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Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replicationdefective recombinant vesicular stomatitis virus, rVSVDG * EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 10 5 -10 8 , and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (r s 50.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins. Received 21 November 2015 Accepted 22 February 2016Puumala virus (PUUV) is a member of the genus Hantavirus in the family Bunyaviridae and is associated with a mild form of haemorrhagic fever with renal syndrome (HFRS), also known as nephropathia epidemica (NE), with about 5000 reported infections in Europe annually (European Centre for Disease Prevention and Control, 2011). The PUUV genome consists of three segments: large (L), medium (M) and small (S), which respectively encode the RNA dependent RNA polymerase (RdRp), glycoprotein precursor (GPC) and nucleocapsid protein (Plyusnin & Morzunov, 2001;Schmaljohn & Hjelle, 1997). The GPC is expressed as a 1133-1158 aa residue polyprotein, which is co-translationally cleaved after a conserved WAASA amino acid sequence by the host cellular signal peptidase complex in the endoplasmic reticulum (Firth et al., 2012; Löber et al., 2001). The resultant N-and C-terminal fragments respectively mature to viral glycoproteins Gn and Gc (Hepojoki et al., 2012). The nascent Gn and Gc fold cooperatively and, apparently, the cytoplasmic tail of Gn and the downstream Gc signal peptide guide the transfer and localization to the Golgi (Pensiero et al., 1988, Pensiero & Hay, 1992Ruusala et al., 1992;Shi & Elliott, 2004). Localization to the Golgi is essential for the maturation of Gn and Gc (Antic et al., 1992;Johansson et al., 2004;Schmaljohn et al., 1986) and for budding of virions (Rowe et al., 2008;Shi & Elliott, 2002). Mature and properly folded Gn and Gc are the specific targets of neutralizing antibodies and thus they have been utilized in the study of glycoprotein folding (Custer et al., 2003;Hooper et al., 1999Hooper et al., , 2001Schmaljohn et al., 1990 (Lyles & Rupprecht, 2007). One of the unique features of VSV is its phenotypic mixing or pseudotype formation capability, i.e. the virus is able to incorporate heterologous glycoproteins in its envelope when budding (Buonocore et al....

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion's surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential.Viruses 2020, 12, 395 2 of 16 Boid Inclusion Body Disease (BIBD), initially recognized in the 1970s, affects mainly captive boas and pythons [11]. BIBD is characterized by the formation of electron dense intracytoplasmic inclusion bodies (IB) [12] in various cell types (including blood cells) and tissues [13][14][15]. BIBD spreads efficiently within collections, and the recommendation to euthanize snakes with BIBD can lead to loss of entire collections [11]. Clinical manifestations of BIBD are variable and include central nervous system (CNS) signs such as head tremors, loss of coordination, and regurgitation [11]. Death due to secondary bacterial, fungal or protozoal infections and neoplastic diseases is common in snakes with BIBD [16], suggesting that BIBD might be associated with an impaired immune response.The causative agent for BIBD remained unknown until the early 2010s, when novel arenaviruses were identified in snakes with BIBD [13,15,17], which extended arenaviruses host range to species other than the well-established rodent reservoirs [1-3]. These findings led to establishment of two genera, Mammarenavirus and Reptarenavirus, within the family Arenaviridae [18,19]. We and others have documented that snakes with BIBD are often co-infected with several reptarenaviruses [20,21], and we discovered another novel arenavirus in snakes, Haartman Institute Snake virus-1 (HISV-1) [20], which later became the type species of a third arenavirus genus, Hartmanivirus [19,22]. The finding that IBs contain reptarenavirus NP further supported the link between reptarenavirus infection and BIBD [15]. Experimental infection of boas and pythons with purified reptarenaviruses provided une...

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