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PubMed1996

Cowpox virus genome encodes a second soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF but not LT alpha.

We show the cowpox genome (Brighton Red strain) contains a single copy gene, crmC, expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The approximately 25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LT alpha, TNF beta) and lacks the conserved (but nonhomologous) approximately 150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF.

Amino Acid Sequence↗
PubMed1998

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.

Amino Acid Sequence↗
PubMed2000

The cowpox virus SPI-3 and myxoma virus SERP1 serpins are not functionally interchangeable despite their similar proteinase inhibition profiles in vitro.

The myxoma virus (MYX) serpin SERP1 is a secreted glycoprotein with anti-inflammatory activity that is required for full MYX virulence in vivo. The cowpox virus (CPV) serpin SPI-3 (vaccinia virus ORF K2L) is a nonsecreted glycoprotein that blocks cell-cell fusion, independent of serpin activity, and is not required for virulence of vaccinia virus or CPV in mice. Although SPI-3 has only 29% overall identity to SERP1, both serpins have arginine at the P1 position in the reactive center loop, and SPI-3 has a proteinase inhibitory profile strikingly similar to that of SERP1 [Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R., and Moyer, R. W. (2000) Virology 272, 267-280]. To determine whether SPI-3 and SERP1 were functionally equivalent, a CPV variant was constructed where the SPI-3 gene was deleted and replaced with the SERP1 gene regulated by the SPI-3 promoter. Cells infected with CPVDeltaSPI-3::SERP1 secrete SERP1 and show extensive fusion, suggesting that SERP1 is unable to functionally substitute for SPI-3 in fusion inhibition. In the reciprocal experiment, both copies of SERP1 were deleted from MYX and replaced with SPI-3 under the control of the SERP1 promoter. Cells infected with the MYXDeltaSERP1::SPI-3 recombinant unexpectedly secreted SPI-3, suggesting either that the cellular secretory pathway is enhanced by MYX or that CPV encodes a protein that prevents SPI-3 secretion. MYXDeltaSERP1::SPI-3 was as attenuated in rabbits as MYXDeltaSERP1::lacZ, indicating that SPI-3 cannot substitute for SERP1 in MYX pathogenesis.

Animals↗
PubMed1997

Characterization of a cowpox-like orthopox virus which had caused a lethal infection in man.

In August 1990 an orthopox virus (OPV) had been isolated from a severe case of a generalized infection with lethal outcome in an immunosuppressed 18-year-old man. In this communication we present a detailed characterization of the causative virus strain. Based on distinct epitope configurations detected by various monoclonal antibodies the isolate could be differentiated from other OPV species and was classified as a cowpox virus (CP). This classification was confirmed by a species-specific PCR assay and by establishing physical maps for the restriction enzymes HindIII and XhoI. Based on serological data of neutralization assays, blocking-ELISAs and Western blotting analysis evidence is provided that the infection had been acquired from a stray cat.

Adolescent↗
PubMed1992

Acute inflammatory response to cowpox virus infection of the chorioallantoic membrane of the chick embryo.

The chick embryo chorioallantoic membrane was used to study the acute inflammatory response in the absence of contributions from the immune system. In preliminary experiments, lesions of wild-type cowpox virus strain Brighton (CPV-BR) and a 38K gene deletion mutant of CPV-BR (CPV-BR.D1) were compared with vaccinia virus (strains WR and Copenhagen), fowlpox virus, laryngotracheitis virus, and infectious tenosynovitis virus, and were ranked for degree of induced inflammation. The maximal and minimal inflammatory responses were observed with CPV-BR.D1 and CPV-BR viruses, respectively. CPV-BR.D1 lacks a 38K gene which encodes an anti-inflammatory 38-kDa protein that has homology to SERPINs. The kinetics and character of the inflammatory response were examined further in the wild-type CPV-BR and mutant CPV-BR.D1 infections using cell counts, electron microscopy, and assays for inflammatory cell activation. CPV-BR virus infection rapidly spread through the ectoderm, uniformly infecting all cells with the production of large amounts of virions and viral-induced cytopathic effect, but evoking little or no inflammatory response until 144 hr p.i. The CPV-BR.D1 infection, on the other hand, was rapidly contained by a dexamethasone-sensitive inflammatory response mainly of activated heterophils which was advanced by 36 hr p.i. Both infections resulted in disseminated disease with similar numbers of liver lesions and only a slight difference in the LD50, with the CPV-BR.D1 values being higher than that for CPV-BR virus. In this model, the acute inflammatory response alone is unable to prevent disseminated disease and associated mortality.

