Introduction to virulence

Viral virulence can be evaluated at different levels: at the host level by how severe the resulting viral disease symptoms are; at the cellular level by the capacity of a virus to grow easily in cell culture. Various virulence factors exist all over the different steps of a virus infecting a target cell/organ/organism. They can influence key steps of the virus cycle itself such as the adsorption to a specific receptor(s), the mechanism for cell penetration (fusion, decapsidation), the strategy for genome expression and replication, the budding out of the cell to propagate infection, etc. They can also target the defences developed by the cell or the organism and counteract their control action (innate and adaptative immunity, etc). In particular, the in vitro or in vivo induction and secretion of IFNs is a central event in the early innate immune response against virus infection (Haller, Kochs, and Weber, 2006). The IFNs induce the expression and activation of a wide range of antiviral proteins against a number of RNA viruses, including arboviruses. Pro-inflammatory cytokines are expected to play a predominant role in controlling the initial events of the virus infections. However,  most RNA viruses have evolved all type of subversive mechanisms and strategies that allow to escape the IFN system and favour their own survival. They bind and inactivate secreted IFN molecules, block IFN activated signalling, disturb the action of IFN-induced antiviral proteins, etc (Weber and Haller, 2007). The molecular mechanisms involved range from a broad host cell shut off to a fine-tuned elimination of key actors of IFN system.

The absence of proofreading mechanism for the polymerase of RNA viruses (in particular for RVF, CCHF, WN) provides them with a powerful tool for genetic flexibility and adaptation capacity. This allows a permanent evolution of the viral population into the cell/host in response to various constraints. The evolution process includes abilities to live longer, reproduce in higher numbers or in contrast to detection threshold, escape to antibodies, invade new cell/body compartments that the virus does not normally infiltrate, etc. The “beneficial” mutations increase the fitness of the pathogen in a specific constrained environment and will be submitted to natural selection inside a host and rise to high frequency. They can be more detrimental to the host causing much harm and virulence.

Thus, rapid and precise evaluation of virulence on circulating virus strains is extremely important for the judgement of public health risk, early warning if a more pathogenic strain is coming up, identification of appropriate vaccine candidates or other convenient prophylactic measures if any, estimation of clinical prognosis in patients and decision-making regarding veterinary intervention measures.
 

Virulence data for RVFV, WNV and CCHFV

Various experimental models, both in vitro and in vivo have been developed on the three viruses focusing the interest of ARBOZOONET.
Rift Valley Fever Virus (RVFV) (Soldan and Gonzalez-Scarano, 2005) replicates efficiently in most common cell cultures, including insects cells (Billecocq et al., 2000), with a rather important cytopathic effect. The most usual epizootic host is the sheep in which RVFV provokes an acute illness leading to death with important hepatic necrosis (Clements et al., 2007; Flick and Bouloy, 2005). However, RVFV also infects a wide variety of laboratory and domestic animals in which it replicates at high titer and is frequently lethal while targeting mainly the liver (focal necrosis) and the brain (necrotic encephalitis) (Gowen and Holbrook, 2008). In laboratory rodents, mice have been extensively used for studying pathogenesis because they are highly susceptible and develop acute fatal hepatitis. More variation exists in inbred rats depending on the strain used. Some are resistant and only show a subclinical immunizing infection, some develop lethal fulminant hepatitis and others an inflammatory encephalitis. RVFV isolates also vary dramatically in their virulence for laboratory rodents. Isolates from Egypt-1977 were virulent for rats whereas isolates from sub-Saharan Africa were milder. Peripheral injection to suckling mice allowed to show difference in virulence between plaque-purified clones of the Egyptian isolates, and the most virulent clones were less sensitive to rat interferon and formed plaques in rat hepatocytes. RVFV can also be neuro-adapted by serial intra-cerebral passage in mice, the neuro-adapted strain showing reduced hepatotropism and lethality when injected by the peripheral route but high virulence by the intracerebral route. Infection of rhesus macaques with RVFV reproduces reasonably infection in humans with about 20% of the monkeys having a severe hemorrhagic fever symptoms. 
West Nile Virus (WNV) (Granwehr et 2004) grows and produces cytopathic effect in a wide variety of primary cell culture as well as on continuous cell lines of human, primate, swine, rodent and amphibian origins (Granwehr et 2004). It also multiplies in Aedes aegypti and Aedes albopictus cells as well as in Drosophilia cells and it is regularly grown in C6/36 insect cells. C-type lectin CD209 has been implicated in WNV binding to the cells (Davis et al., 2006). Integrins have been involved as functional receptors associated with the signaling for WNV entry into vertebrate cells. Furthermore, TLR3 facilitates entry into the CNS.
While the intracerebral infection is lethal for mice and hamsters of all ages, causing meningoencephalitis with poliomyelitis-like features that mimick the severe human disease, resistance to peripheral inoculation increases with age although some strains are pathogenic for adult animals and lethal oral infection has been described in adult mice. After subcutaneous inoculation, virus spreads sequentially to draining lymph nodes, spleen, and serum, producing a high titer viraemia before disseminating to the CNS. Hamsters transmit the virus through the milk. Rat succumb to intracerebral infection only. Guinea pigs, cotton rat and rabbits control disease through antibodies independently of the route of inoculation. Rhesus and bonnet macaques develop fatal encephalitis after intracerebral of intranasal inoculation. Most bird species develop viremia and are rarely subject to death or encephalitis, while chick embryo is highly susceptible to WNV.
Crimea Congo Hemorrhagic Fever virus (CCHFV) has been less extensively studied, probably because of its classification as a BSL4 agent (Ergonul, 2006). Hovewer, CCHFV replicates in a variety of cell lines including LLC-MK2, Vero, BHK-21, and SW-13.4. Virus isolation can be achieved in 2–5 days, but cell cultures lack sensitivity, and usually only allow detection of the relatively high viraemia encountered during the first 5 days of illness. Although more simple and rapid to perform, isolation in cell culture is is less sensitive than traditional methods such as intracranial inoculation to newborn mice. In suckling mice, CCHFV kills by intracerebral injection, with high virus titer in brain and liver, and widespread disseminates after subcutaneous or intranasal inoculation. The animal models are very limited and none of them is satisfactory as a model of the human disease (Whitehouse, 2004; Gowen and Holbrook, 2008)).
In summary, if there is a substantial accumulation of virulence data on RVFV, WNV and CCHFV, these are very dispersed, and not comparable between different laboratories. In addition, the factors determining the virulence and pathogenicity remain poorly understood, as for many other RNA-viruses (Halstead, 1988; Schnittler and Feldmann, 2003). Therefore there is a clear need for characterisation and identification of reference virus strains and standardization of procedure to evaluate the virulence of new viral isolates.

