Production, quantification, and infection of Amazonian Phlebovirus (Bunyaviridae)

Background

In Brazil, approximately 210 different types of arboviruses have been isolated, of which 196 were found in the Brazilian Amazon (Rosa et al., 1998; Azevedo et al., 2007), one of the largest reserves of arboviruses in the world. The Bunyaviridae family is composed of more than 350 viruses divided into five genera, among which Orthobunyavirus, Phlebovirus, Nairovirus, and Hantavirus contain species pathogenic to humans and animals, while the genus Tospovirus is pathogenic only to plants (Walter and Barr, 2011; Ly and Ikegami, 2016).

In most human cases, Phlebovirus cause a wide spectrum of symptoms from mild febrile illness to hemorrhagic fever and death (Bird et al., 2009; Ikegami and Makino, 2011; Brisbarre et al., 2015). A specific member of Phlebovirus, the Rift Valley Fever virus, is potentially dangerous to pregnant domesticated animals such as cattle, goat, and sheep, with infection resulting in fetal malformation and abortion (Swanepoel and Coetzer, 2004). In this work, we used the Icoaraci Phlebovirus (BeAN 24262), which is prevalent in small forest animals (Causey and Shope, 1965). All Phlebovirus are morphologically similar: 80-120 nm in diameter, icosahedral symmetry, and enveloped with two surface glycoproteins (Gn and Gc) (Freiberg et al., 2008). The single-stranded RNA (ssRNA) is segmented and packaged within ribonucleoprotein particles (RNPs) by protein N and associated with an RNA-dependent RNA polymerase (RdRp). The small (S) segment uses the ambisense coding strategy, coding for a nucleocapsid protein (N) and non-structural protein (NSs) (Simons et al., 1990; Giorgi et al., 1991).

Studies in animal models suggest a protective effect of type I interferon (IFN) in infection caused by Phlebovirus (Mendenhall et al., 2009). Most viruses have developed the ability to express proteins capable of preventing the immune response (Habjan et al., 2009). This antagonistic activity has been observed for several non-structural viral proteins (Van Knippenberg et al., 2010) by mechanisms that explore different targets in IFN signaling pathways. Although Phlebovirus exhibit different levels of pathogenicity, the NSs protein is considered an important virulence factor for all members of the genus, as well as other members of the Bunyaviridae family (Habjan et al., 2009; Ikegami et al., 2009).

Several studies have shown the importance of the relationship between viruses and Leishmania parasites in the establishment of reciprocal adaptive advantages that promote survival and persistent infection (Ives et al., 2011; Hartley et al., 2012; Vivarini et al., 2015; Eren et al., 2016; Rath et al., 2018). Our group previously demonstrated that the induction of antiviral pathways by L. amazonensis increases macrophage infection (Pereira et al., 2010; Vivarini et al., 2015). Different Leishmania species associated with host immunological factors determine distinct clinical manifestations of leishmaniasis (Alvar et al., 2012). The production of leishmanicidal mediators, such as IL-12, IL-6, and reactive oxygen species (ROS), by innate immune cells contributes to infection control (Dayakar et al., 2019), but Leishmania parasites are able to subvert the signaling pathways that trigger these mechanisms and instead induce pathways favoring the infectious process. Using Icoaraci Phlebovirus as a model, we demonstrated that this virus potentiates infection by L. amazonensis in macrophages. Increased macrophage infection requires toll-like receptor 3 (TLR3) and protein kinase R (PKR) pathways due to the production of type I IFNs induced by the Leishmania and amplified during co-infection with the virus (Rath et al., 2019). Our results highlight the importance of complex and multifactorial analysis of infectious processes, especially in endemic areas where overlap occurs, as observed between L. amazonensis and Icoaraci Phlebovirus.

Given the socioeconomic impact of Phlebovirus infection and the potential co-infection in cutaneous leishmaniasis, the present protocol for the production, quantitation, and infection of Icoaraci Phlebovirus aims to address the molecular mechanisms associated with the interaction between sand fly-transmitted arboviruses and Leishmania parasite infection in vitro and in vivo in the cell host. Subsequent research using this methodology may reveal important aspects related to the host immune response, helping to outline the pathophysiological phenomena and clinical conditions and clarifying scenarios that may indicate how an antiviral response to double-stranded RNA (dsRNA) can mediate immune-inflammatory and immunopathogenic processes in Phlebovirus infection and co-infections.