The genomes of three North American orthopoxviruses.

The complete genomes of a skunkpox, volepox, and raccoonpox virus were sequenced and annotated. Phylogenetic analysis of these genomes indicates that although these viruses are all orthopoxviruses, they form a distinct clade to the other known species. This supports the ancient divergence of the North American orthopoxviruses from other members of the orthopoxviruses. Only two open reading frames appear to be unique to this group of viruses, but a relatively small number of insertions/deletions contribute to the varied gene content of this clade. The availability of these genomes will help determine whether skunkpox and volepox viruses share the characteristics that make raccoonpox a useful vaccine vector.

Safety and immunogenicity of orally administered poxvirus vectored constructs in the white-footed mouse (Peromyscus leucopus).

Globally, zoonotic spillover is becoming more frequent and represents a growing public health concern. Reservoir-targeted vaccination offers an intriguing alternative to traditional vaccine practices by establishing protection in wild populations that maintain the natural pathogen cycle. As an important pathogen reservoir, Peromyscus leucopus Rafinesque or the white-footed mouse has been the target of several experimental vaccines. However, strategies are limited by the method of administration, need for repeated dosing, or safety of constructs in the field. To address these concerns, we evaluated two highly attenuated poxviruses, raccoonpox virus (RCN) and modified vaccinia Ankara (MVA) virus as potential oral vaccine vectors in white-footed mice. Following oral administration, P. leucopus showed no adverse signs. A single oral dose elicited robust immune responses in mice to the foreign influenza hemagglutinin protein expressed by poxvirus vaccine vectors. Serum hemagglutinin inhibition antibody titers were detected by day 7 post immunization and persisted until study termination (77 days post immunization). This study establishes the safety and immunogenicity of recombinant MVA and RCN poxviruses in P. leucopus and demonstrates the suitability of these vectors as part of a reservoir-targeted vaccine strategy for white-footed mice.

Ophthalmic Features and Implications of Poxviruses: Lessons from Clinical and Basic Research.

Amidst the ongoing monkeypox outbreak, global awareness has been directed towards the prevention of viral transmission and case management, with the World Health Organization declaring the outbreak a public health emergency of international concern. Monkeypox virus is one of several species in the Orthopoxvirus genus, with other species of the genus including the variola, cowpox, mousepox, camelpox, raccoonpox, skunkpox, and volepox viruses. Although the nomenclature of these species is based on the animal host from which they were originally isolated, transmission from animals to humans has been reported with several species. The progression of disease, following an incubation period, typically consists of a prodromal phase with systemic flu-like symptoms. Various organ systems may be affected in addition to the formation of pathognomonic skin lesions. As monkeypox poses a continued public health concern, the ophthalmic sequelae of monkeypox virus, especially those leading to vision loss, warrant consideration as well. This review provides a comprehensive summary of the ophthalmic implications of poxviruses in clinical and laboratory settings reported in the literature, as well as areas of unmet need and future research.

Poxvirus Safety Analysis in the Pregnant Mouse Model, Vaccinia, and Raccoonpox Viruses.

Poxviruses cause many diseases in humans and animals worldwide, and there is a need for vaccines with improved safety and good efficacy. In addition, poxvirus vectors are widely used as recombinant vaccines for various infectious diseases and as recombinant and oncolytic vaccines for cancer. One concern with poxvirus vaccine vectors is that some poxviruses can infect a developing fetus and cause fetal loss or congenital disease. This can be an issue both for patients receiving a vaccine and for pregnant health care providers, including doctors, nurses, and veterinarians, who might receive accidental exposure to the poxvirus by injection or during patient care. We describe here a method for analyzing the safety of virus exposure in pregnant mammals using a mouse model testing vaccinia, canarypox, and raccoonpox virus vectors.

Responses of Juvenile Black-tailed Prairie Dogs ( Cynomys ludovicianus ) to a Commercially Produced Oral Plague Vaccine Delivered at Two Doses.

We confirmed safety and immunogenicity of mass-produced vaccine baits carrying an experimental, commercial-source plague vaccine (RCN-F1/V307) expressing Yersinia pestis V and F1 antigens. Forty-five juvenile black-tailed prairie dogs ( Cynomys ludovicianus ) were randomly divided into three treatment groups (n=15 animals/group). Animals in the first group received one standard-dose vaccine bait (5×107 plaque-forming units [pfu]; STD). The second group received a lower-dose bait (1×107 pfu; LOW). In the third group, five animals received two standard-dose baits and 10 were left untreated but in contact. Two vaccine-treated and one untreated prairie dogs died during the study, but laboratory analyses ruled out vaccine involvement. Overall, 17 of 33 (52%; 95% confidence interval for binomial proportion [bCI] 34-69%) prairie dogs receiving vaccine-laden bait showed a positive anti-V antibody response on at least one sampling occasion after bait consumption, and eight (24%; bCI 11-42%) showed sustained antibody responses. The STD and LOW groups did not differ (P≥0.78) in their proportions of overall or sustained antibody responses after vaccine bait consumption. Serum from one of the nine (11%; bCI 0.3-48%) surviving untreated, in-contact prairie dogs also had detectable antibody on one sampling occasion. We did not observe any adverse effects related to oral vaccination.

