ASFV is a member of the
For years, complex interactions between the virus and the host’s organism, as well as a wide range of ASFV genes, have been preventing the development of an effective vaccine against the disease (26). Several studies have shown promising results regarding vaccination, but the process of developing an effective and safe vaccine to the stage of being ready for the market seems to be excessively long (4, 7, 14).
In Poland, ASF has been spreading consistently since 2014 (19). In active surveillance during epizootics in Poland, wild boar which are PCR negative but seropositive with no visible clinical symptoms or gross lesions may be found (12). These animals probably belong to a convalescent group (30), which is of special interest, since they were able to survive ASFV infection. However, the mechanism for effective combat of ASFV by an ASF-survivor’s immune system is not clearly understood. The importance of cellular and humoral immunity in protection against ASF has been previously indicated by several authors (24, 28, 37), but the role of anti-ASFV antibodies in the neutralisation of the virus remains the subject of discussion (11).
In this study, we analysed five selected sera belonging to ASF survivors to investigate their ability to neutralise the virus
At the end of that experiment, pig#1 recovered partially, presenting a better feed intake, lower fever and no pathological lesions during necropsy. No clinical findings except a moderate fever and enlargement of submandibular lymph nodes noted during necropsy, were observed in pig#2. Pig#1 and pig#2 survived the infection and were euthanised on the 32nd and 25th day post infection, respectively (32). No pathological lesions were observed among the selected wild boar. The characteristics of the animals are summarised in Table 1.
Characteristics of the animals from which the sera used in the study were obtained
Serum/Sample | Species | Sex | Age | Clinical symptoms | Gross lesions |
---|---|---|---|---|---|
Pig#1 | Domestic pig | Male | 9 weeks | Fever, dyspnoea joint swelling, | n/d |
Pig#2 | Domestic pig | Male | 9 weeks | Moderate fever | Enlargement/hyperaemia of submandibular lymph nodes |
WB#1 | Wild boar | Male | 18 months | n/a | n/d |
WB#2 | Wild boar | Female | 18 months | n/a | n/d |
WB#3 | Wild boar | Male | 24 months | n/a | n/d |
n/d – not detected; n/a – not applicable
Poland) containing an anticoagulant (K2‐EDTA). Wild boar blood was collected during hunting into 4 mL plastic tubes and retained for further analyses. An aliquot of 200 μL of each diluted (1:10 PBS, v/v) wild boar and pig blood sample was submitted for DNA extraction.
Cells with the medium supplemented with a serum were placed into 24-well plates, in three replicates, infected at the multiplicity of infection (MOI) ~1.0, and incubated at 37℃ in 5% CO2. The cells and medium were collected at 0, 2, 4 and 7 dpi for real-time PCR analysis. In parallel, for each tested serum, a negative control was prepared to exclude the serum’s ability to infect. Negative controls contained a growth medium, A/A and selected serum.
The pigs’ blood contained detectable ASFV DNA. The mean Cq value (from the first day of detected viraemia to the day of euthanasia) was estimated at 32.3(±1.7) for pig#1 and 31.7(±1.4) for pig#2. Viral DNA was not detected in blood collected from hunted wild boar. In serum, viral DNA was detected only in the case of pig#2. All sera presented a high antibody titre (>5 log10/mL). The highest titres were observed in sera of WB#1 and WB#2. The results are summarised in Table 2.
ASFV DNA detection and anti-ASFV antibody detection and titre in selected samples
Serum/Sample | Blood qPCR Cq (±SD) | Serum qPCR Cq | Antibodies (ELISA) | Antibody titre (log10/mL) |
---|---|---|---|---|
Pig#1 | 32.3 (±1.7) | NEG | POS | 5.01 |
Pig#2 | 31.7 (±1.4) | 34.7* | POS | 5.31 |
WB#1 | NEG | NEG | POS | 5.51 |
WB#2 | NEG | NEG | POS | 5.51 |
WB#3 | NEG | NEG | POS | 5.21 |
qPCR – quantitative (real-time) PCR; WB – wild boar; POS– positive; NEG –negative; * – serum was inactivated for further analysis
Haemadsorption assay results in the presence of selected sera at 20% concentration
dpi | |||||||
---|---|---|---|---|---|---|---|
Serum/sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
FBS | + | + | ++ | ++ | ++ | ++ | ++ |
Pig#1 | − | − | − | − | − | − | − |
Pig#2 * | − | + | + | ++ | ++ | ++ | ++ |
WB#1 | − | − | − | − | − | − | − |
WB#2 | − | − | − | − | − | − | − |
WB#3 | − | − | − | − | − | − | − |
dpi – day post inoculation of cell culture; − – lack of haemadsorption; + – single cell haemadsorption; ++ – multiple cell haemadsorption; * – inactivated
Despite slight differences observed in the growth kinetics of the virus, it was clearly visible that the virus was able to replicate (Fig. 2). Replication of ASFV could be observed both from a decrease of the Cq value in real-time PCR on successive days and by recording the cytopathic effect (CPE) under a light microscope. No clearly visible differences were observed in the virus growth rate between samples containing 10% of a serum and those containing 20%.
ASFV DNA was not detectable during any experiments in the negative control samples, except as denoted by the stable Cq value (~Cq 37.00) of a sample containing the inactivated serum of pig#2. Neither CPE nor haemadsorption were observed in any negative controls.
For each serum sample and control sample (FBS), the difference in the Cq value was recorded between 0 dpi and 7 dpi (Fig. 3). In most cases, no statistically significant differences were noted in the decrease of this value between the samples and the respective control, which indicated similar growth rates of the virus in the presence of FBS or the survivor’s serum. Statistically significant differences were recorded in the case of the 10% concentration of WB#3 serum and the 20% concentration of pig#1 serum set against their respective controls. Surprisingly, enhanced growth rates of ASFV could be observed in the presence of the 10% and 20% concentrations of WB#1 serum, but they were not significantly different from the growth rate in the relevant controls (Figs 2 and 3).
The role of anti-ASF antibodies in effectively combating the disease is not clearly revealed and still remains the subject of discussion (11). Antibody-mediated neutralisation of ASFV has been reported previously (3, 10, 35), but our study showed that anti-ASFV antibodies alone cannot inhibit the replication of ASFV
Since we observed HAI with evidence of simultaneous replication of the virus, this study confirmed that the haemadsorption phenomenon is not essential for virus replication – even for haemadsorbing ASFV isolates. This is in line with research presented by Dixon
Inhibition of haemadsorption showed that the amount of sera used in this experiment was sufficient to opsonise target cells, but not to inhibit internalisation of the virus. This suggests that ASFV does not need the target receptor in order to be internalised, and internalisation may occur passively,
Currently, there is no available treatment for ASFV and for epidemiological reasons and legal regulations, an attempt at ASF treatment is not permitted. Nevertheless, a study conducted to identify a potential target for an antiviral anti-ASFV drug has been published before (17). Several authors have previously reported a beneficial effect of passively acquired antibodies against ASFV in