Determination method of pathogen non-epidemic strain

Abstract

The invention relates to the technical field of infectious disease epidemiology, in particular to a pathogen non-epidemic strain identification method which comprises the following steps: A, detecting an antigen amino acid sequence of a pathogen strain; b, determining an antigen precision difference with a reference strain according to the antigen amino acid sequence of the pathogen strain; c, determining an epidemic time interval between the pathogen strain and the reference strain according to the antigen precision difference; and D, if the epidemic time interval is a negative value, determining that the pathogen strain is a non-epidemic strain.

Description

Technical Field

[0001] The present invention relates to the technical field of infectious disease epidemiology, and in particular to a method for identifying a non-epidemic strain of a pathogen. Background Art

[0002] Pathogens are viruses, bacteria and parasites that cause outbreaks around the world. Some pathogens cause large, even global, epidemics, while others spread only locally and endemic. For example, in the same poxvirus family, smallpox (Variola virus / VARV) caused a global pandemic. Monkeypox was locally sporadic in Africa before May 7, 2022, but has since become epidemic outside of Africa. Smallpox is the only human disease that has been eliminated from the cowpox vaccine. We need to learn from smallpox and cowpox to eliminate monkeypox, and even coronavirus.

[0003] Existing technologies can only identify epidemic strains of O1 and O139 cholera groups. Existing technologies can only be used for epidemic strains of Vibrio cholerae, and cannot identify other bacteria, epidemic strains, and local sporadic virus strains.

[0004] Another aim of this study was to provide a method for identifying strains of pathogens causing epidemic and endemic diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying non-epidemic strains of pathogens.

[0006] Another object of the present invention is to provide a method for determining the epidemic time of pathogens.

[0007] The third object of the present invention is to provide a method for identifying strains of pathogens causing epidemic outbreaks and local sporadic diseases.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for identifying a non-epidemic strain of a pathogen, comprising:

[0010] A. Detect the antigenic amino acid sequence of the pathogen strain;

[0011] B. Determine the difference in antigenic precision between the pathogen strain and the reference strain based on the antigenic amino acid sequence of the pathogen strain;

[0012] C. determining the epidemic time interval between the pathogen strain and the reference strain based on the antigen precision gap;

[0013] D. If the epidemic time interval is a negative value, the pathogen strain is a non-epidemic strain.

[0014] Epidemic: refers to a situation where a disease increases significantly and persists in a region or a wide area. Epidemics are larger in scale and last longer than outbreaks, and often involve a wider geographical area and population. The pathogen strain that causes an epidemic is called an epidemic strain.

[0015] Outbreak: refers to a disease that occurs in a region or community with a significantly higher incidence than usual within a certain period of time. Because outbreaks are usually highly explosive, sudden, and localized, timely measures need to be taken to control them. Non-epidemic strains and epidemic strains are properties of the virus itself, and both can cause outbreaks. An outbreak is a property of non-epidemic strains and epidemic strains that cause illness in the population. Both epidemic strains and non-epidemic strains can have outbreak periods and dormant periods.

[0016] The present invention can identify the strains whose difference from the outbreak date of the reference strain calculated by the mathematical model is a negative number as non-epidemic strains. Both the cowpox virus and the HKU coronavirus isolated from bats are non-epidemic strains.

[0017] The epidemic strain is the strain with a significantly higher incidence rate than the incidence rate in previous years. The non-epidemic strain is the strain with a non-epidemic incidence rate that is not significantly higher than the incidence rate in previous years. If the incidence rate in previous years is close to 0, the incidence rate of the non-epidemic strain is also close to 0.

[0018] Preferably, in step B, the calculation of the antigen precision gap includes: firstly determining the antigen precision of the pathogen strain and the reference strain respectively, and then determining the antigen precision gap.

[0019] Preferably, in step B, the calculation of the antigen precision includes: determining the superantigen, calculating the standard deviation of the amino acid molecular weight of the superantigen, and obtaining the antigen precision.

[0020] Preferably, the method for determining the superantigen comprises: taking glycine as a node, cutting the antigen amino acid sequence to obtain a plurality of longest amino acid peptide chains, which are the superantigen.

[0021] Preferably, among the superantigens, the shortest amino acid peptide chain is the leading superantigen, and the longest amino acid peptide chain is the last superantigen.

[0022] Preferably, the method for determining the superantigen determinant cluster includes: taking glycine as a node, cutting the antigen amino acid sequence to obtain the longest 4 amino acid peptide chains, which are the superantigen; among the superantigens, the shortest amino acid peptide chain is the first superantigen, the second shortest amino acid peptide chain is the second superantigen, the third shortest amino acid peptide chain is the third superantigen, and the longest amino acid peptide chain is the fourth superantigen.

[0023] Preferably, the pathogen strain comprises a poxvirus strain.

[0024] Preferably, the reference strains include one or more of smallpox, cowpox, and monkeypox.

[0025] Preferably, in step C, the popular time interval calculation includes regression equation calculation, and the regression equation includes:

[0026] z=13.762x^2–109.376x-63.290y+221.197

[0027] Wherein, z represents the epidemic time interval, x represents the difference in antigen precision, and y represents the amount of tryptophan contained in the superantigen of the pathogen strain.

[0028] Preferably, the correlation coefficient of the regression equation is R=1.000000.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] In order to determine whether the date of the outbreak is related to their superantigens, a mathematical model needs to be established between them. The method adopted by the present invention includes: A. Collecting the outbreak dates of monkeypox and smallpox, and determining the outbreak time interval z between the pathogen strain and the reference strain SARSIID614. B. Detecting the antigen amino acid sequence of the pathogen strain to determine the superantigen. C. Calculating the precision of the superantigen, determining the increase in antigen precision x between the pathogen strain and the reference strain; y represents the number of tryptophan (W) contained in the superantigen. D. Determining the correlation between the outbreak time interval z, the increase in antigen precision x, and the number of W y contained in the superantigen.

[0031] Results: The regression equation was z = 13.762x^2-109.376x-63.290y+221.197, and the correlation coefficient was R = 1.000000. After statistical testing, the probability of type I error was P = 0.008.

[0032] Conclusion: The method of the present invention can predict the time when monkeypox, smallpox and other pox viruses may become widespread, providing a theoretical basis for timely intervention and prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of a multi-amino acid peptide chain.

[0034] Figure 2 This is an equation diagram showing that the relative stability of superantigen determinants is highly correlated with antigen precision or immunogenicity.

[0035] Figure 3 This is the regression equation graph of the time interval (number of months) between epidemics. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Example 1

[0038] 1. Collect the outbreak dates of monkeypox and smallpox viruses and determine the outbreak time interval between the pathogen strains and the reference strain SARS ID614

[0039] The first human case of monkeypox infection was reported in the Democratic Republic of Congo in 1970. The patient was a 9-month-old boy, the only member of his family who had not been vaccinated against smallpox. Before May 7, 2022, monkeypox was only prevalent in Africa.

[0040] During the smallpox epidemic in Yugoslavia from February 16 to April 11, 1972, a total of 175 people became ill, of whom 35 (20%) died. The time interval from February 16, 1972 to the reference date of December 12, 2019 is 573.862 months.

[0041] On May 7, 2022, monkeypox began to spread in the UK, and showed a trend of spreading from the African epidemic to a larger area around the world.

[0042] The outbreak time intervals between the pathogen strains and the reference strain SARS ID614 are shown in Table 1.

[0043] Table 1. Increased precision of superantigens and stability of superantigens (compared with reference superantigen SARS IID614)

[0044]

[0045] 2. Detect the antigenic amino acid sequence of pathogen strains to determine superantigens

[0046] The sequence of protein AGR36452 contains the cadherin-like domain protein in monkeypox virus.

[0047] The AAA69465 protein sequence in smallpox virus contains a cadherin-like domain protein.

[0048] In order to explore the molecular mechanism by which vaccinia vaccine successfully protects humans from smallpox, the protein sequence of CAD90748, a protein containing cadherin-like domain in vaccinia virus, was analyzed.

[0049] The amino acid sequence of the spike protein QHD43416 is located in the wild SARS IID614.

[0050] For the negative control, the amino acid sequence of the spike protein [Bat SARS coronavirus HKU3, ACCESSION QND76034] was used to represent a strain that has never infected humans.

[0051] Taking glycine as the node, the amino acid sequence of the antigen is cut with glycine to obtain the longest multi-amino acid peptide chain, i.e., the superantigen. Figure 1 shown.

[0052] For smallpox, cowpox, monkeypox and coronavirus, the shortest amino acid peptide chain is the primary superantigen, the second shortest amino acid peptide chain is the secondary superantigen, the third shortest amino acid peptide chain is the tertiary superantigen, and the fourth shortest amino acid peptide chain is the quaternary superantigen.

