Monkeypox diagnostic raw materials, diagnostic enzymes, antigens, antibodies
After the Covid-19, the monkeypox virus once again detonated the world. Human monkeypox was first identified in a 9-year-old boy in the Democratic Republic of Congo in 1970 and is prevalent in Africa. Recently, with the continuous increase of non-monkeypox endemic areas and the number of infected people, monkeypox has once again entered the public eye. Scientists from many countries have called on the public not to panic, but to be vigilant against monkeypox and take effective epidemic prevention measures. After obtaining the monkeypox gene sequence, Jiangsu Watson Bio Ltd immediately formulated a monkeypox project development plan, and established a special R&D team to develop monkeypox diagnostic materials to help prevent and control the monkeypox epidemic.
Introduction to Monkeypox Virus
Monkeypox virus (MPV) belongs to the Orthopoxvirus genus and is an enveloped double-stranded DNA virus. MPV can effectively infect human primary monocytes, by inhibiting CD4+ and CD8+ T cell activation, eliminating local T cell responses, avoiding systemic immunosuppression and immune monitoring. The results of gene sequence alignment of monkeypox virus (MPV-ZAI) and variola virus (VAR-IND and VAR-GAR) showed that there were significant differences in the regions encoding virulence and host range factors at the ends of MPV and VAR genomes. The results of phylogenetic tree analysis showed that MPV and VAR were not direct “relatives”. The MPV IFN resistance gene is mutated to encode the IL-1β-binding protein gene, and these sequence differences may lead to reduced MPV pathogenicity and infection rate. However, it is not excluded that MPV will undergo human adaptation through spontaneous or recombination.
Genes Comparation of Monkeypox Virus And Smallpox Virus
Scientists including Sergei N. Shchelkunov of the Russian National Research Center for Virology and Biotechnology “Vectors” sequenced a 197 kb genome of MPV isolated from a patient during a massive monkeypox outbreak in Zaire in 1996. By aligning the genome sequences of MPV with those of the severe disease-causing Indian 1967 strain (VAR-IND) and the relatively mild disease-causing smallpox Garcia-1966 strain (VAR-GAR), it was found that the middle part of the MPV genome encodes essential The nucleotide sequences of enzymes and structural proteins were 96.3% homologous to variola virus (VAR). The regions at the ends of the genome that encode virulence and host-range factors vary considerably. Mutations in two interferon (IFN) resistance genes in MPV and the presence of interleukin-1β (IL-1β) inhibition may contribute to the differences in the properties of these two viruses and may also limit the use of monkeypox as a model of smallpox. Although extensive genetic differences confirm that MPV is not an immediate ‘relative’ of VAR, future adaptations of MPV to humans are not excluded.
- Full-length gene: The full-length of MPV-ZAI gene is 196858 bp, including ≥60 amino acid residues and 190 basically non-overlapping ORFs. Its structural characteristics and GC content (31.1%) are similar to other orthopoxviruses. The full-length coding sequence of MPV-ZAI gene is 195118 bp longer than that of VAR. The longer length of MPV DNA is mainly due to repeats of the left 4 terminal ORFs as part of terminal inverted repeats (TIRs), whereas the VAR genome is a very short geneless TIR that lacks ORF repeats (Fig. 1).
Fig.1 Schematic diagram of species-specific variable genomic regions at the ends of MPV-ZAI and VAR-IND (Note 1)
- Central genome: The central genomic region of orthopoxviruses mainly contains highly conserved essential genes. The central genomic region of MPV-ZAI DNA has a total of 101 466 bp, which is delineated by the C10L and A25R ORFs, and the homology to VAR-IND is 96.3%. The amino acid sequence of the virion protein encoded by this region of MPV-ZAI was 91.7-99.2% similar to that of VAR-IND.
- Terminal regions: The two terminal regions of the MPV-ZAI and VAR-IND genomes exhibited considerable variation caused by deletions (Fig. 1) and ORF truncations (Tables 1 and 2) of one DNA relative to the other. The amino acid sequence identities of MPV-ZAI and VAR-IND deduced virulence and immunomodulatory factors were 83.5-93.6%.
