Virulence mismatches in index hosts shape the outcomes of cross-species transmission Nardus Mollentzea,b,1, Daniel G. Streickera,b, Pablo R. Murciab, Katie Hampsona, and Roman Bieka aInstitute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow G12 8QQ, United Kingdom; and bMedical Research Council–University of Glasgow Centre for Virus Research, Glasgow G61 1QH, United Kingdom Edited by Nils Chr. Stenseth, University of Oslo, Oslo, Norway, and approved September 25, 2020 (received for review April 27, 2020) Whether a pathogen entering a new host species results in a single ratio of any viral disease (9, 10). Rabies virus naturally infects infection or in onward transmission, and potentially an outbreak, multiple carnivore and bat species, which each perpetuate species- depends upon the progression of infection in the index case. specific maintenance cycles (11). Although most cross-species Although index infections are rarely observable in nature, exper- transmission events do not lead to onward transmission, each imental inoculations of pathogens into novel host species provide maintenance cycle represents a rare past cross-species transmis- a rich and largely unexploited data source for meta-analyses to sion event that established transmission in a novel host. Dead-end identify the host and pathogen determinants of variability in in- cross-species transmissions and historical host shifts are detectable fection outcomes. We analyzed the progressions of 514 experi- in rabies virus phylogenies, and epidemiological surveillance re- mental cross-species inoculations of rabies virus, a widespread veals that nascent host shifts remain commonplace (11–13). As zoonosis which in nature exhibits both dead-end infections and such, rabies virus exhibits extensive variation in the epidemiolog- varying levels of sustained transmission in novel hosts. Inocula- ical outcomes of cross-species transmission. Here, we exploit tions originating from bats rather than carnivores, and from cross-infection studies conducted over several decades, in which warmer- to cooler-bodied species caused infections with shorter diverse mammalian species were inoculated with rabies viruses of incubation periods that were associated with diminished virus ex- bat and carnivore origin, to investigate the individual-level out- cretion. Inoculations between distantly related hosts tended to comes of index infections. result in shorter clinical disease periods, which are also expected The potential for onward transmission of rabies virus is likely to impede onward transmission. All effects were modulated by to depend on the incubation period (from bite to the appearance ECOLOGY infection dose. Taken together, these results suggest that as host of clinical signs) and the duration of clinical signs prior to death species become more dissimilar, increased virulence might act as a (here, the clinical period) of infected hosts. Longer incubation limiting factor preventing onward transmission. These results can periods are associated with greater distribution of virus through explain observed constraints on rabies virus host shifts, describe a previously unrecognized role of host body temperature, and pro- the central nervous system (14), spread to a wider range of tis- vide a potential explanation for host shifts being less likely between sues (15), and higher virus titers in the salivary glands, all of genetically distant species. More generally, our study highlights which should facilitate onward transmission (16). Conversely, meta-analyses of experimental infections as a tractable approach faster progression of infection has been associated with lower to quantify the complex interactions between virus, reservoir, and virus excretion, and in extreme cases animals die before the virus novel host that shape the outcome of cross-species transmission. reaches the salivary glands, making transmission highly unlikely cross-species transmission | virulence | virus | rabies Significance ross-species transmission is an important source of emerging Emerging disease epidemics often result from a pathogen Cand endemic disease. Viruses such as West Nile virus, rabies establishing transmission in a novel host species. However, for virus, and Lassa virus cause tens of thousands of human infec- reasons that remain poorly understood, most cross-species tions annually through transmission from animal reservoirs transmissions fail to establish in the newly infected species. (1–3). Cross-species transmission is also the first step toward host Examining experimental cross-species inoculations of rabies shifts, where pathogens establish transmission cycles in novel virus, we show that host and viral factors predict differences in hosts (4). While the broader-scale epidemiological and ecologi- disease progression in ways that are expected to impact the cal factors driving cross-species transmission are beginning to be likelihood of onward transmission. Disease progression was understood (reviewed in refs. 5–7), we remain unable to antici- accelerated and virus excretion decreased when the reservoir pate whether cross-species transmission will cause “dead-end” and novel host were physiologically or genetically more dis- infections or transmit onward. Infection dynamics at the cross- similar. These insights may help to explain and predict host species interface, specifically the probability of infection given shifts in rabies and other zoonotic viruses and highlight meta- exposure and the progression of index infections in novel hosts, analyses of experimental inoculation data as a powerful and are generally unobservable in nature. This is a crucial gap given generalizable approach for understanding the dynamics of that the outcomes of cross-species infections have profound index infections. implications for host shifts and disease emergence. Author contributions: D.G.S., P.R.M., K.H., and R.B. designed research; N.M. and R.B. Cross-infection studies, in which viruses from a natural res- performed research; N.M. analyzed data; and N.M., D.G.S., P.R.M., K.H., and R.B. wrote ervoir are experimentally inoculated into novel host species, the paper. provide a rare view into the dynamics of index infections. Since The authors declare no competing interest. the dose, route, timing, and origin of viral exposure are known, This article is a PNAS Direct Submission. these factors can be controlled for to identify the biological and This open access article is distributed under Creative Commons Attribution License 4.0 evolutionary rules that govern the outcomes of cross-species trans- (CC BY). mission. We focus on Rabies lyssavirus (family Rhabdoviridae)asa 1To whom correspondence may be addressed. Email: [email protected]. model pathogen for understanding cross-species transmission (8). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Rabies virus is a primarily bite-transmitted zoonotic RNA virus that doi:10.1073/pnas.2006778117/-/DCSupplemental. infects all mammals and, untreated, has the highest case fatality www.pnas.org/cgi/doi/10.1073/pnas.2006778117 PNAS Latest Articles | 1of8 Downloaded by guest on September 27, 2021 (14–17). Further, the clinical period of rabies coincides with the physiological features affecting disease outcome (26), a poten- period of greatest infectivity, when excretion of virus in the saliva tial explanation for the occurrence of host shifts over wide often coincides with clinical signs such as aggression which phylogenetic scales. promote transmission (18). Testing for shifts in incubation and clinical period durations, and in the amount of virus excreted, Here, we test these hypotheses by conducting a meta-analysis of individual-level data from 514 published experimental cross- allows us to examine what plausible factors may constrain on- species infections involving rabies virus. We show that features of ward transmission of rabies in index hosts following cross-species the virus and of the inoculated host species interact with the transmission. initial conditions of exposure to influence the outcome of cross- Based on previous work on rabies virus and in other host– species transmission in ways expected to affect the likelihood of pathogen systems, several mechanisms are hypothesized to in- onward transmission in the novel host species. fluence infection dynamics and the outcome of cross-species transmission (Fig. 1): Results 1) Features of exposed host species (host effects), irrespective Our meta-analysis of cross-species inoculation experiments of the infecting virus. For example, larger-bodied species may recorded results from 65 experiments in total. In agreement with be more resistant to infection and thus require either higher observations from natural infections (27, 28), not all inoculations infectious doses or a longer period of virus replication before resulted in clinical rabies. Only 1,054 (63%) of the 1,672 inoc- symptoms become apparent. More generally, evolutionarily ulated animals for which any data were available developed ra- conserved similarities in host physiology mean that groups of bies during the observation periods of the included experiments. related taxa might have similar susceptibility or clinical out- The proportion of animals which developed rabies was similar comes of infection (19). among both within-species (525/822) and cross-species inocula- 2) Features inherent to the virus lineage involved (virus effects), tions (529/850). irrespective