Cowpox with Severe Generalized Eruption, Finland

Cowpox with a severe, generalized eruption was diagnosed in an atopic 4-year-old girl by electron microscopy, virus isolation, polymerase chain reaction, and immunoglobulin (Ig) M and low-avidity IgG antibodies. The hemagglutinin gene of the isolate clustered with a Russian cowpox virus strain, and more distantly, with other cowpox and vaccinia virus strains. The patient’s dog had orthopoxvirus-specific antibodies, indicating a possible transmission route

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.This work was outlined and supported by Project Grant #632785 of the National Health and Medical Research Council of Australia and The Australian Research Council Federation Discovery Project #1096623. CBW was supported by a National Health and Medical Research Council of Australia Senior Research Fellowship #571905. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Vaccinia protein C16 blocks innate immune sensing of DNA by binding the Ku complex

VACV gene C16L encodes a 37-kDa protein that is highly conserved in orthopoxviruses and functions as an immunomodulator. Intranasal infection of mice with a virus lacking C16L (vΔC16) induced less weight loss, fewer signs of illness and increased infiltration of leukocytes to the lungs compared with wild-type virus. To understand C16’s mechanism of action, tandem affinity purification and mass spectrometry were used to identify C16 binding partners. This revealed that Ku70, Ku80 and PHD2 interact with C16 in cells. Ku70 and Ku80 constitute the Ku heterodimer, a well characterised DNA repair complex. MEFs lacking Ku, or the other component of the DNA-dependent protein kinase (DNA-PK) complex, the catalytic subunit of DNA-PK (DNA-PKcs), were shown to be deficient in the upregulation of IRF-3-dependent genes such as Cxcl10, Il6 and Ifnb in response to transfection of DNA, but not poly (I:C). Furthermore, following infection of MEFs with VACV strain MVA the activation of Cxcl10 or Il6 transcription was dependent on DNA-PK. Therefore, DNA-PK is a DNA sensor capable of detecting poxvirus DNA and activating IRF-3-dependent innate immunity. C16 inhibited the binding of Ku to DNA, and therefore inhibited DNA-mediated induction of Cxcl10 and Il-6 in MEFs. The role of C16 in vivo was also examined: infection with vΔC16 led to increased production of Cxcl10 and Il-6 following intranasal infection of mice compared with wild-type virus. C16 is therefore an inhibitor of DNA-PK-mediated DNA sensing and innate immune activation. C16 was also shown to bind to PHD2, an enzyme involved in regulation of hypoxic signalling. VACV was found to activate the transcription of hypoxia-related genes, and C16 expression in cells was also capable of doing this. The role of hypoxic signalling in VACV infection remains poorly understood

Smallpox Then and Now

Smallpox has claimed millions of lives during its existence. Deaths from the disease far out numbered that of the bubonic plague, the Black Death of the Middle Ages, and the wars of the 20th century all combined. Survivors of the disease acquired immunity but suffered a variety of long-term afflictions, such as permanent scarring, disfiguration, and blindness (Tucker, 2001). Although smallpox was eventually eradicated from the wild, it left a long trail of sickness and deaths that afflicted people of every race, class and social status. Even as recently as 1967, the disease continued to leave a devastating impact on the world causing an estimated two million deaths (Tucker, 2001). After the declaration of freedom from smallpox occurred in 1980, the terror of the disease faded from our awareness (Tucker, 2001). While some would consider smallpox to be a disease of the past and long forgotten, such consideration would be premature.Master of Public Healt

Vaccinia Virus Gene B7R Encodes an 18-kDa Protein That is Resident in the Endoplasmic Reticulum and Affects Virus Virulence

AbstractThis paper presents a characterisation of vaccinia virus (VV) gene B7R that was predicted to encode a polypeptide of 182 amino acids with an N-terminal signal peptide. In vitro transcription and translation analysis showed the B7R gene product was a 21-kDa protein that, in the presence of microsomes, was processed into an 18-kDa mature form. The 18-kDa form associated with the microsomal membranes and was within the lumen of the vesicle where it was inaccessible to exogenous protease or an antibody raised against the B7R C terminus. Within VV-infected cells, the 18-kDa form of B7R was detected late during infection in the endoplasmic reticulum where it colocalised with protein disulphide isomerase. The B7R protein was detected neither in the culture supernatant nor associated with virus particles. A virus deletion mutant lacking the B7R gene and a revertant virus were constructed. Compared to wild-type and revertant viruses, the deletion mutant replicated normally in cell culture and had unaltered virulence in a murine intranasal model of infection. However, the deletion mutant was attenuated in a murine intradermal model where it induced a smaller lesion than the control viruses

Reassessing the foundations : Worldwide smallpox eradication, 1957-67

An expansive, worldwide smallpox eradication programme (SEP) was announced by the World Health Assembly in 1958, leading this decision-making body to instruct the World Health Organization Headquarters in Geneva to work with WHO Regional Offices to engage and draw in national governments to ensure success. Tabled by the Soviet Union’s representative and passed by a majority vote by member states, the announcement was subject to intense diplomatic negotiations. This led to the formation, expansion and reshaping of an ambitious and complex campaign that cut across continents and countries. This article examines these inter-twining international, regional and national processes, and challenges long-standing historiographical assumptions about the fight against smallpox only gathering strength from the mid-1960s onwards, after the start of a US-supported programme in Western Africa. The evidence presented here suggests a far more complex picture. It shows that although the SEP’s structures grew slowly between 1958 and 1967, a worldwide eradication programme resulted from international negotiations made possible through gains during this period. Significant progress in limiting the incidence of smallpox sustained international collaboration, and justified the prolongation and expansion of activities. Indeed, all of this bore diplomatic and legal processes within the World Health Assembly and WHO that acted as the foundation of the so-called intensified phase of the SEP and the multi-faceted activities that led to the certification of smallpox eradication in 1980