Rabies is an acute progressive encephalomyelitis caused by negative-sense single-stranded RNA viruses from the genus Lyssavirus
, family Rhabdoviridae
. At present, the genus includes 12 established viral species and 3 viruses awaiting taxonomic assessment (1–4
). Of these, classic rabies virus (RABV) is the most broadly distributed, causing >99% of human lyssavirus cases worldwide (1
). In China, RABV causes 2,000 to 3,000 reported human deaths per year. Almost all Chinese lyssavirus isolates belong to RABV species originating from carnivores (5
). However, several human rabies cases of bat origin, in which the viruses were not identified, were reported (6
). In 2012, for the first time, a lyssavirus was isolated from a greater tube-nosed bat (Murina leucogaster
) in the Jilin Province of China. It was identified as Irkut virus (IRKV) (isolate IRKV-THChina12) (6
). Phylogenetic analysis demonstrated that structural proteins of this virus shared high sequence identity (>98%) with IRKV isolate Ozernoe, from a human case of rabies following a bat bite in Russia in 2007 (7
). Several studies have investigated the serological cross-reactivity of lyssaviruses and the protective ability of commercially available RABV biologics against non-RABV lyssaviruses. In general, it has been shown that rabies biologics provide sufficient protection against lyssaviruses from phylogroup I (8
). However, a more recent study demonstrated that rabies biologics available in the United States elicited only partial protection against several non-RABV lyssaviruses, including IRKV (9
). The need existed, therefore, to further assess the serological cross-reactivity between RABV and IRKV and to evaluate whether rabies biologics available in China and elsewhere provide reliable protection against IRKV infection. In this study, we conducted a series of preexposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) experiments in an animal model, to evaluate the protective effects of several commercially available and experimental biologics against IRKV infection.
For the first time, an IRKV was recently isolated from a bat in China through the active rabies surveillance program. Although Chinese IRKV belongs to the same phylogroup, 1, as the widely distributed RABV, it is more closely related phylogenetically to European bat lyssavirus type 1 and Duvenhage virus than to RABV (6
). Phylogenetic relationships and genetic distances between lyssaviruses partially reflect the extent of serological cross-reactivity. For example, it was suggested that 72 to 74% amino acid sequence identity within glycoprotein ectodomains provides sufficient cross-neutralization between lyssaviruses (8
). The same can be inferred for the protection elicited by rabies biologics (which all are based on several well-characterized RABV strains) against non-RABV lyssaviruses. Commercial rabies biologics have been fully efficacious against a phylogenetically related Australian bat lyssavirus (20
). However, the protection elicited by several rabies biologics against IRKV in animal models was limited (9
Additional attention should be paid to conservation of antigenic sites on lyssavirus glycoproteins. This is particularly important as several virus-neutralizing monoclonal antibodies (MAbs) were offered for replacement of conventional HRIG in human rabies PEP (21–28
). For example, a linear epitope with the key residues LCGV within antigenic site I serves for binding of MAbs CR57 and 62071-3 (22
). Similarly, conserved conformational antigenic sites II and III contain binding epitopes for several other MAbs (24–28
). Amino acid differences in these epitopes may alter MAb binding and thus abolish neutralization of non-RABV lyssaviruses, including IRKV. Therefore, MAbs selected for antibody cocktails for use in PEP must be scrutinized for their ability to neutralize non-RABV lyssaviruses.
In this study, the efficacy of rabies biologics available in China against IRKV was determined in routine and modified PrEP and PEP experiments, as described previously (9
). Although a single dose of rabies vaccine did not induce adequate protection against IRKV infection, routine PrEP (three vaccine doses, on days 0, 7, and 28) induced strong protection against RABV and IRKV infection.
In the PEP experiments, however, only very high doses of RABV immunoglobulins conferred partial protection of animals against IRKV infection, whereas the routine PEP regimen (20 IU/kg body weight of HRIG injected only once, followed by the 2-1-1 vaccine regimen) did not protect animals against IRKV infection at all. Moreover, combination of HRIG with vaccines decreased the VNA titers and survival rates of hamsters, compared with the groups that were given HRIG only, likely because of interference between the vaccines and the immunoglobulins. Short incubation periods in a hamster model may not provide sufficient time for the development of active immune responses, with protection against infection being based solely on antibodies delivered passively. These antibodies may fail to protect animals against IRKV infection based on limited cross-neutralization activity (Tables 2
). It is possible that PEP models with longer incubation periods or situations involving real-life exposures would allow sufficient time for the development of active immune responses to vaccination and would protect animals and humans against IRKV infection, as may be inferred from our PrEP experiments. However, short incubation periods may occur after real-life exposures as well (29
), and this increases the demand for the development of novel immunoglobulin preparations (including MAbs) for protection against IRKV and other non-RABV lyssaviruses.
Interferon can inhibit rabies virus activity in vivo
and does not interfere with circulating VNAs (30
). This was confirmed in our PEP experiment. Perhaps addition of interferon to PEP regimens would increase the protection of humans against non-RABV lyssaviruses, although this must be further evaluated in higher-mammalian models and clinical trials.
A replication-deficient vector derived from human adenovirus type 5 was chosen for the cross-immunogenicity experiments, since it has been well studied for expression of RABV proteins and has been evaluated in animal models (31
). In our experiments, a recombinant vaccine expressing IRKV glycoproteins provided significantly better results in animals challenged with IRKV than did a recombinant vaccine expressing RABV glycoproteins. However, it did not provide reliable protection against RABV challenge. Chimeric lyssavirus glycoproteins from partial RABV and IRKV genes should be considered, to achieve protection against these two viruses (32
Surveillance for non-RABV lyssaviruses in China is very limited, as is diagnostic capacity across the country. The threat of IRKV to animals and humans is difficult to estimate. Although at least two bat-associated human rabies cases in China have been reported (6
), IRKV was not identified in those cases, as the diagnoses were based on clinical signs and exposure history only.
In conclusion, commercially available rabies biologics do not provide protection against IRKV infection as reliable as that against RABV infection. Before the development of new biologics (e.g., chimeric vaccines and MAbs), the combination of higher doses of RABV immunoglobulins with interferon may be effective treatment for exposure to IRKV and other non-RABV lyssaviruses. In addition, active field surveys should be performed to investigate more thoroughly the prevalence and circulation patterns of IRKV in China, to assess properly the public health and veterinary implications.