Contemporary outbreaks of different avipoxviruses in Humboldt penguins of wild animal park Planckendael and in chickens of commercial poultry farms in Belgium

In the present study, the first outbreak of a penguinpox virus (PPV) in Humboldt penguins (Spheniscus humboldd) and four outbreaks of fowlpox virus (FPV) in layer chickens are reported. Clinically, cutaneous wart-like growths were observed around the eyes in four juvenile Humboldt penguins and cutaneous nodular lesions in the comb, wattles, around the eyes and other unfeathered skin parts of layer chickens. Histopathology (FPV and PPV), electron microscopy (PPV), virus isolation (FPV) and PCR amplification (FPV and PPV) confirmed that both isolates were avipoxviruses (APVs). According to the phylogenetic analysis of the partial P4b core protein gene, the Belgian Humboldt PPV clustered with sequences of free-range (domestic and synanthrope bird species) and wild bird species of the United States and Europe (99-100% homology), and all four Belgian FPV isolates clustered with FPV isolates of chickens, turkeys, canary and FPV attenuated live vaccines from all over the world (100% homology)

Rapid evolution of protein kinase PKR alters sensitivity to viral inhibitors.

Protein kinase PKR (also known as EIF2AK2) is activated during viral infection and phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, and these differences were traced to positively selected residues near the eIF2alpha binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in nonhuman model systems

Population, sexual and reproductive health, rights and sustainable development: forging a common agenda.

This article suggests that sexual and reproductive health and rights activists seeking to influence the post-2015 international development paradigm must work with sustainable development advocates concerned with a range of issues, including climate change, environmental issues, and food and water security, and that a way of building bridges with these communities is to demonstrate how sexual and reproductive health and rights are relevant for these issues. An understanding of population dynamics, including urbanization and migration, as well as population growth, can help to clarify these links. This article therefore suggests that whether or not sexual and reproductive health and rights activists can overcome resistance to discussing "population", become more knowledgeable about other sustainable development issues, and work with others in those fields to advance the global sustainable development agenda are crucial questions for the coming months. The article also contends that it is possible to care about population dynamics (including ageing and problems faced by countries with a high proportion of young people) and care about human rights at the same time. It expresses concern that, if sexual and reproductive health and rights advocates do not participate in the population dynamics discourse, the field will be left free for those for whom respecting and protecting rights may be less of a priority

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

The extraordinary evolutionary history of the reticuloendotheliosis viruses

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

Background: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-gamma ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl beta-galactosidase assay with primary isolates of HIV-1. Results: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl b-galactosidase assay. Conclusions: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques

Diminished Intracellular Invariant Chain Expression Following Vaccinia Virus Infection

Vaccinia virus (VV) has been used as a vaccine to eradicate smallpox and as a vaccine for HIV and tumors. However, the immunoevasive properties of VV, have raised safety concerns. VV infection of APC perturbs MHC class II-mediated Ag presentation. Exposure of human B cell lines to VV induced a dramatic reduction in cellular expression of the class II chaperone, invariant chain (Ii) during the late stages (i.e. 8–10 h) of infection. Yet, cell viability and surface expression of MHC class II molecules were maintained up to 24 h after exposure to virus. Reductions in Ii and class II mRNA levels were detected as early as 6 h after VV infection of APC. To examine whether VV was acting solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation, cycloheximide (CHX). Within 1 h of B cell CHX treatment, Ii protein expression decreased coupled with a loss of class II presentation. Analysis of Ii degradation in VV or CHX treated cells, revealed on-going Ii proteolysis contributing to reduced steady state Ii levels in these APC. Yet in contrast with CHX, VV infection of APC altered lysosomal protease expression and Ii degradation. Virus infection induced cellular cathepsin L expression while reducing the levels of other lysosomal proteases. These results demonstrate that at late stages of VV infection, reductions in cellular Ii levels coupled with changes in lysosomal protease activity, contribute in part to defects in class II presentation

Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapoxand orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera