ABSTRACT
Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.
Subject(s)
Flavivirus , Yellow Fever , Zika Virus Infection , Zika Virus , Animals , Chlorocebus aethiops , Child , Humans , Zika Virus/genetics , Vero Cells , Yellow Fever/genetics , Extracellular Signal-Regulated MAP Kinases , Mitogen-Activated Protein Kinase Kinases , Virus Replication/physiologyABSTRACT
Viruses depend on cells to replicate and can cause considerable damage to their hosts. However, hosts have developed a plethora of antiviral mechanisms to counterattack or prevent viral replication and to maintain homeostasis. Advantageous features are constantly being selected, affecting host-virus interactions and constituting a harsh race for supremacy in nature. Here, we describe a new antiviral mechanism unveiled by the interaction between a giant virus and its amoebal host. Faustovirus mariensis infects Vermamoeba vermiformis, a free-living amoeba, and induces cell lysis to disseminate into the environment. Once infected, the cells release a soluble factor that triggers the encystment of neighbor cells, preventing their infection. Remarkably, infected cells stimulated by the factor encyst and trap the viruses and viral factories inside cyst walls, which are no longer viable and cannot excyst. This unprecedented mechanism illustrates that a plethora of antiviral strategies remains to be discovered in nature.IMPORTANCE Understanding how viruses of microbes interact with its hosts is not only important from a basic scientific point of view but also for a better comprehension of the evolution of life. Studies involving large and giant viruses have revealed original and outstanding mechanisms concerning virus-host relationships. Here, we report a mechanism developed by Vermamoeba vermiformis, a free-living amoeba, to reduce Faustovirus mariensis dissemination. Once infected, V. vermiformis cells release a factor that induces the encystment of neighbor cells, preventing infection of further cells and/or trapping the viruses and viral factories inside the cyst walls. This phenomenon reinforces the need for more studies regarding large/giant viruses and their hosts.
Subject(s)
Amoebozoa/virology , Giant Viruses/physiology , Virus Replication/physiology , Viruses, Unclassified/physiologyABSTRACT
Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus.IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle.
Subject(s)
Acanthamoeba castellanii/virology , Mimiviridae/physiology , Virus Internalization , Virus Replication/physiology , Virus Uncoating/physiology , Acanthamoeba castellanii/metabolism , PhagocytosisABSTRACT
Viruses display a wide range of genomic profiles and, consequently, a variety of gene expression strategies. Specific sequences associated with transcriptional processes have been described in viruses, and putative promoter motifs have been elucidated for some nucleocytoplasmic large DNA viruses (NCLDV). Among NCLDV, the Marseilleviridae is a well-recognized family because of its genomic mosaicism. The marseilleviruses have an ability to incorporate foreign genes, especially from sympatric organisms inhabiting Acanthamoeba, its main known host. Here, we identified for the first time an eight-nucleotide A/T-rich promoter sequence (AAATATTT) associated with 55% of marseillevirus genes that is conserved in all marseilleviruses lineages, a higher level of conservation than that of any giant virus described to date. We instigated our prediction about the promoter motif by biological assays and by evaluating how single mutations in this octamer can impact gene expression. The investigation of sequences that regulate the expression of genes relative to lateral transfer revealed that the promoter motifs do not appear to be incorporated by marseilleviruses from donor organisms. Indeed, analyses of the intergenic regions that regulate lateral gene transfer-related genes have revealed an independent origin of the marseillevirus intergenic regions that does not match gene-donor organisms. About 50% of AAATATTT motifs spread throughout intergenic regions of the marseilleviruses are present as multiple copies. We believe that such multiple motifs are associated with increased expression of a given gene or are related to incorporation of foreign genes into the mosaic genome of marseilleviruses.IMPORTANCE The marseilleviruses draw attention because of the peculiar features of their genomes; however, little is known about their gene expression patterns or the factors that regulate those expression patterns. The limited published research on the expression patterns of the marseilleviruses and their unique genomes has led us to study the promoter motif sequences in the intergenic regions of the marseilleviruses. This work is the first to analyze promoter sequences in the genomes of the marseilleviruses. We also suggest a strong capacity to acquire foreign genes and to express those genes mediated by multiple copies of the promoter motifs available in intergenic regions. These findings contribute to an understanding of genomic expansion and plasticity observed in these giant viruses.
Subject(s)
Acanthamoeba/virology , DNA Viruses/genetics , DNA, Intergenic , Genome, Viral , Nucleotide Motifs , Promoter Regions, Genetic/genetics , Base Sequence , Computational Biology , DNA Viruses/pathogenicity , DNA, Viral , Genomics , PhylogenyABSTRACT
We investigated possible vaccinia virus (VACV) in urban house cats in Brazil. Serum samples from 6 cats were positive for VACV by PCR, indicating likely VACV circulation among house cats in urban areas of Brazil. This finding highlights the importance of epidemiologic surveillance to avoid outbreaks among urban human populations.
