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Cowpox: a re-evaluation of the risks of human cowpox based on new epidemiological information.

Human cowpox is a rare but relatively severe infection of interest because of its links with Edward Jenner and the introduction of smallpox vaccine and, more recently, because of re-evaluation of the epidemiology of the infection. This indicates that cowpox is not enzootic in cattle, relegates the cow to a minor role, and emphasizes the importance of feline cowpox as a source of human infection and of wildlife as virus reservoirs. The evidence available suggests that the virus is of low infectivity for humans and should not become an increasing problem despite the cessation of smallpox vaccination and increasing numbers of immunocompromised individuals.

Animals

Effect of double infection of cowpox virus-infected cells with paramyxovirus (Sendai virus) on formation of cowpox virus-specific cell surface antigen.

The formation of cowpox virus-specific cell surface antigen (CPV S-ag) was significantly enhanced by double infection with HVJ (Sendai virus). Simultaneous double infection, superinfection with HVJ and superinfection with CPV of cells persistently infected with HVJ similarly enhanced the formation of CPV S-ag, while pre-infection with HVJ was ineffective. To be effective, cells must be infected at a m.o.i. of greater than or equal to 1.0 and HVJ gene functions had to be expressed. The HVJ-infected cell extracts had an ability to accelerate uncoating (or degradation) of CPV, causing an early increase and a subsequent decrease in the infectivity of CPV. This activity reached a maximum 4--6 hr after HVJ infection, the increase paralleling enhancement of the total activity of several cellular enzymes. Addition of puromycin abolished the increase of these activities and the formation of CPV S-ag. Thus, the double infection with HVJ of CPV-infected cells induces an enhancement of CPV S-ag formation presumably as a consequence of activation of cellular enzymes which in turn accelerates uncoating of CPV.

Antigens, Surface

Inflammatory responses and the generation of chemoattractant activity in cowpox virus-infected tissues.

Histological examination of the lesions produced on the chick chorioallantois infected with cowpox virus shows extensive haemorrhage but there are few inflammatory cells. However, infection with a deletion mutant, white cowpox virus, results in little haemorrhage but there is massive polymophonuclear cell infiltration. Extracts from tissues infected with the parental, red cowpox virus contained little or no chemoattractant activity detectable in micropore filter assays. However, white cowpox virus-infected tissue extracts, including cellular extracts from infected tissue cultures, had a marked effect in vitro on the migration of both neutrophils and monocytes. The chemoattractant activity induced in ovo by white cowpox virus infection has sedimentation characteristics in sucrose density gradients that are similar to leukotactic factors shown previously to be present in the allantoic fluid of chick embryos infected with paramyxoviruses. Induction of chemoattractant activity did not occur after infection of chick chorioallantois with a recombinant white cowpox virus constructed to express a protein that is responsible for the haemorrhagic character of red cowpox virus. This gene product has been shown previously to have homology with various serine protease inhibitors. The significance of these studies to the immunogenicity and pathogenicity of vaccinia recombinant viruses is discussed.

Allantois

Characteristics of four cowpox virus isolates from Norway and Sweden.

We report the first isolation of cowpox virus from a domestic cat in Norway, and the first confirmed isolation of cowpox virus from a human case in Norway. These two Norwegian cowpox virus isolates, as well as two Swedish human isolates, were partially characterized and compared with each other and with cowpox virus Brighton and vaccinia virus strain Western Reserve. Restriction enzyme analysis of the genomes revealed differences between all six viruses examined, but suggested that the two Norwegian isolates are closely related, as are the two Swedish isolates. Restriction endonuclease digestion of genomic DNA demonstrated that one of the Swedish isolates and the two Norwegian isolates have larger genomes than vaccinia virus strain Western Reserve, but smaller than cowpox Brighton. All four Scandinavian isolates lacked a 72 base-pair region within the A-type inclusion body protein gene which is present in the prototype cowpox virus Brighton.

Adolescent

Studies on the polypeptides of poxvirus. II. Comparison of virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses.

Virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses were examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. At least 42 vaccinia virus-induced polypeptides were identified among the polypeptides of cells pulse-labeled with [35S]-methionine and/or of fractionated cells labeled with [14C]-leucine for 24 hr. They consisted of 15 polypeptides (early polypeptides) which were synthesized even in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl, and 27 polypeptides (late polypeptides) which were synthesized only in the absence of cytosine-1-beta-D-arabinofuranosyl-HCl. By the same procedure at least 40 cowpox virus-induced polypeptides (14 early polypeptides and 26 late polypeptides) and at least 31 Shope fibroma virus-induced polypeptides (13 early polypeptides and 18 late polypeptides) were identified. Comparative studies of virus-induced polypeptides on the basis of migration in SDS-polyacrylamide gel electrophoresis revealed that 11 polypeptides were early polypeptides common to both vaccinia and cowpox viruses; 21 were late polypeptides common to both vaccinia and cowpox viruses; 4 were early polypeptides common to both vaccinia and Shope fibroma viruses; 7 were late polypeptides common to both vaccinia and Shope fibroma viruses; 5 were early polypeptides common to both cowpox and Shope fibroma viruses; 9 were late polypeptides common to both cowpox and Shope fibroma viruses; 4 were early polypeptides common to all three viruses; and 7 were late polypeptides common to all three viruses.

Cell Line

The mode of death of pig kidney cells infected with cowpox virus is governed by the expression of the crmA gene.

Pig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing the crmA gene; recombinant cowpox virus A602, lacking the crmA gene; or cowpox virus A604, a revertant of virus A602, expressing the crmA gene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that the crmA gene is necessary to inhibit processes of apoptosis induced in LLC-PK1 cells by infection with cowpox virus. Thus in cells of certain types, the crmA gene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replication in vivo, potentially facilitating both virus trafficking and interference with antiviral immune defenses.

Animals

Comparison of thymidine kinase and A-type inclusion protein gene sequences from Norwegian and Swedish cowpox virus isolates.

During the last decades, cowpox virus, a member of the genus Orthopoxvirus within the Poxviridae family, has appeared as a pathogen in domestic cats, zoo animal species, and humans. At the same time, vaccinia virus, another orthopoxvirus, has been used as a recombinant vaccine vector with foreign genes inserted in the thymidine kinase (TK) gene. By PCR and cycle sequencing, we have determined the nucleotide sequences of the TK gene and the A-type inclusion protein (ATIP) gene of virus isolates from two human cowpox cases in Sweden, as well as a human and a feline case from Norway. We also obtained the corresponding sequences from ectromelia virus (strain Moscow), cowpox virus (strain Brighton) and vaccinia virus (strain Western Reserve). The new virus isolates differed from ectromelia virus and vaccinia virus, and were confirmed to be cowpox virus strains. Isolates originating from the same country had nearly identical TK sequences and fully identical ATIP sequences. They probably represent local geographical strains of cowpox virus.

Animals

The existence of an envelope on extracellular cowpox virus and its antigenic relationship to the vaccinia envelope.

Virus released from cowpox infected cells was demonstrated by electron-microscopy to be surrounded by an envelope not present on mature intracellular virus. Enveloped cowpox had an isopycnic density of 1.23 g/ml, was infectious and neutralized by antibodies specific for the envelope antigens of vaccinia virus but not neutralized by antibodies specific for intracellular naked vaccinia virus. Only 1 per cent of the total intracellular virus yield was released as enveloped virus. The major cowpox glycoprotein (76 K) and a 44 K glycoprotein did not comigrate with any vaccinia glycoproteins whereas cowpox glycoproteins at 42 K and 20-23 K did coelectrophores with vaccinia glycoproteins. All of the mentioned cowpox glycoproteins were precipitated by vaccinia antiserum.

Animals

A comparison of the antigens present on the surface of virus released artificially from chick cells infected with vaccinia virus, and cowpox virus and its white pock mutant.

Antisera prepared against vaccinia and cowpox viruses were absorbed with purified suspensions of vaccinia virus, red cowpox and white cowpox viruses. They were then tested for their ability to neutralize the viruses, and to precipitate the virus soluble antigens.The results showed that some virus specific antigens were not virus surface components and that some components were present on the surface of all three viruses. However, certain components were detected on the surface of vaccinia virus but not on the surface of cowpox virus, and vice versa. Some evidence for the existence of a vaccinia-specific surface component was also obtained.Comparisons between results of cross-neutralization tests and immunodiffusion tests on the absorbed sera indicated that antibody to a number of antigens, including the classical LS, and the cowpox-specific d antigen play no part in the process of poxvirus neutralization.

Animals

Restriction endonuclease analysis of red cowpox virus and its white pock variant.

