Reverse Genetics System for Shuni Virus, an Emerging Orthobunyavirus with Zoonotic Potential

Abstract

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.

Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host

“…RVFV virions were detected with antibody 4-D4 48 targeting the Gn glycoprotein in combination with Alexa Fluor 488-conjugated secondary antibodies. SBV virions were detected with serum from an immunized rabbit 52 targeting the Gc glycoprotein in combination with FITC-conjugated secondary antibodies. Cell nuclei (cyan) were visualized with DAPI.…”

Shuni Virus Replicates at the Maternal-Fetal Interface of the Ovine and Human Placenta

“…The virus was passaged twice in Vero E6 cells before use in the described experiments. Virus titer was determined by end-point dilution assay using Vero E6 cells and subsequent staining with a SHUV-specific antiserum targeting the head domain of SHUV glycoprotein C (Gc) (SHUV-Gc head [ 22 ]) and calculated using the Spearman-Kärber algorithm [ 23 , 24 ].…”

“…Titrations were performed by end-point dilution assay on Vero cell monolayers in 96 well plates, followed by immunostaining of SHUV antigen using a rabbit polyclonal antiserum [ 22 ]. Titers were determined using the Spearman-Kärber algorithm and expressed as 50% tissue culture infective dose (TCID50)/mL.…”

Visualizing the RNP content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host

“…Supplementary Figs. 3b, 4) were detected with serum from an immunized rabbit 52 Supplementary Fig. 3a).…”

“…FISH probes were added at a final concentration of 250 nM for RVFV and 125 nM for SBV. Hybridoma 4-D4 48 supernatant (1:160 dilution) and serum from an immunized rabbit 52 (1:4,000 dilution), were used as primary antibodies against RVFV and SBV, respectively. Following hybridization and incubation with primary antibodies, cells were extensively washed at 37°C (twice with pre-hybridization buffer for 30 min and twice with 2x SSC for 15 min).…”