Detection of African Swine Fever Virus Protein VP73 in Tissues of Experimentally and Naturally Infected Pigs

Abstract

African swine fever (ASF) is caused by a DNA virus that targets cells of the mononuclear phagocyte system (MPS). ASF virus (ASFV) antigens have been detected in tissue sections using purified polyclonal sera from hyperimmune pigs by immunofluorescence or immunoperoxidase techniques. These investigations have been performed to study the pathogenesis of the disease. In this respect, the use of immunoperoxidase techniques on paraffin-embedded tissue samples represents a considerable step forward in the study of the disease because it facilitates the identification of different cell populations and links the presence of viral antigen with cell and tissue lesions. However, monoclonal antibodies (MAbs) have not been used for the detection of ASFVspecific proteins in immunohistologic studies. The purpose of the present investigation was to study the distribution of ASFV protein VP73 in tissue sections of different organs from pigs experimentally infected with the moderately virulent ASFV isolate E75 and in naturally infected pigs with characteristic lesions of acute, subacute, and chronic ASF by both avidin-biotin-peroxidase and alkaline phosphatase-conjugated biotin-streptavidin immunohistochemical techniques using an MAb. VP73 distribution was also studied in asymptomatic seropositive pigs. Three 3-month-old miniature pigs weighing 12 kg each and free from parasitic and infectious diseases at the beginning of the study were inoculated intramuscularly with 10 50% hemadsorbing doses of the moderately virulent and hemadsorbent ASFV E75 isolate. Two miniature pigs were used as negative controls. Test animals were euthanized by exsanguination after azaperone and thiobarbital treatment at 10 and 12 days postinoculation (dpi). One of the pigs scheduled for euthanasia at 8 dpi was found dead at 7 dpi. In addition, 27 naturally infected Iberian pigs from southwestern Spain that had anti-ASFV antibodies detected in serum by an indirect enzyme-linked immunosorbent assay ELISA were also used. These pigs were euthanized by the Agricultural Ministry of Spain and the Junta of Andalucia between 1985 and 1991. Six Large White × Landrace pigs, which served as controls in a previous experience, were employed as negative controls in this study. Tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, and stained with hematoxylin and eosin. For the immunohistochemical analysis, 3-μm-thick tissue sections were predigested with protease