The purpose of WP5: establisment of virulence standards
The primary goal of the WP5 will be to establish a global census, among the collaborating teams of:

• the viral strains and isolates of RVFV, WNV and CCHFV used or available; 
• the various cell  used or available for propagating/producing the viruses and studying their tropism and susceptibility: primary cell lines prepared from various animal species and tissues; mammalian cell lines like VERO E6 , BKH-21, hepatocytes, neurones, cells “knocked out” in various elements of the IFN system (IFNAR, STAT, etc); insect cell lines such as C6/36, AP61, etc;
• the available standardized tests to characterize the virulence of new isolates of WNV, RVFV, CCHFV.
• the available animal models.
• the level of security (BSL3, BSL4) accessible in each team of the consortium

All these available materials, methods and competences will serve as a basis for completing the principal objective of WP5: “Standardization of experimental procedures for evaluating the virulence of strains and isolates of WNV, RVFV and CCHFV” what will include the following steps:

• Consensual identification (working groups) of one or two reference strain(s) per virus species (CCHFV, RVFV, WNV), in particular those classically used in National or WHO Collaborative Reference Centres, or showing typical growth/virulence features.
• Distribution of the selected strain to all other WP participants by the laboratory of origin together with the parameters for specific in vitro amplification (cell line and culture media, temperature, multiplicity of infection, time up to harvesting);
• The selected strains will be screened by each WP participant by its own routine laboratory methods to characterise the markers of virulence of according to “indoor” in vitro or in vivo standards. In vitro systems include: permissiveness of different cell lines, virus growth curve in permissive versus IFN-KO cell cultures, virus susceptibility/resistance to antiviral molecules, heat treatment, irradiation, 95% ethanol or other chemicals. In vivo systems concern different animal models, strains (including transgenic animals), stage of maturity (suckling to adults), routes of infection (intra-cerebral, intra-muscular, intra-nasal, and intra-peritoneal) that will evidence differences in infectivity, viremia or pathogenicity to different viral strains;
• Results will be synthesized to select one or two reference methods and WP participants will have to reproduce virulence data on reference viruses using reference methods.
• The standardized virulence tests will be used to characterize the virulence of other laboratory strains as well as new isolates of WNV, RVFV, CCHFV. Data will be published and recorded in an electronic database hosting virulence characteristics of the strains/isolates (+ isolation place and conditions, sequence, etc.)