African swine fever (ASF) is an infectious disease of domestic and wild pigs of all breeds and ages caused by a virus of Asfivirus genus, Asfarviridae family. Since its introduction in 2007 to Georgia ASF outbreaks have been reported in more than 50 countries of Europe, Asia, Africa and the Caribbean Basin region [1-3].

As regards the degree of virulence, ASFV strains can be divided into high, moderate, low virulence or non-virulence. Depending on the strain /isolate virulence the clinical course of ASF can be hyperacute, acute (highly virulent isolates/strains), subacute (highly and moderately virulent strains), chronic (moderate and low virulent strains), subclinical and asymptomatic (non-virulent strains) [4]. Superacute ASF kills pigs on days 3–5 post infection, either without clinical manifestations, or with short-term hyperthermia (> 41.0 °C), without loss of activity and appetite. Acute ASF is manifested by an early increase in body temperature (> 41.0 °C), loss of appetite, dullness (animals are recumbent most of time), rapid breathing, cyanosis of ear, underbelly, hind limbs and perineum skin, laboured breathing, leg paresis and paralysis, sometimes constipation or bloody diarrhea may be present. Death occurs on days 6–14 post infection. Hyperacute and acute forms of ASF result in 100% mortality rate. Pigs infected with subacute forms of ASF demonstrate clinical signs similar to the acute form, but they are less pronounced. Recurrent hyperthermia, depression, loss of appetite are reported, joint swelling and severe respiratory disorders occur at later stages of the disease. Most animals (about 70%) die within 15–30 days post infection [3][5][6]. Chronic form lasts for more than 30 days with periodic relapses. Animals demonstrate intermittent hyperthermia, exhaustion, stunting, arthritis of varying severity, respiratory disorders, and necrotic skin ulcers. The mortality rate is about 30% [7][8]. Subclinical and asymptomatic forms of ASF are observed in wild indigenous pigs (warthogs, giant forest hogs, bushpig) in African endemic countries, in wild boars in Sardinia, as well as in domestic pigs and wild boars experimentally infected with some ASFV attenuated strains [9-11].

In recent years, some progress has been made in the research and development of ASF modified live virus (MLV) first-generation vaccine candidates, which are used in some Southeast Asian countries (Vietnam, Philippines) [12]. A serious obstacle to the possible use of already developed and future ASF MLV vaccine candidates is the lack of internationally and nationally agreed parameters (requirements) for the evaluation of their purity, activity, safety and efficacy. In the world’s leading laboratories, various methods are used to evaluate the safety and efficacy of the vaccine candidates, which hinders the scientific community from side-by-side comparison and assessment of the results obtained, and government agencies and institutions authorized to issue permits for their use do not have such grounds.

Currently, the Biological Standards Commission of the World Organization for Animal Health is approving the updated Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, which includes harmonized standards and recommendations for ASF MLV vaccine candidates. Similar work is being done in the Russian Federation.

The main parameters of the safety and efficacy of ASF MLV vaccine candidates are: clinical signs, viremia, survival rate after immunization and virulent ‘homologous’ challenge [13]. To assess the clinical signs in pigs infected with ASF virus of various pathogenicity, a number of authors proposed using clinical scoring systems that cover thirteen signs (Table 1). The least number of clinical signs (4) was covered by the scoring system proposed by E. B. Howey et al. [14]; the most extensive scoring system (11) was created by A. S. Olesen et al. [15], which totaled 12 and 42 points, respectively.

The purpose of this work was to develop a clinical symptom-based scoring system to be used for characterizing of newly recovered ASFV isolates causing various forms of the disease, as well as for the determination of safety and efficacy of ASF MLV vaccine candidates.

The results of our own experiments conducted with ASF virus strains of various virulence were used in the study:

– highly virulent strains: Stavropol 01/08, Vladimir-Vyazniki/2017, Bryansk-21 (VIII serotype, II genotype), Lisbon-57 (I, I), Mozambique-78 (III, V), France-32 (IV, I);

– moderately virulent: Novgorod-2019 (VIII, II), PSA-1-NH (IV immunotype, I);

– non-virulent (attenuated): Katanga-350 (I, I), MK-200 (III, V), FC-32/135 (IV, I), Stavropol 71/2017 (VIII, II) [22-29].

Twenty-five to thirty kilograms large white pigs were received from the Unit of experimental animal preparation of the Federal Research Center for Virology and Microbiology. Experiments on pigs were performed in accordance with the “Guidelines for keeping and use of laboratory animals” [30]. The observed ASF clinical signs were recorded daily. Body temperature was measured rectally using mercury thermometers, other signs were determined visually.

A detailed description of porcine leukocyte (PL) cell culture preparation and determination of ASFV infectivity were performed as described earlier [22][31]. The results were evaluated by hemadsorption or cytopathogenic effect during 7 days. Virus titers were calculated using Kärber method, modified by I. P. Ashmarin, and expressed as 50% hemadsorbing units (HAE50/cm3) or tissue culture infectious doses (TCID50/cm3) [32].

