Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Comparison of the genome DNA sequences of Bangladesh-1975 and India-1967 variola viruses.

The nucleotide sequences of genome DNAs and the deduced amino acid sequences of proteins from potential open reading frames (ORFs) of variola smallpox viruses from outbreaks in India in 1967 and in Bangladesh in 1975 have been compared and the analyses of the sequences are updated. Alignment of the DNAs revealed 99.3% base sequence identity. Of the 200 potential encoded proteins of each virus, 122 were identical, 42 showed substitution of a single amino acid, 11 showed two residues changes, and the remainder were more diverged. The variant proteins were encoded mainly in the near-terminal regions of each genome. The most conserved region between the variola DNAs included ORFs A33L to A49R, which is a relatively poorly conserved region compared with vaccinia virus.

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses.

Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.

Specific detection of monkeypox virus by polymerase chain reaction.

The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72-95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences.

Human monkeypox in Kasai Oriental, Zaire (1996-1997).

Monkeypox is an orthopoxvirus with enzootic circulation in the rainforests of central and western Africa; the virus can be transmitted to humans and cause a syndrome clinically similar to smallpox (e.g., pustular rash, fever, respiratory symptoms, and in

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.

Formulation and evaluation of baits for oral rabies vaccination of raccoons (Procyon lotor).

Captive raccoons were offered a variety of vaccine containers and bait components in a series of three-choice tests. Paraffin wax ampules were the most readily accepted vaccine container. Preferred bait components included corn and shellfish oils, deep fried corn meal batter, and egg, apple and buttermilk flavorings. These results, together with factors including ease of bait formulation, cost, and suitability for field use, were used to develop an experimental delivery system for an oral rabies vaccine. The developed system was composed of a polyurethane sleeve (1.5 x 5.5 cm) dipped in a commercial food batter mix together with corn meal, milk and egg. The sleeve was deep fried in corn oil and a 2.0 ml ampule containing a recombinant rabies vaccine was then inserted into the sleeve bait. These baits were presented to 10 captive raccoons. Nine of the 10 animals developed high levels of rabies virus neutralizing antibodies. Field tests are needed to determine if the delivery system developed also is effective for wild raccoons.

[First appearance of monkey pox in human beings in Gabon]. / Première apparition au Gabon de monkey-pox chez l'homme.

The authors report on the first coming out in Gabon of human monkey-pox. Four children in the same family were simultaneously attacked. In one of the two fatal cases, diagnosis of monkey-pox was confirmed by the isolation of the virus. In the three other cases, diagnosis was based on clinical and epidemiological findings. The coming out of monkey-pox in Gabon squares with sporadic coming out of this zoonosis observed in the humid tropical forests of west and Central Africa. Two fatal cases presented some symptoms of hemorrhagic fever and cutaneous symptoms did not dominate the clinical feature. As a consequence of it, monkey-pox has to be envisaged in similar cases. The attack of liver and spleen in man has not been documented up till now, as it has been for monkeys. Classical diagnosis is based on the isolation of the virus from a cutaneous lesion. In the present case, the virus was isolated from a blood sample. Epidemiological study did not reveal any interhuman transmission evidence, nor the source of infection.

Detection of poliovirus antigens and antibodies: microindirect haemagglutination and haemagglutination inhibition tests for poliovirus types I, II, and III.

Microtitre indirect haemagglutination (IHA) and IHA-inhibition (IHAI) procedures were adapted to determine the reactivities of type I, II, and III poliovirus antibodies and antigens. Glutaraldehyde-fixed sheep erythrocytes were sensitized for these tests with concentrated, partially purified preparations of type I, II, and III poliovirus. Antibody titres by IHA were generally 10 to 100 times greater than serum microneutralization (SN) titres. The SN and IHA reactivities of three kinds of sera were compared. Of these sera, virus type specific antibodies, in monospecific guinea pig sera one week after immunization and in sera from hyperimmunized horses, could be readily differentiated and measured; antibodies in human diagnostic specimens, however, showed some intertypic cross reactivity. Monovalent one-week immune guinea pig sera reacted specifically in the IHAI test to differentiate viruses, and could be used for virus typing and differentiating strains of poliovirus type III.

Orthopoxvirus DNA: a comparison of restriction profiles and maps.

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAs of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.

Sequences of the raccoon poxvirus hemagglutinin protein.

Primers based on sequences flanking the vaccinia virus (VV) strain IHD hemagglutinin protein (HA) open reading frame (ORF) enabled amplification of HA DNA segments from the genome of raccoon poxvirus (RCN) and VV strain WR. The amplified segments produced unequal cross-hybridization signal intensities against each other, indicating sequence differences between the HA of RCN (in HindIII-G) and that of VV-WR (in HindIII-A). About 1.5 kb of sequences in the HA region were then determined from clones pRCN/HindIII-G and pVV/BamHI-32, a subclone of VV-WR HindIII-A. Pairwise analyses of the RCN and VV-WR HA nucleotide sequences showed 82, 66, and 86% homology, respectively, between the putative promoter, ORF, and transcript terminator regions and 53% homology between the deduced amino acid sequences of the HA of RCN (310 residues) and those of VV-WR (314 residues). The deduced HA amino acid sequences showed a putative signal peptide and transmembrane-anchor moiety of 70 and 62% homology and a rather distinct central domain (residues 146 to 254) of 32% homology. Additional hybridizations with the amplified segments described above enabled location of the HA gene in the HindIII-A fragment of the orthopoxviruses volepox virus (VPX) and skunk poxvirus (SKP); however, amplified DNA of either the entire HA ORF of RCN or that of VV-WR, or a portion, from the center to right end, did not hybridize with VPX or SKP, suggesting that the HA of RCN, VV, VPX, and SKP are rather diverged from each other. The VV HA was found to be closely related to that of ectromelia and variola viruses. The data are consistent with reports of hemagglutination-inhibition partial cross-reactivity between RCN, VPX, VV, and other orthopoxviruses and might lead to an explanation of the basis of syncytia formation by RCN, VPX, and SKP.