Acute Disease↗
PubMed1992

Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein.

Supernatants from vaccinia virus (VV)-infected CV-1 cells were examined and found to contain a 33 kd protein capable of binding murine interleukin-1 beta (mIL-1 beta). A VV open reading frame (ORF) that exhibits 30% amino acid identity to the type II IL-1 receptor was expressed in CV-1-EBNA cells and shown specifically to bind mIL-1 beta. A similar ORF from cowpox virus was expressed and also specifically bound mIL-1 beta. A recombinant VV was constructed in which this ORF was disrupted (vB15RKO). Supernatants from vB15RKO-infected cells did not contain an IL-1-binding protein. Supernatants from VV-infected CV-1 cells were capable of inhibiting IL-1-induced murine lymphocyte proliferation in vitro while supernatants from vB15RKO infected cells did not. Intracranial inoculation of mice with vB15RKO suggests that this ORF is involved in VV virulence. The possible role of a virus-encoded IL-1-binding protein in the pathology of a poxvirus infection and its relationship to other poxvirus-encoded immune modulators is discussed.

Animals↗
PubMed2006

Distinct thymidine kinases encoded by cowpox virus and herpes simplex virus contribute significantly to the differential antiviral activity of nucleoside analogs.

Orthopoxviruses and herpesviruses are both large enveloped DNA viruses, yet these virus families exhibit very different susceptibilities to antiviral drugs. We investigated the activation of nucleoside analogs by the types I and II thymidine kinase (TK) homologs expressed by herpes simplex virus type 1 (HSV-1) and cowpox virus (CV). Antiviral activity against TK(-) and TK(+) strains of HSV-1 and CV was determined, and the ratio of the EC(50) values was used as a measurement of TK dependence. As to HSV-1, most of the selected compounds were markedly less effective against the TK(-) strains, suggesting that this enzyme was required for the activation of these nucleoside analogs. This differs from the results for CV where only idoxuridine and bromodeoxyuridine appeared to be activated, putatively by the type II TK expressed by this virus. These data confirm that the type II TK encoded by CV exhibits a more limited substrate specificity than the type I TK encoded by HSV-1. These data suggest that the inefficient activation of nucleoside analogs by the orthopoxvirus TK significantly limits their activity. Additional screening against orthopoxviruses will be required to identify nucleoside analogs that are efficiently activated by their type II TK.

Amino Acid Sequence↗
PubMed2006

In vitro and in vivo evaluation of isatin-beta-thiosemicarbazone and marboran against vaccinia and cowpox virus infections.

It has been reported previously that some thiosemicarbazone compounds have prophylactic activity against smallpox disease and therapeutic activity against vaccinia virus (VV) infections. In these studies, isatin-beta-thiosemicarbazone (IBT) and marboran were administered once daily by intraperitoneal (ip) injection to mice using 30, 10 or 3 mg/kg for 5 days beginning 24, 48 or 72 h after inoculation with VV or cowpox virus (CV). Both compounds were highly effective (p < 0.01) at preventing mortality due to VV even when treatment was delayed up to 72 h postinfection. In CV-infected mice, neither IBT nor Marboran were effective in preventing mortality at any dosage tested when administered at 24 h postinoculation. Viral replication in liver, spleen and kidney was delayed or reduced by 100-to 10,000-fold by 10 mg/kg of marboran, but not IBT, in VV infections. Neither compound was effective against CV infection. Neither IBT nor marboran treatment of mice cutaneously infected with VV or CV reduced viral replication or clinical disease. These results suggest that this class of compound has little therapeutic potential for orthopoxvirus infections since the in vivo activity against CV, a surrogate virus for variola, is lacking.

Animals↗
PubMed2006

Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different.

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.

Amino Acid Sequence↗
PubMed2004

Recombinant viruses obtained from co-infection in vitro with a live vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus display different plaque phenotypes and loss of the transgene.

Some poxviruses are very attractive as transgenic vaccine vectors for humans, domestic animals and wildlife. Poxviridae family members circulate in different ecosystems and parts of the world, providing a pool of possible recombination partners for released or escaped genetically modified poxviruses. We performed in vitro double infections with a vaccinia virus strain Ankara (MVA) vectored influenza vaccine and a cowpox virus isolate from Norway, isolated hybrids, and further analyzed three hybrid viruses with different plaque phenotypes. One of the hybrids was genetically unstable, and during adaptation to new host cells its MVA derived influenza gene was deleted at a high frequency. This is significant in a risk assessment context, since the transgene would be the only logical tag for monitoring unwanted spread and non-target effects of a vaccine virus. Putative recombination events involving genetically modified and naturally occurring viruses should be included in health and environmental risk assessments.

