Evaluation of Hemostatic Changes in Wistar-Furth and Lewis Rats Infected with Rift Valley Fever Virus Strain ZH501

Abstract

Rift Valley Fever virus (RVFV) is a member of the genus Phlebovirus, family Bunyaviridae. Members of this family are characterized by a tripartite, negative sense RNA genome with potential for genetic reassortment. RVFV has caused epizootics in domestic animal populations in African countries, and has been found to affect man, causing acute febrile disease and occasional death due to hemorrhagic fever and encephalitis. Investigators have noted hemostatic changes in primates experimentally infected with RVFV, suggesting that a correlation of hemostatic dysfunction to the human system is possible. This potential for comparative analysis of RVFV-infection in man creates a need for a cost-efficient, readily available model system to study the virus-induced abnormalities in the hemostatic system of man. Wistar-Furth rats prove to be extremely susceptible (LD₅₀, 5 PFU) to infection after subcutaneous inoculation of the Egyptian strain ZH501 of RVFV, and die with extensive liver necrosis 3-5 days post-inoculation. Lewis rats are in contrast largely resistant (LD₅₀, >10⁶ PFU) to the lethal effects of RVFV infection. In utilizing these available rat strains, we examined the general factor deficiencies of the intrinsic and extrinsic pathways of hemostasis which may be acquired secondarily to viral infection, such as liver dysfunction, which is characteristic of RVFV infection. This was accomplished by inoculating adult WF and LEW inbred rats subcutaneously with 5 x 10² plaque forming units of the ZH501 strain of RVFV. Groups of 5 animals were assayed serially at 12 hour intervals. Levels of antibody to RVFV ZH501 in the serum of infected animals were determined by enzyme-linked immunosorbent assay (ELISA). Sera, in addition to liver, spleen, and adrenal gland tissue homogenates were assayed for viral titer, and samples of these tissues were taken for PTAH staining to determine fibrin polymerization. Liver function of the infected animals was assessed by glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT) colorimetric enzyme assay and compared to values found in uninfected control rats. Complete blood counts (CBCs) and cell differentials were determined in both the virally infected and control animals. Abnormalities of the intrinsic pathway (Factors VIII, IX, XI, XII) were determined by performing a modified activated partial thromboplastin time test (APTT), where as the extrinsic pathway (Factors II, V, VII, X) deficiencies were monitored by a modified prothrombin time test (PT). Variations in clotting times were determined in the infected rats and again compared to the normal values of uninfected control rats. RVFV infection with the Egyptian strain of RVFV, ZH501, was confirmed in both the RVFV-susceptible Wistar-Furth and the RVFV resistant Lewis inbred strains of Rattus norvegicus by virus re-isolation from tissue homogenates of liver, spleen, and adrenal gland, and by the detection of high viremia in the sera of inoculated animals. The acute onset of fatal disease in the susceptible WF strain did not allow sufficient time for detectable IgG production. Hepatic injury was observed by examination of stained tissue sections, and further indicated by the high correlation between viremia, tissue titers, and the prolonged PT and APTT clotting times. Rift Valley Fever virus infection does induce hemostatic change in both the Wistar-Furth and Lewis rat strains to varying degrees, but by which specific mechanisms remains unclear. The results shown here suggest a number of possible mechanisms for hemostatic disruption due to RVFV infection. Severe degrees of hepatic injury lead to impairment of the hemostatic system, not only in its ability to synthesize the coagulation factors, but also in its ability to produce the anti-. coagulation enzymes, which function in the clearance of the activated coagulation factors. These results appeared to be determined by rat genotype. The use of the Wistar-Furth and Lewis rat models is not recommended for the evaluation of Rift Valley Fever virus induced hemostatic change.