Subject(s)
Cat Diseases/epidemiology , Cat Diseases/virology , Urban Health , Vaccinia virus , Vaccinia/veterinary , Animals , Animals, Domestic , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Brazil/epidemiology , Cat Diseases/immunology , Cats , Phylogeny , Public Health Surveillance , Vaccinia virus/classification , Vaccinia virus/genetics , Vaccinia virus/immunology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunologyABSTRACT
Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain.
Subject(s)
Antibodies, Viral/blood , Cattle Diseases/epidemiology , DNA, Viral/blood , Disease Outbreaks , Marsupialia/virology , Rodentia/virology , Vaccinia/epidemiology , Animals , Brazil/epidemiology , Cattle , Cattle Diseases/blood , Cattle Diseases/transmission , Disease Reservoirs/virology , Incidence , Molecular Typing , Vaccinia/blood , Vaccinia/transmission , Vaccinia/veterinary , Vaccinia virus/classification , Vaccinia virus/genetics , Vaccinia virus/pathogenicityABSTRACT
UNLABELLED: Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an â¼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. IMPORTANCE: Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of â¼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.
Subject(s)
Acanthamoeba/virology , Cytoplasmic Vesicles/virology , Giant Viruses/physiology , Virus Internalization , Animals , Capsid/chemistry , Capsid/metabolism , Capsid Proteins/genetics , Cytoplasmic Vesicles/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Genome, Viral , Giant Viruses/ultrastructure , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Phagocytosis , Phylogeny , Virion/genetics , Virion/physiology , Virion/ultrastructure , Virus ReplicationABSTRACT
We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out.
Subject(s)
Cattle Diseases/epidemiology , Cattle Diseases/virology , Vaccinia virus/genetics , Vaccinia/veterinary , Animals , Cattle , Disease Outbreaks , Genes, Viral , Geography, Medical , RNA, Viral , South America/epidemiology , Uruguay/epidemiology , Vaccinia virus/classification , Vaccinia virus/isolation & purification , ZoonosesABSTRACT
Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.
Subject(s)
Acanthamoeba/enzymology , Acanthamoeba/virology , Host-Parasite Interactions , Mimiviridae/growth & development , Serine Proteases/biosynthesis , Spores, Protozoan/growth & development , Acanthamoeba/growth & developmentABSTRACT
UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the â¼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.
Subject(s)
Acanthamoeba/virology , Glycoproteins/metabolism , Mimiviridae/physiology , Viral Proteins/metabolism , Virus Attachment , Acanthamoeba/physiology , Acanthamoeba/ultrastructure , Acetylglucosamine/metabolism , Analysis of Variance , Mannose/metabolism , Microscopy, Electron, Transmission , Species Specificity , Virus Replication/physiologyABSTRACT
BACKGROUND: Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus, a virus from Orthopoxvirus genus (OPV) that affects mainly cattle herds and humans in rural areas in Brazil. Because most studies have focused on outbreaks situations, data on BV epidemiology is limited. A cross sectional study in Brazilian rural areas during 2012-2013 was conducted to determine the neutralizing antibodies seroprevalence and risk factors for BV. METHODS: A structured questionnaire was applied to elicit demographics data and farming practices considered risk factors for BV exposure. Neutralizing anti-OPV antibodies were investigated using plaque reduction neutralization test. The neutralizing antibodies prevalence rates were calculated and the risk factor analysis was performed using multivariate logistic regression. RESULTS: Two hundred and forty participants were enrolled in this study with a prevalence of neutralizing antibodies of 30.8 % (95 % confidence interval [CI], 25.3-36.9). In multivariate analysis, age > 35 years (Odds Ratio [OR] = 18.2; CI 95 % = 7.7 - 43.2) and previous outbreak in property (OR = 3.9; C I95 % = 1.2 - 12.6) were independently associated with anti-OPV neutralizing antibodies. CONCLUSIONS: In this study, anti-OPV protective immunity (neutralizing antibody titers) was assessed in an endemic BV Brazilian rural area. Our findings indicate that epidemiological surveillance is required and should be applied by public health authorities to create interventions and/or prevention strategies to avoid viral spread causing future outbreaks among individuals who are under risk of infection.
