10⁶ TCID₅₀ of the genotype II lower lethal variant HLJ/HRB1/20 and monitored daily for 28 days post-challenge. Clinical scores (A and B) were recorded and summed daily based on a harmonized scoring system described previously [23]. Rectal temperature (C and D) and survival (E and F) of all pigs were assessed daily after the challenge. The dashed black lines in panels C and D indicate the threshold of normal rectal temperature. Clinical signs including depression, arthroncus, cutaneous necrosis, and phyma were recorded (G).

Figure 4. Viral shedding and replication in HLJ/18-7GD-vaccinated pigs challenged with the genotype II lower lethal variant HLJ/HRB1/20. HLJ/18-7GD-vaccinated and control pigs were challenged with 10⁶ TCID₅₀ of the genotype II lower virulent variant HLJ/HRB1/20. Oral and rectal swabs and blood were collected at the indicated days post-challenge. The indicated tissue samples were collected from the dead pigs and surviving pigs that were euthanized on day 28 post-challenge. Viral DNA in these samples was extracted and detected using qPCR. LN1, intestinal lymph node; LN2, inguinal lymph node; LN3, submaxillary lymph node; LN4, bronchial lymph node.

Table 2. Clinical signs of HLJ/18-7GD vaccinated pigs challenged with the lower lethal ASFVs.

Administration		Pig No.	Earliest time disease signs appeared (days post-challenge)
Virus	Group		Fever	Arthroncus	Phyma	Cutaneous necrosis	Depression	Death
HLJ/HRB1/20 (Genotype II)	Control	3244	7	22	-^a	-	11	/^b
		3248	-	-	-	-	-	/
		4211	4	21	19	20	13	22
		2309	9	-	-	-	8	11
	Vaccinated	2915	-	-	-	-	-	/
		2903	-	-	-	-	-	/
		2461	-	-	-	-	-	/
		2462	-	-	-	-	-	/
		2477	-	-	-	-	-	/
SD/DY-I/21 (Genotype I)	Control	3983	6	4	22	-	14	/
		3995	9	17	25	19	14	/
		3996	4	14	22	24	11	/
		19	12	17	22	-	10	/
	Vaccinated	4077	9	-	-	-	-	/
		4010	11	-	-	-	-	/
		3437	9	-	-	-	-	18
		4089	14	-	-	-	-	/
		3447	12	-	-	-	-	/

^a no signs of disease.

^b the pig survived the infection.

Autopsy was carried out upon death or euthanasia. The gross lesions of all organs and lymph nodes were observed. Some pigs in the control group developed tissue lesions including trichocardia, hydropericardium, seroperitoneum, tonsil damage, and hyperaemia of the lymph nodes as previously described [4], whereas the pigs in the HLJ/18-7GD-vaccinated group only showed mild lymphadenomegaly and lymph node hemorrhage (data not shown). Histopathological analysis of the lung, spleen, kidney, tonsil, inguinal lymph node, and submaxillary lymph node showed that vaccination with HLJ/18-7GD prevented their histopathological damage (Figure S3). Tissues from non-immunized piglets showed significant alterations including thickening of pulmonary alveolar walls and dense infiltration of inflammatory cells in the lung, a reduction in lymphocyte numbers and accumulation of red blood cells in the spleen, multifocal interstitial hemorrhaging in the kidney, diffuse lymphocyte reduction in the tonsil, and severe congestion, lymphocyte necrosis and lymphopenia in inguinal lymph nodes and submaxillary lymph nodes (Figure S3). However, the histological features of the immunized piglet tissues remained normal, except for reactive lymphocyte hyperplasia in submaxillary lymph nodes (Figure S3).

High levels of viral DNA were detected in tissues and lymph nodes from all four control pigs (Figure 4), but very low levels of viral DNA were detected in only four lymph node samples from three vaccinated pigs (Figure 4). These results indicate that HLJ/18-7GD induced effective protection against challenges with naturally occurring lower virulent variants in pigs, decreasing virus shedding and replication in vivo and preventing the development of chronic clinical signs of depression, arthroncus, phyma, and cutaneous necrosis.

HLJ/18-7GD vaccination induced poor cross-protection against genotype I low virulent ASFV challenge in pigs

To investigate whether HLJ/18-7GD could induce cross-protection against genotype I NH/P68-like ASFVs, five 7-week-old SPF pigs were i.m. immunized with 10⁶ TCID₅₀ of HLJ/18-7GD, and then were i.m. challenged with 10⁶ TCID₅₀ of genotype I isolate SD/DY-I/21 on day 28 p.im.. Four age-matched unvaccinated pigs were similarly challenged as a control.

