Lesions and cellular tropism of natural Rift Valley fever virus infection in sheep

Abstract

Rift Valley fever (RVF) is a viral haemorrhagic disease caused by a segmented single stranded RNA virus of the Bunyavirales order, Phenuiviridae family. The virus causes severe disease in both humans and animals and epidemics have been reported in most countries in Africa and the Arabian Peninsula. The virus is primarily transmitted by mosquitoes, mainly of the genera Aedes and Culex. Characteristically RVF virus (RVFV) causes abortion storms in sheep during which approximately 10 to 20% of adult ewes die. In the summers of 2010-2011, South Africa had an extensive outbreak of RVF affecting livestock and humans. The diagnosis of RVF was confirmed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), and in selected cases also by immunohistochemistry (IHC). Sheep necropsied during the outbreak were examined by histopathology and IHC. The first chapter is a review of research conducted regarding RVF infection in sheep, including descriptions of the pathology and immunohistochemical findings. Relevant information concerning the pathogenesis of RVFV and other viral haemorrhagic diseases with a similar pathogenesis was also summarized. The aim of the second chapter was to describe the tissue tropism and target cells of RVFV in 99 adult sheep from the 2010 South African RVF outbreak. A further aim was to determine the extent to which virus could be detected in different organs and summarize gross, histopathological and immunohistochemical findings relative to results from previous research. Multifocal-random, necrotizing hepatitis was the most distinctive lesion of RVF cases in adult sheep. Of cases where liver, spleen and kidney tissues were available 45/70 (64%) had foci of acute renal tubular epithelial injury in addition to necrosis in both the liver and spleen. In some cases, acute renal injury was the most significant RVF lesion. Concerning the diagnostic specificity of lesions in the liver, early apoptotic hepatocytes (Councilman bodies) and late apoptotic bodies with nuclear fragments and condensed cytosol were diagnostically useful. In contrast, eosinophilic intranuclear inclusions were of limited diagnostic value since they were only identified in 7/99 (7%) adult sheep. Immunolabelling for RVFV was most consistent and unequivocal in liver followed by spleen, kidney, lung and skin. It was determined in this study that the minimum set of specimens to be submitted for histopathology and IHC to confirm or exclude a diagnosis of RVF are liver, spleen and kidney. Skin from areas with visible crusts and lung could be useful additional samples. RVF is much more acute in young lambs than in older sheep, and there appear to be important differences in the lesions and tropism. Therefore, the principle aim of the third chapter was to describe the quantitative and qualitative pathomorphology of RVF in 71 young lambs and contrast this with results for adult sheep. A further aim was to determine the diagnostic significance of different tissues and specific histologic features of RVF in young lambs. The liver lesion of RVF was much more severe in young lambs, where it caused a diffuse necrotizing hepatitis with widespread labelling of virtually every hepatocyte. A striking diagnostic feature in lambs was multifocal liquefactive hepatic necrosis (primary foci) against a background of diffuse hepatocellular death. Primary foci were present in 59 of 71 (86%) cases, with viral antigen noticeably sparse within them. Intranuclear inclusion bodies were also diagnostically useful, and present in 27 of 71 (38%) cases. Additionally, cell death morphology in the liver in RVFV infected lambs was more heterogeneous than previously suspected whereby many hepatocytes displayed histomorphological features of lytic cell death mechanisms (cell swelling, rupture of the plasma membrane and cellular collapse) as well as apoptosis. Whereas, apoptosis was demonstrated in RVFV infected mice using terminal deoxynucleotidyl transferase dUTP nick-end labelling, the involvement other cell death mechanisms in RVF, including pyroptosis and necroptosis, should be further explored. In young lambs, lymphocytolysis was present in all lymphoid organs except for the thymus. The kidney lesion was also much less severe than in adult sheep but viral antigen was easier to find and generally widespread. Histopathological lesions in the lungs of lambs mirrored that in adults. However, viral antigen was easy to find in lambs compared to adult sheep where viral antigen was very difficult to find in the lungs. As in adult sheep, splenic lymphocytolysis was a prominent feature, but due to a developmentally normal lack of germinal centres this was more prominent at the edge of the periarteriolar lymphoid sheaths and the red pulp in lambs. Labelling was also often seen in capillaries and small blood vessels either as non-cell-associated antigen, or as antigen in endothelial cells or intravascular cellular debris. Additionally, in the kidney viral antigen was in the peri-macular cells including the juxtaglomerular and granular extraglomerular mesangial cells. Liver and spleen specimens were the most consistently positive for RVFV antigen and were adequate to confirm or exclude a diagnosis of RVF in most cases in young lambs using IHC. Specimens from the lungs and kidneys were useful additional samples due to their characteristic histologic lesions. There is a perception that RVF disappears in the interepidemic periods and only reappears suddenly when heavy rainfall occurs. However, RVFV infection in pregnant ewes causes a wide variety of outcomes for both ewes and foetuses and it is difficult to exclude RVF from the list of possible differential diagnoses. Diagnosing the cause of abortions in endemic areas are further complicated by the observation that the virus seemingly does not always cross the placenta to cause lesions in the foetus. Additionally, the tropism and lesions of the placenta have not been thoroughly described. Therefore, the principle aim of the fourth chapter was to describe RVF in naturally infected foetal tissues and determine the diagnostic significance of the lesions. A total of 72 foetuses were studied of which 58 were confirmed positive for RVF.