What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Structural mechanism of ER retrieval of MHC class I by cowpox.

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.

A-type inclusion bodies: a factor influencing cowpox virus lesion pathogenesis.

The family Poxviridae comprises the most complex animal DNA viruses. During some poxvirus infections, A-type inclusion bodies (ATIs), codified by the ati gene, are produced. Although some studies have compared poxviruses that encode these inclusion bodies with those that do not, the biological function of ATIs is poorly understood. A recombinant ati-deleted cowpox virus was constructed and compared with the wild-type virus in in vitro experiments including electron microscopy and plaque and viral growth assays. No significant differences were observed in vitro. This reinforces the conclusion that the inclusion body is not essential for in vitro viral replication and morphogenesis. Additionally, different lesion progressions in vivo were observed by macroscopic and histological analysis, suggesting that the presence or absence of ATIs could result in different healing dynamics. This is the first time that the role of ATIs during viral replication has been studied based solely on one variable, the presence or absence of ATIs.

Comparison of the susceptibility of the red fox (Vulpes vulpes) to a vaccinia-rabies recombinant virus and to cowpox virus.

Sylvatic rabies can be efficiently controlled by vaccination of foxes with a vaccinia-rabies recombinant virus. However, the risk of recombination between the engineered vaccine virus and other orthopoxviruses endemic in wildlife, such as cowpox virus, still needs to be investigated. In this study, foxes inoculated orally and intradermally with cowpox virus were found to be not very susceptible to cowpox virus, which was isolated from only the oropharynx and tonsils, at low titre and for only five days after inoculation. Thus the risk of recombination between these viruses in foxes is very low.

Frame-shift mutations within the vaccinia virus A-type inclusion protein gene.

The genetic basis for the failure of vaccinia virus (strain WR) to form a full-length 150 kiloDalton (kDa) A-type inclusion protein was determined by sequencing a 4.1-kb pair segment of DNA and analyzing its transcription products. Open reading frames predicted to encode slightly overlapping 84.5- and 27.1-kDa proteins homologous to contiguous N-terminal segments of the A-type inclusion protein of cowpox virus were found. A putative deletion of two adjacent nucleotides occurring within several consecutive AG repeats and an insertion of 8 nucleotides accounted for the first and second reading frame shifts, respectively. Additional small mutations affecting reading frames were present in the C-terminal region of the gene. The vaccinia and cowpox virus mRNAs encoding the disparate size A-type inclusion proteins were similar in length, had equivalent 5' and 3' ends, and were expressed late in infection indicating the absence of mutations affecting transcriptional signals.