Animals↗
PubMed2004

The cowpox virus host range gene, CP77, affects phosphorylation of eIF2 alpha and vaccinia viral translation in apoptotic HeLa cells.

Host restriction of vaccinia virus has been previously described in CHO and RK13 cells in which a cowpox virus CP77 gene rescues vaccinia virus growth at the viral protein translation level. Here we investigate the restrictive stage of vaccinia virus in HeLa cells using a vaccinia mutant virus (VV-hr) that contains a deletion of 18-kb genome sequences resulting in no growth in HeLa cells. Insertion of CP77 gene into VV-hr generated a recombinant virus (VV-36hr) that multiplied well in HeLa cells. Both viruses could enter cells, initiate viral DNA replication and intermediate gene transcription. However, translation of viral intermediate gene was only detected in cells infected with VV-36hr, indicating that CP77 relieves host restriction at the intermediate gene translation stage in HeLa cells. Caspase-2 and -3 activation was observed in HeLa cells infected with VV-hr coupled with dramatic morphological alterations and cleavage of the translation initiation factor eIF4G. Caspase activation was reduced in HeLa cells infected with VV-36hr, indicating that CP77 acts upstream of caspase activation. Enhanced phosphorylation of PKR and eIF2alpha was also observed in cells infected with VV-hr and was suppressed by CP77. Suppression of eIF4G cleavage with the caspase inhibitor ZVAD did not rescue virus translation, whereas expression of a mutant eIF2alpha protein with an alanine substitution of serine at amino acid position 51 (eIF2alphaS51A) partially restored viral translation and moderately increased virus growth in HeLa cells.

Animals↗
PubMed2005

The cowpox virus fusion regulator proteins SPI-3 and hemagglutinin interact in infected and uninfected cells.

The serpin SPI-3 and the hemagglutinin (HA) encoded by cowpox virus (CPV) block cell-cell fusion, and colocalize at the cell surface. wtCPV does not fuse cells, but inactivation of either gene leads to fusion. SPI-3 mAb added to wtCPV-infected cells caused fusion, confirming that SPI-3 protein at the cell surface prevents fusion. The SPI-3 mAb epitope mapped to an 85-amino acid region at the C-terminus. Removal of either 44 residues from the SPI-3 C-terminus or 48 residues following the N-terminal signal sequence resulted in fusion. Interaction between SPI-3 and HA proteins in infected cells was shown by coimmunoprecipitation. SPI-3/HA was not associated with the A27L "fusion" protein. SPI-3 and HA were able to associate in uninfected cells in the absence of other viral proteins. The HA-binding domain in SPI-3 resided in the C-terminal 229 residues, and did not include helix D, which mediates cofactor interaction in many other serpins.

Amino Acid Sequence↗
PubMed2003

Plasma membrane localization and fusion inhibitory activity of the cowpox virus serpin SPI-3 require a functional signal sequence and the virus encoded hemagglutinin.

The cowpox virus (CPV) glycoprotein serpin SPI-3, a functional protease inhibitor, and the viral hemagglutinin (HA) are required to prevent fusion of wt CPV infected cells. SPI-3 and HA from CPV infected cells co-localize to the plasma membrane and are found in extracellular enveloped virus (EEV). We also show that an N-terminal SPI-3 signal sequence, but not glycosylation, is required for membrane localization and fusion inhibition. In the absence of HA (CPVDeltaHA), no SPI-3 is found on the membrane and infected cells fuse. Conversely, HA from both wt CPV and CPVDeltaSPI-3 infections is on the membrane, indicating a requirement of HA for SPI-3 plasma membrane localization. In the absence of HA, secretion of SPI-3 or SPI-3 N-glyc(-) was markedly enhanced, suggesting HA serves to retain SPI-3 on the plasma membrane,thereby preventing cell fusion.

Animals↗
PubMed1998

The inflammation modulatory protein (IMP) of cowpox virus drastically diminishes the tissue damage by down-regulating cellular infiltration resulting from complement activation.

