Cytokine Storm Combined with Humoral Immune Response Defect in Fatal Hemorrhagic Fever with Renal Syndrome Case, Tatarstan, Russia

Abstract

Hemorrhagic fever with renal syndrome (HFRS) is endemic in Tatarstan, where thousands of cases are registered annually. Puumalaorthohantavirus is commonly detected in human case samples as well as in captured bank voles, the rodent hosts. The pathogenesis of HFRS is still not well described, although the cytokine storm hypothesis is largely accepted. In this study, we present a comprehensive analysis of a fatal HFRS case compared with twenty four non-fatal cases where activation of the humoral and cellular immune responses, pro-inflammatory cytokines and disturbed blood coagulation were detected using immunological, histological, genetic and clinical approaches. Multiple organ failure combined with disseminated intravascular coagulation syndrome and acute renal failure was the cause of death. Decreased Interleukin (IL)-7 and increased IL-18, chemokine (C-C motif) ligand (CCL)-5, stem cell growth factor (SCGF)-b and tumor necrosis factor-beta (TNF-β) serum levels were found, supporting the cytokine storm hypothesis of hantavirus pathogenesis.

Keywords: CCL5; HFRS; IL-18; Puumala orthohantavirus; SCGF-b; TNF-β; cytokine; fatal case.

Figure 1

Multi-slice CT of fatal HFRS lung.

Figure 2

PUUV neutralizing antibodies in the fatal and non-fatal HFRS sera. Blue – non-fatal HFRS; red – fatal HFRS.

Figure 3

Phylogenetic analysis of PUUV RNA from fatal HFRS case. The sequence of the fatal case is marked by a blue diamond. The nucleotide alignment and phylogenetic analysis of PUUV strains was based on partial S segment sequences of 217 nucleotides in length (nucleotides 378–594). The tree branch lengths were calculated using the average pathway method and have a scale representing the number of changes in the sequence. Red – RUS lineage; Blue – FIN lineage.

Figure 4

Histochemistry and immunofluorescence analysis of the lung tissue from fatal HFRS. Lung tissues (0.5–3-μm thick) were deparaffinized before histochemistry and immunofluorescence analysis. A and B. Tissue samples were stained with hematoxylin and eosin (H&E). Arrow points at exudate in the lung alveolus and bronchiole. C. hantavirus nucleocapsid protein (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-mouse Alexa 488 (1:1000; Invitrogen, Waltham, MA, USA); D. CD4+ leukocytes (polyclonal anti-CD4 (1:100, Abcam, Cambridge, MA, USA) and anti-rabbit Alexa647 (1:1000; Invitrogen, Waltham, MA, USA); E. CD20+ leukocytes (monoclonal anti-CD20 (1:50, Abcam, Cambridge, MA, USA) and anti-mouse Alexa 488 (1:1000; Invitrogen, Waltham, MA, USA); DAPI – used to visualize nucleus. White arrows show positive CD20+ infiltrating leukocytes.

Figure 5

Histochemistry and immunofluorescence analysis of kidney from fatal HFRS. Kidney tissues (0.5–3-μm thick) were deparaffinized before histochemistry and immunofluorescence analysis. A and B. Tissue samples were stained with hematoxylin and eosin (H&E). Arrows in the figure show whit changes in kidney structure. White arrows—kidney glomeruli exhibited wrinkling epithelium. Black arrows mark the plasmorrhexis of kidney. C. hantavirus nucleocapsid protein (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-mouse Alexa 488 (1:1000; Invitrogen, Waltham, MA, USA). D. CD4+ (1:100, Abcam, UK) and anti-rabbit Alexa 647 (1:1000; Invitrogen, Waltham, MA, USA). E. CD8+ (1:100, Abcam, UK) and anti-mouse Alexa 647 (1:1000; Invitrogen, Waltham, MA, USA) F. clusterin (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-mouse Alexa 647 (1:1000; Invitrogen, Waltham, MA, USA). DAPI was used to visualize the nucleus.