High resolution polyacrylamide gradient gel electrophoresis.

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e.g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%) and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickettsii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.

Enterovirus type 70 virion and intracellular proteins.

The proteins of purified enterovirus type 70 grown in rhabdomyosarcoma cells and the intracellular proteins at 4 to 5 h after infection have been examined by polyacrylamide gel electrophoresis. Virions contained four proteins: P-1 (35,000, daltons) P-2 (28,000, daltons) P-3 (27,000, daltons) and P-4 (9,000 daltons). Further, addition of ZnCl2 to infected cultures inhibited virus plaque development and interfered with post-translational cleavage.

Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses.

Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.

Gene homology between orf virus and smallpox variola virus.

About 47% identity was observed between the deduced amino acid sequences of a protein encoded by a gene of the parapoxvirus orf virus (OV) strain NZ2 and a 6 kDa protein of unknown function reported to be produced by an open reading frame expressed early after infection by the orthopoxvirus Western Reserve vaccinia virus (VAC); the open reading frame is absent from VAC strain Copenhagen. Examination of sequences reported for variola virus (VAR) strains Bangladesh, India, Congo- 1970, Somalia- 1977 and Garcia- 1966 revealed each encoded a correlate 58 amino acid protein. The open reading frame was not reported in the original analyses of these sequences because a lower limit of 60 amino acids was used to identify potential encoded proteins. Inspection of partial reading frames reported for cowpox virus (CWV) and ectromelia virus (EMV) suggested that these viruses might also code for a correlate of the VAC WR protein. DNA sequencing of cloned fragments of CWV and EMV confirmed that both these orthopoxviruses encode closely related, full length variants of the VAC and VAR open reading frames. The OV homologue is coded in the OV strain NZ2 BamHI-E fragment E2L open reading frame, which we reported is transcribed early postinfection; moreover, analysis of an NZ2 variant showed E2L was absent, indicating that E2L, like the VAC cognate, is nonessential for virus replication in cell culture. The parapoxvirus and orthopoxvirus correlates have about 20% amino acid sequence resemblance to African swine fever virus DNA binding protein p10, suggesting an ancestral relation of genes.

Raccoon poxvirus feline panleukopenia virus VP2 recombinant protects cats against FPV challenge.

An infectious raccoon poxvirus (RCNV) was used to express the feline panleukopenia virus (FPV) open reading frame VP2. The recombinant, RCNV/FPV, was constructed by homologous recombination with a chimeric plasmid for inserting the expression cassette into the thymidine kinase (TK) locus of RCNV. Expression of the VP2 DNA was regulated by the vaccinia virus late promoter P11. Southern blot and polymerase chain reaction (PCR) analyses confirmed the cassette was in the TK gene of the RCNV genome. An immunofluorescent antibody assay using feline anti-FPV polyclonal serum showed the expressed viral antigen in the cytoplasm of infected cells. Radioimmunoprecipitation with the same antiserum detected a 67-kDa VP2 protein which exactly matched the migration of the authentic FPV VP2 protein by SDS-polyacrylamide gel electrophoresis. Nine five-month-old cats were vaccinated and 21 days later were boosted with the recombinant virus. Peroral FPV challenge 2 weeks after the booster showed that the cats were fully protected as measured by examining clinical signs and total white blood cell counts in peripheral blood. Cats not immunized developed low to very low leukocyte counts following peroral FPV challenge. The nine vaccinated cats showed high FPV neutralization antibody prior to challenge, whereas nonvaccinated cats formed anti-FPV antibodies only after challenge.

Topography of variola smallpox virus inverted terminal repeats.

We examined the nucleotide sequences of the inverted terminal repeat (ITR) regions adjacent to the covalently closed hairpin end sequences of three variola major and four minor strains from smallpox outbreaks in Europe, Asia, Africa, and South America. The ITR regions ranged in size from 581 to 1051 base pairs (bp) and contained no apparent open reading frames. Two nonrepetitive sequence elements, NR1 and NR2, were conserved and resembled nonrepetitive elements in the ITRs of other orthopoxviruses. Depending on strain, the terminally positioned NR1 and the more internal NR2 flanked a direct repeat region containing from none to four copies of a 69-bp sequence and one copy of a 54-bp related sequence partial repeat. A distinctive pattern of ITR topography of NR1 and NR2 flanking a single copy of the 69-bp unit characterized each of three examined alastrim variola minor strains. A nonalastrim African minor strain from the last natural case of smallpox in Somalia in 1977 showed the largest ITR region of the examined viruses because of a second direct repeat cluster following NR2.

Serological relatedness of monkeypox, variola, and vaccinia viruses.

Closely related human and monkey orthopoxviruses were differentiated by serologic techniques. Antiviral sera were tested by immunodiffusion for reactivity against six different viral antigens prepared from either infected cell cultures or infected chorioallantoic membranes (CAMs) of embryonated eggs. Portions of each antiserum were separately absorbed with heterologous antigens from infected CAMs to remove common reactivity. The absorbed sera formed immunodiffusion precipitates with both types of antigen preparation and revealed specific-character differences that made it possible to classify the viruses as variola, vaccinia, or monkeypox. Cross-complement fixation tests were also used to examine the immunologic reactivities of antisera to detergent-treated, purified preparations of three orthopoxviruses. Only common reactivities were detected by this method, however, and differentiating reactivities were not observed.