Vaccinia virus (VV) and other pathogenic poxviruses encode for a complement control protein. The VV complement control protein or VCP, was one of the first soluble microbial proteins postulated to have an active role in the immunomodulation of the host defense. Since then, 2 other poxviruses, including variola virus and cowpox virus (CPV), were found to have corresponding proteins. Based upon earlier studies which demonstrated the role of the CPV complement control protein in modulating the specific tissue responses in BALB/c and congenic-matched C5-sufficient and C5-deficient mice, the CPV equivalent has been renamed the inflammation modulatory protein (IMP), so as to specifically reflect its function. In this study, the in vivo cellular response of mice injected with CPV or a recombinant virus lacking the IMP sequence (CPV-IMP) was examined using a connective tissue air pouch model. Microscopic examination revealed that CPV-IMP caused a significant mononuclear cell infiltration into the connective tissue and adjacent dermal tissue of the skin. To characterize IMP's ability to regulate the observed cellular infiltration through both complement derived and non-complement derived chemotactic factors, footpad and skin connective tissue of C3 knockout mice and footpad of MIP-1alpha knockout mice received injections of CPV and CPV-IMP. In comparison to the matched control, significantly greater footpad specific swelling response was seen in C3 -/- mice injected with CPV. This indicates an important role for C3 in poxvirus pathogenesis. However, MIP-1 alpha -/- mice injected with CPV-IMP recovered earlier than mice injected with CPV alone. This indicates that the function of IMP in vivo in mice with a complete repertoire of immune components is to limit cellular infiltration by down regulating the complement derived chemotactic analphylotoxins, thereby modulating the inflammatory response contributing to a diminished tissue pathology and preservation of viral habitat.

Animals↗
PubMed2006

Differential role played by the MEK/ERK/EGR-1 pathway in orthopoxviruses vaccinia and cowpox biology.

Appropriation of signalling pathways facilitates poxvirus replication. Poxviruses, as do most viruses, try to modify the host cell environment to achieve favourable replication conditions. In the present study, we show that the early growth response 1 gene (egr-1) is one of the host cell factors intensely modulated by the orthopoxviruses VV (vaccinia virus) and CPV (cowpox virus). These viruses stimulated the generation of both egr-1 mRNA and its gene product, throughout their entire replication cycles, via the requirement of MEK [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway. We showed that, upon VV infection, EGR-1 translocates into the nucleus where it binds to the EBS (egr-1-binding site) positioned at the 5' region of EGR-1-regulated genes. In spite of both viruses belonging to the same genus, several lines of evidence, however, revealed a remarkable contrast between them as far as the roles played by the MEK/ERK/EGR-1 pathway in their biological cycles are concerned. Hence (i) the knocking-down of egr-1 by siRNA (small interfering RNA) proved that this transcription factor is of critical relevance for VV biology, since a decrease of about one log cycle in virus yield was verified, along with a small virus plaque phenotype, whereas the gene silencing did not have a detrimental effect on either CPV multiplication or viral plaque size; (ii) while both pharmacological and genetic inhibition of MEK/ERK resulted in a significant decrease in VV yield, both approaches had no impact on CPV multiplication; and (iii) CPV DNA replication was unaffected by pharmacological inhibition of MEK/ERK, but phosphorylation of MEK/ERK was dependent on CPV DNA replication, contrasting with a significant VV DNA inhibition and VV DNA replication-independence to maintain ERK1/2 phosphorylation, observed under the same conditions.

Animals↗
PubMed2000

Cowpox infection: not yet consigned to history.

Two patients presented with hard black eschars covering granulating ulcers. The ulcers healed within 6 weeks to leave small scars. The diagnosis of cowpox was confirmed by serology in both cases and in addition by polymerase chain reaction in the first. The source of infection was likely to be a rodent in the first case whilst the second was more typical in being transmitted by a cat.

Adult↗
PubMed1999

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus.

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-A resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a beta-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a beta-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.

Amino Acid Sequence↗
PubMed1997

The effect of cowpox virus infection on fecundity in bank voles and wood mice.

Although epidemic infectious diseases are a recognized cause of changes in host population dynamics, there is little direct evidence for the effect of endemic infections on populations. Cowpox virus is an orthopoxvirus which is endemic in bank voles (Clethrionomys glareolus), wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis) in Great Britain. It does not cause obvious signs of disease nor does it affect survival, but in this study we demonstrate experimentally that it can reduce the fecundity of bank voles and wood mice by increasing the time to first litter by 20-30 days. The pathogenic mechanisms causing this effect are at present not known, but this finding suggests that natural subclinical infection could have a considerable effect on the dynamics of wild populations.

Animals↗