All pigs were monitored daily for 28 days after challenge. In the control group, some pigs started to show fever from day 4 p.c.. Clinical signs gradually appeared until the end of the observation period, but fever mainly occurred between days 4 and 14 p.c., and the rectal temperature of all pigs was normal from day 15 p.c. (Figure 5). Two control pigs developed cutaneous necrosis from day 19 p.c., and all pigs developed depression, arthroncus, and phyma, but all survived the 28-day observation period (Figure 5 and Table 2). The vaccinated pigs started to show clinical signs such as intermittent cough and anorexia from day 4 p.c., and had higher clinical scores from days 10–19 p.c. (Figure 5). The rectal temperature of all vaccinated pigs increased for 9 days from day 10 p.c., and returned to normal from day 20 p.c. (Figure 5). No vaccinated pig showed any chronic clinical signs of arthroncus, phyma, or cutaneous necrosis (Figure 5 and Table 2). One of five vaccinated pigs died on day 18 p.c., but the remaining pigs survived the 28-day observation period (Figure 5).

Figure 5. Clinical signs and survival of HLJ/18-7GD-vaccinated pigs challenged with the genotype I low virulent virus SD/DY-I/21. HLJ/18-7GD-vaccinated and control pigs were challenged with 10⁶ TCID₅₀ of the genotype I low virulent virus SD/DY-I/21 and monitored daily for 28 days post-challenge. Clinical scores (A and B) were recorded and summed daily. Rectal temperature (C and D) and survival (E and F) of all pigs were assessed daily after the challenge. The dashed black lines in panels C and D indicate the threshold of normal rectal temperature. Clinical signs including depression, arthroncus, cutaneous necrosis, or phyma were recorded (G).

Oral and rectal swabs and blood were collected at different timepoints with a five-day interval after challenge. In the control group, viral DNA was detected in oral and rectal swabs from day 10 p.c., and in blood from day 5 p.c. until the end of the observation period (Figure 6). Viral DNA was detected in oral swabs from two vaccinated pigs from day 15 p.c.; in rectal swabs from one pig on day 15 p.c.; and in blood from day 10 p.c. (Figure 6). Viral DNA was detected in tissues and lymph nodes from all control pigs, but was not detected in the liver, kidney, or intestinal lymph nodes of all vaccinated pigs (Figure 6). Higher levels of viral DNA were detected in the spleen of one vaccinated pig and the lungs of three vaccinated pigs (Figure 6). Vaccination with HLJ/18-7GD suppressed histopathological lung damage (Figure S4). Non-immunized piglets displayed severe localized interstitial pneumonia within the lungs, characterized by complete occlusion of alveolar spaces, dense infiltration of inflammatory cells within the alveoli, and abnormal proliferation of epithelial cells. Of note, the lungs of immunized piglets showed no discernible signs of pathological alterations. Lymphopenia was observed in inguinal lymph nodes from the immunized and control pigs (Figure S4). However, no significant pathological changes were observed in the spleen, kidneys, tonsils, or submaxillary lymph nodes of the immunized or control pigs (Figure S4). The results indicate that HLJ/18-7GD did not induce solid cross-protection against genotype I low virulent ASFV challenge in pigs, but prevented the occurrence of chronic signs.

Figure 6. Viral shedding and replication in HLJ/18-7GD-vaccinated pigs challenged with the genotype I low virulent virus SD/DY-I/21. HLJ/18-7GD-vaccinated and control pigs were challenged with 10⁶ TCID₅₀ of the low virulent virus SD/DY-I/21. Oral and rectal swabs and blood were collected on the indicated days post-challenge. The indicated tissue samples were collected from the dead pigs and surviving pigs that were euthanized on day 28 post-challenge. Viral DNA in these samples was extracted and detected by using qPCR. LN1, intestinal lymph node; LN2, inguinal lymph node; LN3, submaxillary lymph node; LN4, bronchial lymph node.

Discussion

Genotype II ASFV has spread and evolved in the field since 2018 in China. Subsequently, genotype II variants with moderate virulence and genotype I viruses with different virulence have been identified [3, 4, 5, 22]. It is essential to evaluate the protective efficacy of ASF vaccine candidates against the current prevalent ASFVs in the field. In this study, we showed that the ASF vaccine candidate HLJ/18-7GD provides solid protection against a highly virulent variant and a less lethal isolate derived from genotype II Georgia07-like ASFV, but had poor protection against genotype I NH/P68-like low virulent virus. ASFVs continuously circulate and evolve in nature. Therefore, extensive epidemiological studies should be carried out to understand the evolution and variation of ASFVs. In addition, the protective effect of vaccine candidates on the emerging variants should be evaluated promptly.

Several attenuated gene-deleted ASFVs have been developed and shown to provide protection against virulent virus challenges [16, 20, 27, 28]. However, most of these attenuated ASFVs could not induce sterilizing immunity against homologous and heterologous virulent virus challenge, but substantially suppressed viral replication and shedding, and prevented animal death and clinical signs [15, 16, 28, 29]. Just recently WOAH published the Draft Standards for ASF Modified Live Virus (MLV) Vaccines for Domestic and Wild Pigs in its website (www.oie.org), which designates MLV vaccines as the first generation of ASF vaccine. The minimum efficiency of MLV vaccines should include the protection against mortality, reduction of typical acute disease signs caused by ASF, and reduction of horizontal disease transmission. Here, HLJ/18-7GD vaccination similarly prevented pig death from infection with the genotype II variants HuB/628/20 and HLJ/HRB1/20, and decreased viral replication in blood and tissues. However, low levels of viral DNA were detected in some lymph nodes from vaccinated pigs after challenge. A small amount of temporary virus shedding was also detected in oral and rectal swabs during the observation period. Proper utilization of live attenuated ASF vaccine candidates in local areas may prevent disease progression and pig death, and greatly reduce virus loads in pigs and the environment, which would contribute to ASF control and promote the healthy development of the pig industry. However, new generations of ASF vaccines with good safety and sterile immunity are needed to ultimately eradicate ASF.