Based on our tests using 144 animals and the published criteria for evaluation of ASF clinical signs, seven major clinical signs that were observed in infected pigs were selected: increased temperature, dullness, loss of appetite, skin lesions, joint involvement, respiratory disorders, neurological disorders. Each symptom, depending on the severity, was scored 3 or 4 points (Table 2). Such ASF clinical signs as eye discharges (conjunctivitis), diarrhea, bloody feces, bloody urine, vomiting, were not demonstrated by most infected animals, and they were not included in the scoring system. The system is based on the following principles: a) inclusion of the most characteristic clinical signs; b) increasing score representing a greater severity of clinical manifestation; c) the characteristic of a specific score does not exclude the simultaneous presence of those rated with lower scores.

Hyperthermia, loss of activity and appetite were observed in all pigs with acute, subacute and chronic forms of ASF, and redness and cyanotic skin were noted in 80–95% of the animals. These signs were scored from 0 to 4 points (Table 2). Less often (60–70%) the above mentioned forms of ASF induced incoordination and breathing problems. Joint involvement was observed in 35–50% of pigs with subacute and chronic disease. These three signs were scored from 0 to 3 points. Constipation, diarrhea, and bloody feces were reported only in some animals with acute and sub-acute ASF.

The developed ASF clinical scoring system was evaluated by the experiments on pigs infected with 12 strains of various virulence (Table 3) [22-29].

It should be noted that the minimum and maximum total scores given in Table 3 are not the sum of the minimum and maximum points for each of the seven clinical signs considered; they correspond to individual scores of the test animals in each of these groups.

Highly virulent strains (Stavropol 01/08, Vladimir-Vyazniki/2017, Lisbon-57, Mozambique-78, France-32, Bryansk-21) caused acute and less often subacute disease regardless of the infection routes and doses used. Acute ASF produced hyperthermia (41.0–42.0) °C, partial or complete loss of activity and appetite, redness or cyanosis of the ear, abdominal wall, tail and perineum, severe painful breathing with nasal discharge, leg paresis and paralysis, convulsions were recorded. Bloody diarrhea was observed in only a few animals. Before death, on days 6–14 post infection, the body temperature in pigs decreased to 38.0 °C. The subacute ASF produced the following clinical signs: hyperthermia, loss of activity and appetite, minor redness or cyanosis (tips of the ears, tail). Acute and subacute ASF, regardless of the infection routes or doses, gave the clinical scores in different individuals ranging from 13 to 22.

Chronic ASF was observed in pigs infected intramuscularly with Novgorod-2019 or PSA-1-NH strains. The disease was characterized by intermittent hyperthermia (40.5–41.5) °C, loss of activity and appetite. In 10–14 days, the animals demonstrated cachexia, stunting, different respiratory disorders and joint problems (arthritis). Some animals demonstrated spotted skin redness, which subsequently could become cyanotic and sporadically necrotic (PSA-1-NH strain). The duration of the disease could be different and lasted for more than 30 days. “Clinical recovery” was recorded in some animals with subsequent reсurrence, manifested by an in increase in body temperature. The ASF clinical scores in such animals ranged from 6 to 18.

Less pronounced signs of chronic ASF were recorded in 40–60% of pigs after intranasal inoculation of PSA-1-NH strain, as well as in 7% of pigs inoculated with attenuated Katanga-350 strain. Recurrent hyperthermia was observed (from 40.1 to 40.7 °C), a slight decrease in activity (got up quickly when touched) and appetite (50–90% of feed consumed) during 1–4 days, in some cases shortness of breath and slight joint swelling were observed. The maximum ASF clinical scores in these animals reached 6–10.

Most pigs infected intramuscularly with attenuated Katanga-350 strain, attenuated but not protective Stavropol 71/2017 strain, as well as intranasally with a low dose (103 TCID50) of naturally attenuated PSA-1-NH strain demonstrated subclinical ASF: body temperature in 40–60% of animals not exceeded 40.5 °C during 1–4 days, 90–100% of the feed was consumed. No other changes were recorded. The total clinical score ranged from 0 to 5.

The asymptomatic ASF was recorded in 100% of pigs inoculated intramuscularly with strain FK-32/135, in 75–90% of pigs inoculated with strain MK-200, in 40–60% with strain Katanga-350. ASF clinical signs in such animals were not recorded, the total scores were 0.

It should be noted that the ASF clinical scoring system is not applicable for isolates causing peracute disease, since the death of pigs occurs 3–5 days after infection without most clinical signs manifested due to intramuscular administration of virulent virus isolates in large doses.

Over the past 5–7 years, circulation of ASF genotype II virus isolates of various virulence, from highly virulent to naturally attenuated, have been evidenced in Europe and Asia [2][3][6][9][11][33].

According to our observations, the dynamics of ASF clinical signs varies depending on the virulence of the isolate/strain, to a lesser extent on the dose and method of the virus inoculation [26-28]. The developed and tested ASF clinical scoring system is suitable both for the characterization of newly recovered isolates and for the evaluation of MLVs and MLV vaccine candidates. Based on our experience, for candidate strains, the total clinical score should not exceed 4–5 points. Ideally, when the score is 0, as for example after vaccination of pigs with ASFV FK-32/135 strain [26].

It should be noted that for the evaluation of MLV vaccine safety, in addition to clinical signs, it is proposed to study the levels and duration of viremia, virus shedding, post-mortem changes, persistence of the vaccine and virulent (infecting) viruses in tissues, potential transmission and reversion to virulence, safety in the field and some others [13]. However, if, after the inoculation of a candidate vaccine strain to pigs, the total clinical score exceeds 5, it is not reasonable to conduct other studies.