(1) Virulence factors: Notably, the MPV-ZAI mutation is the translation of two IFN resistance genes that are intact intracellular proteins in VAR and other orthopoxviruses. One of them (C3L in VAR-IND, Table 1) is a homolog of eukaryotic translation initiation factor 2α (eIF-2α) that inhibits the antiviral activity of double-stranded RNA-dependent protein kinase (PKR) by acting as a decoy. The MPV (strains ZAI and CNG) genomes do not encode this protein. The study showed that mutant VAC lacking this gene exhibited IFN sensitivity, with approximately 100-fold reduction in viral yield compared to the parental virus. Another IFN resistance gene (E3L in VAR-IND, Table 1), present in VAR and other orthopoxviruses, was expressed in long or short form by methionine 1 or 2, respectively (Fig. 2A). In VAC, the N-terminal domain of the long-shaped protein encoded by this gene mediates Z-DNA binding, nuclear localization and PKR interaction, and is a protein required for VAC toxicity. The C-terminal domains of short- or long-form proteins bind double-stranded RNA, inhibit IFN-induced activation of PKR and 2-5A synthase, and are required for IFN resistance and VAC host range. The first translation initiation codon of MPV and downstream nonsense mutations are evident, so only the short form is translated (Fig. 2A). Mutations in these two IFNs may result in less human-to-human transmission of MPV than VAR. MPV encodes a complementary binding protein with only 3 short repeats compared with 4 in other orthopoxviruses (Fig. 2B). In addition, MPV encodes a secreted IL-1β-binding protein and 3-l-hydroxy-δ-5 steroid dehydrogenase, and there is no complete ORF in the VAR strain (Table 1). Notably, the VAC gene, encoding the IL-1β-binding protein, was associated with fever and pathogenicity. Therefore, the presence of IL-1β-binding protein in MPV may be one of the reasons for its lower pathogenicity than VAR.
Fig.2 Amino acid sequence alignment of MPV and VAR IFN resistance factor and complementary binding protein (Note 2)
Table 1. Comparison of MPV and VAR virulence factors
(2) Ankyrin: ORFs encoding proteins containing ankyrin repeats, some of which have host-range functions (orthopoxvirus homologous MPV-ZAI D7L and C1L), are the largest orthopoxvirus gene family. Among the 10 genes belonging to this family, the gene corresponding to VAR-IND B19R in the MPV-ZAI genome was deleted, and the gene corresponding to the MPV-ZAI gene D1L was deleted in both VAR strains. In addition, four VAR genes in this family (D6L, D7L, C1L, and O3L in VAR-IND) were truncated relative to their MPV homologs. For now, we can only speculate on how these differences might affect host range or virulence.
Table 2. MPV and VAR ankyrin alignment
- Phylogenetic tree analysis: To better understand genetic relationships, a phylogenetic analysis of the terminal variable genomic regions of four human pathogenic orthopoxviruses was performed using 117,600 bp of DNA (Fig. 3). The major and minor subspecies of VAR are closely related, and MPV is slightly further away from VAR than VAC.
Fig.3 Phylogenetic tree analysis of MPV, VAR, CPV, VAC terminal variable genome sequences
In conclusion, the results of the genome comparison of MPV and VAR indicated that there were multiple differences in the virulence genes of MPV with the major and minor strains of VAR. MPV and VAR likely evolved independently of orthopoxvirus ancestors. Genetic differences between VAR and MPV raise considerable questions about the validity of this MPV smallpox model. However, MPV itself causes severe disease, and we need to closely monitor MPV infection rates to ensure that it does not spontaneously or recombine human adaptation in unvaccinated populations with high HIV prevalence.
Note 1: TIRs are indicated by arrows and short tandem terminal repeat regions are indicated by rectangles. Coincident sequences are represented by wide black blocks; deletions of one genome relative to the other are shown as lines. The boundaries of variable genomic regions are marked by the number of nucleotides corresponding to their positions in the genome.
Note 2: Fig. 2A is an alignment of the amino acid sequences of the orthopoxvirus E3L IFN resistance factor encoded by the corresponding genes of VAR-IND, VAR-GAR and MPV-ZAI. The region containing the N-terminal adenosine deaminase Z-α domain (marked in grey) and the C-terminal double-stranded RNA-binding motif is shown. Identical amino acid residues are marked with dots; amino acid deletions are marked with dashes. The first and second methionine residues starting a long or short form of the protein are marked with an asterisk above the sequence. Fig. 2B is an amino acid sequence alignment of orthopoxvirus complementary binding proteins. The ORFs of VAR-IND and MPV-ZAI are displayed. Conserved cysteine residues are marked with black vertical blocks, other conserved residues are marked with grey vertical blocks. The numbers above the squares indicate the four canonical repeat regions of complementary control proteins.
|Monkeypox Molecular Diagnostic Enzyme|
|Serial Number||Product||Article Number|
|1||Hot Start Enzyme||M106S2|
|Monkeypox Virus Antigen|
|Purity||90%, assessed from R250-stained SDS-PAGE gels||90%, assessed from R250-stained SDS-PAGE gels||90%, assessed from R250-stained SDS-PAGE gels||90%, assessed from R250-stained SDS-PAGE gels|
|Buffer||1x PBS，pH 7.4||1x PBS，pH 7.4||1x PBS，pH 7.4||1x PBS，pH 7.4|
|Storage||Store at -80°C and avoid repeated freezing and thawing||Store at -80°C and avoid repeated freezing and thawing||Store at -80°C and avoid repeated freezing and thawing||Store at -80°C and avoid repeated freezing and thawing|