Burrow Dusting or Oral Vaccination Prevents Plague-Associated Prairie Dog Colony Collapse.

Plague impacts prairie dogs (Cynomys spp.), the endangered black-footed ferret (Mustela nigripes) and other sensitive wildlife species. We compared efficacy of prophylactic treatments (burrow dusting with deltamethrin or oral vaccination with recombinant "sylvatic plague vaccine" [RCN-F1/V307]) to placebo treatment in black-tailed prairie dog (C. ludovicianus) colonies. Between 2013 and 2015, we measured prairie dog apparent survival, burrow activity and flea abundance on triplicate plots ("blocks") receiving dust, vaccine or placebo treatment. Epizootic plague affected all three blocks but emerged asynchronously. Dust plots had fewer fleas per burrow (P < 0.0001), and prairie dogs captured on dust plots had fewer fleas (P < 0.0001) than those on vaccine or placebo plots. Burrow activity and prairie dog density declined sharply in placebo plots when epizootic plague emerged. Patterns in corresponding dust and vaccine plots were less consistent and appeared strongly influenced by timing of treatment applications relative to plague emergence. Deltamethrin or oral vaccination enhanced apparent survival within two blocks. Applying insecticide or vaccine prior to epizootic emergence blunted effects of plague on prairie dog survival and abundance, thereby preventing colony collapse. Successful plague mitigation will likely entail strategic combined uses of burrow dusting and oral vaccination within large colonies or colony complexes.

Mucosal administration of raccoonpox virus expressing highly pathogenic avian H5N1 influenza neuraminidase is highly protective against H5N1 and seasonal influenza virus challenge.

We previously generated recombinant poxviruses expressing influenza antigens and studied their efficacy as potential highly pathogenic avian influenza (HPAI) vaccines in mice. While both modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) expressing hemagglutinin (HA) provided strong protection when administered by parenteral routes, only RCN-neuraminidase (NA) showed promise as a mucosal vaccine. In the present study we evaluated the efficacy of RCN-NA constructs by both intradermal (ID) and intranasal (IN) routes. Surprisingly, while RCN-NA completely protected mice when administered by the IN route, it failed to protect mice when administered by the ID route. After challenge, significantly less virus induced pathology was observed in the lungs of mice vaccinated with RCN-NA by the IN route as compared to the ID route. Furthermore, IN administration of RCN-NA elicited neutralizing antibodies detected in bronchoalveolar lavage (BAL) samples. We also determined the role of cellular immune responses in protection elicited by RCN-NA by depleting CD4 and CD8 T cells prior to challenge. Finally, we demonstrated for the first time that antibodies against NA can block viral entry in addition to viral spread in vitro. These studies demonstrate the importance of mucosal administration of RCN viral vectors for eliciting protective immune responses against the NA antigen.

Raccoonpoxvirus safety in immunocompromised and pregnant mouse models.

Numerous poxviruses infect humans and animal hosts, and a poxvirus vaccine with an improved safety profile is needed as the current vaccinia virus vaccine is contraindicated in individuals that have a history of eczema or heart disease, or are immunocompromised or pregnant. In addition, poxviruses make excellent vaccine vectors for other infectious diseases and cancer. Raccoonpoxvirus is a naturally occurring attenuated North American poxvirus, and thus it is of interest as a vaccine vector platform. This study explores the effects of raccoonpoxvirus in SCID and Nude immunocompromised and pregnant mouse models to assess its virulence and probable safety for human and animal populations. We also analyzed the safety of recombinant raccoonpox carrying a gene expressing a foreign antigen, rabies virus glycoprotein, designed for heterologous vaccine protection. Our data show that recombinant raccoonpoxviruses are avirulent in many cases and are much safer than vaccinia virus (strain WR). Raccoonpoxviruses also have the advantage of being able to replicate in mammalian cells. This allows increased immunogenicity and production efficiency, giving an advantage over non replicating vectors such as Modified Vaccinia Ankara MVA or canarypoxvirus.