The genetic variation and complexity of ASFVs affect the protection and cross-protection afforded by attenuated ASF vaccines. During ASF epidemiologic study, genetic analysis of viral genes that are susceptible to mutation helps to understand the evolution of ASFVs. Evaluating the genetic changes of virulence-determinant genes such as EP402R and MGF genes help to predict whether the virulence of the emerging mutants changes or not. However, currently the key genes associated with ASFV immune protection are largely unknown, and it is impossible to predict whether vaccine candidates still provide immune protection against the emerging mutants based on genetic changes of viral genes, which needs to be examined by animal experiments. Therefore, in this study, to clarify the immune protection of HLJ/18-7GD against emerging variants, pigs were vaccinated with HLJ/18-7GD, and then were challenged with different emerging variants on day 28 p.im.. It is necessary to explore and identify important genes for immune protection, and to establish a more convenient and rapid system for ASF vaccine evaluation in near future.

Genotype I attenuated ASFVs induce efficient protection against challenges of homologous and heterologous viruses including genotype II and X isolates [16, 30–32]. However, there is limited information on the cross-protection provided by attenuated genotype II ASFVs. Here, our studies showed that HLJ/18-7GD is protective against the genotype II emerging prevalent virulent variant HuB/628/20 and the lower virulent variant HLJ/HRB1/20. HLJ/18-7GD vaccination prevents pigs from developing ASF-specific clinical signs and death, decreases viral shedding via the anal and oral routes, and suppresses viral replication. For genotype I low virulent virus infection, HLJ/18-7GD vaccination prevents the development of chronic clinical signs including arthroncus, phyma, and cutaneous necrosis, and decreases viral shedding via the anal and oral routes. But HLJ/18-7GD could not induce solid cross-protection against heterologous genotype I low virulent virus challenge. Vaccinated pigs still developed fever similar to the control animals after challenge, although their temperature increase was delayed by six days. Surprisingly, one of the five vaccinated pigs died on day 18 p.c., and higher viral titers were detected in several spleen and lung samples from the vaccinated pigs. It is unclear whether this phenomenon was caused by individual pig differences or vaccination, and it should be noted that, in a similar published study, one pig infected with 10³ TCID₅₀ of SD/DY-I/21 died on day 16 post-infection [5].

Cellular immunity plays a dominant role in the immune protection induced by attenuated ASFVs [33, 34]. Our study showed that the genotype II ASF vaccine candidate HLJ/18-7GD induces less protection against genotype I virus SD/DY-I/21. Genotype I virus SD/DY-I/21 and genotype II virus HLJ/18 share 96% genomic identity. The difference between the two viral genomes likely affects the level of protective cellular immunity. In addition, our recent studies have showed that highly virulent genotype I and II recombinants have emerged in the field in China, and that HLJ/18-7GD could not provide efficient protection against challenges with these lethal recombinants [22]. Currently the mechanism of immune protection induced by attenuated ASFVs remains unclear. Our data on the poor cross-protection of HLJ/18-7GD against genotype I NP/P68-like virus and genotype I and II recombinant provide valuable clues to help unravel the mechanism of immune protection for ASFVs.

In summary, the live attenuated vaccine HLJ/18-7GD induces efficient protection in pigs against challenges with the different pathogenic genotype II ASFVs currently emerging in China. Genotype II ASFVs dominantly circulate in China, and HLJ/18-7GD vaccination thus shows promise as an alternative strategy for preventing and controlling ASF. Importantly, ASF vaccines with efficient cross-protection against different ASFV genotypes are urgently needed.

Author contributions

D.M.Z, and Z.G.B. conceived and designed the project. Z.L.W., J.W.Z., F.L., Z.J.Z., and W.Y.C. performed the experiments. Z.L.W., J.W.Z., F.L., Z.J.Z., W.Y.C., X.F.Z., E.C.S., Y.M.Z., R.Q.L., and X.J.H. analyzed the data. D.M.Z, Z.G.B., and Z.L.W. wrote and finalized the manuscript. All authors read and approved the manuscript.

Acknowledgments

We thank Susan Watson for editing the manuscript.

Data availability

The datasets used and analyzed during the current study are not accessible online, but will be available upon request to the corresponding author. Sequences of the viruses used in this study have been deposited in GenBank (accession numbers: OR126359 for HuB/628/20, and MW656282 for HLJ/HRB1/20).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Heilongjiang Provincial Natural Science Foundation of China (JQ2023C005), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202301), and the National Key R&D Program of China (2021YFD1800101 and 2019YFE0107300).