A novel highly reproducible and lethal nonhuman primate model for orthopox virus infection

Abstract

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1-3 days after onset of symptoms, even when very low infectious viral doses of 5x10(2) pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID(50) (minimal monkey infectious dose 50%) of 8.3x10(2) pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.

Figure 1. Kaplan-Meier plot showing the effect of different doses of calpox virus inoculated intranasally (i.n.) on the survival of marmosets.

Survival post infection (p.i.) is expressed as percentages. Ratios given on the right side indicate numbers of surviving animals versus numbers of animals inoculated. Doses as low as 8.3×10³ pfu led to 100% mortality.

Figure 2. Distribution of calpox virus in different tissues.

Virus was determined in different tissues of infected animals and is shown exemplarily for i.v. infected animals of group I determined by real-time PCR. A) calpox virus genome equivalents (GE) (viral DNA copy number)/10⁶ c-myc GE (DNA copy number of cellular gene marker); B) calpox virus mRNA copies/10⁶ c-myc mRNA copies; calculation of the copy numbers for calpox virus as well as c-myc is based on the mean value of duplicate measurements and a respective plasmid standard for each real-time PCR assay; lines indicate the median value for all 5 i.v.-infected marmosets (group I); GE: genome equivalents.

Figure 3. Analysis of antibodies and virus particles in infected animals.

Section A: Determination of orthopox virus-specific IgM antibodies. IgM antibodies were determined in the serum of infected marmosets at the time of death (day 14 p.i.), calpox virus-infected Hep2 cells were incubated with serum (1∶10 diluted) and visualized with anti-human IgM-FITC labeled antibody, 1∶63 magnification; A1) negative serum; A2) animal V-a; A3) animal VI-a, bars correspond to 20 µm; Section B: Electron micrographs of mature and immature calpox virus particles in different tissues of animal V-b; B1) immature virus particles in liver tissue; B2) mature virus particles in lung tissue and B3) immature virus particles in spleen tissue; arrows indicate virus particles.

Figure 4. Detection of calpox virus in tissues and saliva.

A) comparison of infectious calpox virus titer and calpox virus genome equivalents (GE) per ml tissue homogenate for two animals infected with 3.5×10³ (V-a) and 5.0×10² pfu (VI-a) (line indicates the median value for animals V-a and VI-a); for real-time PCR: calculation of the copy numbers for calpox virus is based on the mean value of duplicate measurements and a respective plasmid standard; B) infectious calpox virus in saliva (normalization approximated by using throat swab as the denominator) compared to GE in blood (during course of infection) for three different infectious doses (group IV: 3.5×10⁴, V: 3.5×10³ and VI: 5.0×10² pfu); pfu: plaque forming unit; p.i.: post infection; GE: genome equivalents.