The DNA of red cowpox virus strain Brighton or its white pock variant was analysed by cleavage with restriction endonucleases HindIII, XhoI, PstI or KpnI. Physical maps were constructed and the genomes compared with that of vaccinia virus strain DIE. The size of the red cowpox genome is 23 to 29 megadaltons greater than that of vaccinia and results from the presence of additional, near terminal sequences. An internal region of about 75 megadaltons appears to be highly conserved between the two viruses. The red cowpox genome contains near terminal, repetitive sequences which have some homology with those of vaccinia virus DNA. Rapid renaturation of red cowpox terminal restriction fragments indicates that these are covalently cross-linked. Viable white pock variants arise continually and map as deletion mutants lacking similar sequences from one specific terminus only of the parental genome. The deletion represents 11 to 12% of the red cowpox DNA and includes the terminal repetition which therefore is not required for replication. The deleted terminus of the white pock variant genome does not appear to be cross-linked.

Base Sequence

A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.

Amino Acid Sequence

[Jenner's cowpox vaccine in light of current vaccinology].

Two hundred years ago Edward Jenner inoculated James Phipps with vaccinia and 181 years later smallpox had disappeared from the surface of the earth as a result of generalized vaccination. Compared to the requirements of modern vaccinology, the procedures used by Jenner and his successors, were extremely primitive because of an almost total lack of knowledge in the field of microbiology and immunology. The active principle of smallpox vaccine is vaccinia virus, which in many respects, differs from that of natural cowpox; the term "cowpox" has been used for more than a century and a half to designate the vaccine; it appears itself to be a misnomer, because it is most probably by a virus of rodents, which only occasionally infects bovines or other species, especially cats. The origin of vaccinia remains doubtful, but a plausible explanation is that it is derived from horse-pox. Jenner was convinced that he was working with a virus of equine origin, which was occasionally transmitted from the horse to the cow by the personnel on the farms. Horse pox has now completely disappeared. Especially during the first years after Jenner's discovery, great confusion was caused by other lesions on the cow's udder, which were called "spurious cowpox". We know today that these lesions could be caused by the viruses of papular stomatitis, pseudo-cowpox or para-vaccinia (milker's nodules), herpes mammilitis and papillomatosis; they could not be differentiated from those of cowpox or vaccinia, in addition lesions due to bacteria or other causes also led to confusion. During the first eighty years the vaccine was being transferred almost exclusively from arm to arm with the risks inherent in this procedure; one of the reasons for applying this method was the fear of "bestialization" thought to be linked with the use of material of animal origin. Several contaminations have been observed as a result of the use of the arm-to-arm procedure: smallpox was transmitted, especially in the beginning, because vaccinations were carried out in a contaminated environment. Syphilis was diagnosed in several countries after the use of vaccine taken from syphilis patients. At least two foci of hepatitis were reported after the use of contaminated human lymph. Transmission of tuberculosis or what was then designated as scrofulosis was unlikely, but was used as one of the main arguments against vaccination by the antivaccinists. Varicella and measles were transmitted from time to time with the vaccine and also bacterial infections, such as staphylococci, streptococci e.a. From the global point of view, however, the number of contaminations remained limited in comparison with the large numbers of vaccinations that were performed. Another problem the early vaccinators were facing, was that of the decline and disappearance of the immunity after a certain number of years. Jenner and his successors believed that the immunity post vaccination would be lifelong as it was after variolation. When in the early part of the 19th century more and more immunity breakdowns occurred, this observation led to total confusion and it took dozens of years of debate and controversy before the only logical and efficacious measure, i.e. revaccination, was generally accepted and implemented. In the last third of the 19th century "human lymph", obtained by arm-to-arm vaccination, was gradually replaced everywhere by animal lymph i.e. vaccine produced on the skin of animals, mainly calves. The determining factor in the switch was the risk of vaccination syphilis. Everywhere vaccine institutes were created, where the vaccinia virus was propagated on the skin of calves. The harvested virus served each time for the inoculation of fresh calves; this resulted in a gradual increase of the number of passages leading to the possible risk of overattenuation. To avoid this risk, passages in man, donkeys, rabbits or other species were performed from time to time.

History, 19th Century

Cowpox in British voles and mice.