Subject(s)
Antibodies, Viral/blood , Orthopoxvirus/immunology , Poxviridae Infections/blood , Zoonoses/blood , Adolescent , Adult , Aged , Aged, 80 and over , Agricultural Workers' Diseases/blood , Agricultural Workers' Diseases/immunology , Agricultural Workers' Diseases/virology , Animals , Antibodies, Neutralizing/blood , Brazil/epidemiology , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Livestock/virology , Male , Middle Aged , Orthopoxvirus/genetics , Orthopoxvirus/isolation & purification , Poxviridae Infections/immunology , Poxviridae Infections/virology , Rural Population , Seroepidemiologic Studies , Young Adult , Zoonoses/epidemiology , Zoonoses/immunology , Zoonoses/virologyABSTRACT
We enrolled 21 patients with laboratory-confirmed yellow fever (YF), hospitalized at Eduardo de Menezes Hospital, Brazil, to be treated with sofosbuvir, a drug approved for hepatitis C. Given the absence of specific YF antiviral treatments, the off-label nonrandomized sofosbuvir treatment aimed to address high disease severity and the risk of fatal outcomes. Patients received a daily dose of 400â mg sofosbuvir from 4 to 10 days post-symptom onset. YF viral load (VL) comparisons were made between treated and nontreated patients who either survived or died. The genomic VL for the treated group steadily decreased after day 7 post-symptom onset, suggesting that sofosbuvir might reduce YF VL. This study underscores the urgent need for YF antiviral therapies, advocating for randomized clinical trials to further explore sofosbuvir's role in YF treatment.
ABSTRACT
Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.
Subject(s)
Cowpox virus/physiology , MAP Kinase Signaling System/physiology , Signal Transduction/physiology , Vaccinia virus/physiology , Virus Replication/physiology , rac1 GTP-Binding Protein/physiology , Animals , BALB 3T3 Cells , Chlorocebus aethiops , Mice , Phosphorylation/physiology , Vero Cells , rac1 GTP-Binding Protein/metabolismABSTRACT
In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil.
Subject(s)
Cattle Diseases/epidemiology , Cattle Diseases/virology , Disease Outbreaks/veterinary , Vaccinia virus/isolation & purification , Vaccinia/veterinary , Animals , Brazil/epidemiology , Cattle , Cell Line , Hemagglutinins, Viral/genetics , Humans , Mice , Phylogeny , Vaccinia/epidemiology , Vaccinia/virology , Vaccinia virus/classification , Vaccinia virus/geneticsABSTRACT
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.
Subject(s)
Cowpox virus/pathogenicity , Cowpox/pathology , Cowpox/virology , Inclusion Bodies/virology , Animals , Chlorocebus aethiops , Cowpox/genetics , Disease Models, Animal , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Sequence Deletion , Vero Cells , Viral Plaque AssayABSTRACT
Apeu virus (APEUV) (family Bunyaviridae, genus Orthobunyavirus) was plaque purified and characterised by serological and molecular analysis. Neutralising assays confirmed cross-reactivity between purified APEUV clones and the Caraparu virus complex of group C orthobunyaviruses. Partial sequencing of the L, M and S segments of one APEUV clone (APEUV-CL5) was carried out. A phylogenetic tree constructed with the L amino acid sequences clustered APEUV-CL5 within the genus Orthobunyavirus, confirming its serological classification. Analysis of M segment sequences clustered APEUV-CL5 in the Caraparu virus complex (Group C), in agreement with serological tests and previous molecular characterisation. However, the sequence of the nucleocapsid gene (N) gave low identity values when compared to those of the group C viruses. The phylogenetic tree based on N nucleotide sequences clustered APEUV-CL5 next to the California and Bwamba groups. This remarkable S nucleotide variability suggests that APEUV-CL5 could be a genetic reassortant and that this evolutionary mechanism is present in the history of the group C viruses.
Subject(s)
Bunyaviridae Infections/virology , Orthobunyavirus/classification , Orthobunyavirus/isolation & purification , Phylogeny , Animals , Antibodies, Viral/immunology , Cell Line , Humans , Mice , Molecular Sequence Data , Orthobunyavirus/genetics , Orthobunyavirus/immunologyABSTRACT
Orthopoxvirus (OPV) has been associated with worldwide exanthematic outbreaks, which have resulted in serious economic losses as well as impact on public health. Although the current classical and molecular methods are useful for the diagnosis of OPV, they are largely inaccessible to unsophisticated clinical laboratories. The major reason for the inaccessibility is that they require both virus isolation and DNA manipulation. In this report, a rapid, sensitive and low-cost semi-nested PCR method is described for the detection of OPV DNA directly from clinical specimens. A set of primers was designed to amplify the conserved OPV vgf gene. The most useful thermal and chemical conditions were selected and minimum non-inhibitory dilutions were determined. More than 100 Brazilian Vaccinia virus (VACV) field clinical specimens were tested using this semi-nested PCR in order to confirm its applicability. Cowpox virus was also detected by PCR from the ear scabs of scarified Balb/c mice. In addition, the method was highly sensitive for the detection of VACV DNA in murine blood and excreta, which are among the suggested reservoirs of OPV. Together, these data suggest that semi-nested PCR can be used for initial screening for OPV and as a routine diagnostic laboratory method.