Serosurveys indicate that bank voles, field voles and woodmice are probably reservoir hosts of cowpox virus in western Europe, although virus has not yet been isolated from these species. In this study, bank voles, field voles, woodmice and laboratory mice were shown to be susceptible to combined intradermal and subcutaneous inoculation with 3-20 plaque-forming units (pfu) of cowpox virus. Bank and field voles, but not laboratory mice, were also susceptible to combined oral and nasal inoculation with 50 pfu. Few clinical signs were seen and virus was generally recovered only from inoculation sites. Bank voles were not susceptible to injection of ectromelia virus (5000 pfu) into the skin (as described above). These results provide information on which further pathogenesis and transmission studies can be based, and support the view that the orthopoxvirus antibody detected in British wild voles and woodmice indicates infection with cowpox virus. However, further investigation of the pathogenesis of cowpox in these species is needed to understand better the epidemiology of the disease.

Administration, Intranasal

A ligase chain reaction targeting two adjacent nucleotides allows the differentiation of cowpox virus from other Orthopoxvirus species.

A ligase chain reaction (LCR) assay was developed to distinguish cowpox virus from other Orthopoxvirus species. The LCR targets two adjacent adenosine residues which are only present in the A-type inclusion protein gene (ATI-gene) of cowpox virus. Two primer pairs were designed with a one base pair overlap at the junction site and one primer of each pair was labeled radioactively. Detection of the ligation product was achieved after denaturing polyacrylamide gel electrophoresis and autoradiography. Prior to LCR, the corresponding region of the ATI-gene was amplified by a consensus primer-directed polymerase chain reaction. All 18 cowpox virus isolates investigated could be clearly discriminated from 10 vaccinia virus strains, 5 camelpox virus isolates, as well as from mousepox and monkeypox virus reference strains. The LCR method allows a fast identification of cowpox virus isolates and is a feasible tool for the analysis of small mutations within viral genes.

Animals

Clinical cowpox cases in Norway.

In 1994, a human and a feline case of cowpox virus infection appeared in the western part of Norway. Cowpox has not been diagnosed with certainty in Norway since the beginning of this century, when it was associated with the use of cowpox virus as a vaccine against smallpox. The human infection manifested as a spontaneously emerged, severe ulceration at the medial angle of the right eye in a 37-y-old woman, and developed into a relatively severe dermatitis. The ulcer healed slowly, leaving a scar. The feline infection was represented by a febrile, dehydrated and anorectic 6-months-old non-pedigree short-hair, with crater-like ulcers all over the body. After antibiotic and fluid therapy, revision of the skin lesions and amputation of a gangrenous toe, the cat recovered. Electron microscopy of the isolates and cultivation of virus on chorioallantoic membrane of chicken embryos confirmed the suspicion of cowpox virus infection.

Adult

Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity.

Soluble receptors for gamma interferon (IFN-gamma) are secreted from cells infected by 17 orthopoxviruses, including vaccinia, cowpox, rabbitpox, buffalopox, elephantpox, and camelpox viruses, representing three species (vaccinia, cowpox, and campelpox viruses). The B8R open reading frame of vaccinia virus strain Western Reserve, which has sequence similarity to the extracellular binding domain of cellular IFN-gamma receptors (IFN-gamma Rs), is shown to encode an IFN-gamma binding activity by expression in recombinant baculovirus. The soluble virus IFN-gamma Rs bind IFN-gamma and, by preventing its interaction with the cellular receptor, interfere with the antiviral effects induced by this cytokine. Interestingly, in contrast to cellular IFN-gamma Rs, which are highly species specific, the vaccinia, cowpox, and camelpox virus IFN-gamma Rs bind and inhibit the biological activity of human, bovine, and rat IFN-gamma but not mouse IFN-gamma. This unique broad species specificity of the IFN-gamma R would aid virus replication in different species and suggests that vaccinia, cowpox, and camelpox viruses may have evolved in several species, possibly including humans but excluding mice. Last, the conservation of an IFN-gamma R in orthopoxviruses emphasizes the importance of IFN-gamma in defense against poxvirus infections.

Cell Line

Is cowpox misnamed? A review of 10 human cases.

Twelve separate outbreaks of confirmed cowpox, 10 involving humans, were reviewed. Six of the patients, including three children, had severe infections and five were admitted to hospital. In three outbreaks both people and cows were affected but it was not known how the infections entered the herds. In seven outbreaks no direct contact with cattle was established and clinical and serological examination failed to show evidence of cowpox in the bovine population. Comparison of these data with information about infections known to be enzootic in cattle leads to the suggestion that cows are not the natural reservoir of cowpox. This should be remembered when diagnosis is considered. The role of small wild animals as hosts and vectors of "cowpox" should be investigated.

Animals