[0053] Through global population studies, the present invention identified 17 such candidate viral superantigens in smallpox, cowpox and monkeypox. All of these were isolated from global populations. The dormancy periods of these candidate viruses, or the time intervals between large-scale epidemics caused by them, were searched and analyzed in real viruses that host and circulate in all populations around the world.

[0054] 3. Calculate the superantigen precision and determine the increase in antigen precision x between the pathogen strain and the reference strain; y represents the content of tryptophan (W) in the superantigen

[0055] The standard deviation of the amino acid molecular weight in the superantigen was calculated to obtain its antigenic precision.

[0056] Each superantigen fragment contains amino acids of different molecular weights. The mean and standard deviation (SD) of the amino acid molecular weights were calculated using Excel. As shown in Table 2, the average molecular weight of the amino acids in the smallpox E60 superantigen is 134.2461, and the standard deviation (SD) of the amino acid molecular weight is 28.0381279. Compared with the reference SD 25.23 of SARS IID614, which appeared later, the SD of the later SARS IID614 decreased and increased by -2.8094310, as shown in Table 1.

[0057] The SD of the amino acid molecular weight is an indicator of the antigen precision, as shown in Table 1.

[0058] y represents the number of superantigens containing tryptophan (W) shown in Table 2.

[0059] Table 2. Molecular weight and standard deviation (SD) or antigen precision of superantigens in smallpox, cowpox and monkeypox

[0060]

[0061] 4. Determine the correlation between the outbreak interval z, the increase in antigen precision x, and the number of superantigens containing W y

[0062] Find the correlation between the stability of superantigen z, the increase in antigen precision x and the tryptophan (W) y it contains.

[0063] Example 2

[0064] 1. Correlation between the stability z of superantigens, the increase in antigen precision x, and the amount of tryptophan (W) they contain y in smallpox, monkeypox, and COVID-19.

[0065] If x represents the increase in the antigenic accuracy of the superantigen, y represents the amount of tryptophan contained in the superantigen, and z represents the distance between the outbreak time of the superantigen-induced epidemic strain and the outbreak time of the reference strain, that is, stability, the regression equation is z = 13.762x^2-109.376x-63.290y+221.197, and the correlation coefficient R = 1.000000, such as Figure 2 As shown. After statistical testing, the probability of type I error was P = 0.008. The improvement of superantigen precision (regardless of whether it contains tryptophan W) and the amount of tryptophan in superantigens are dose-dependent with the stability of superantigens, which is statistically significant.

[0066] 2. If y=1, the regression equation for smallpox and SARS II is z=13.762x^2-109.376x+157.91, and the correlation coefficient R=1.000000.

[0067] In both smallpox and SARS II viruses, there was a dose-dependent relationship between the stability or outbreak interval z between the pathogen strain and the reference strain and the increase in antigenic accuracy x between the pathogen strain and the reference strain.

[0068] As shown in Table 1 and Figure 3 As shown, the antigen SD of the superantigen smallpox E60 is 28.0381279. The precision of the reference superantigen is 25.2286969. The precision of the antigen decreases from smallpox E60 to SARS IID614. The improvement in antigen precision relative to the reference strain is -2.8094310. The conservative time for smallpox is 573.862 months.

[0069] If x represents the improvement of superantigen antigen precision, and z represents the stability of superantigen, that is, the duration of silence or the number of months required from its outbreak date to the reference strain, the regression equation is z = 13.762x^2-109.376x+157.91, and the correlation coefficient R = 1.000000. After statistical testing, the probability of type I error is P = 0.008.

[0070] Example 3

[0071] 1. The 1970 monkeypox epidemic was caused by the chief superantigen D1381, which is 39 amino acids long, 0W.

[0072] In previous studies, local sporadic infectious diseases were dominated by superantigens of 0W. D1381 should be a suitable superantigen, with a length of 39 amino acids and 0W. In 1970, the first case of human monkeypox infection was reported in the Democratic Republic of Congo. The patient was a 9-month-old boy, the only member of the family who had not been vaccinated against smallpox. Previously, the effectiveness of the smallpox vaccine against monkeypox was estimated to be 85%.

[0073] Why the cowpox vaccine can provide humans with smallpox and monkeypox vaccines is a question that needs to be answered. In previous work, it was found that high-precision superantigens produce high-precision antibodies that can neutralize superantigens of lower or equal precision. This rule also applies to smallpox, cowpox, and monkeypox. The superantigen of D1435 is derived from cowpox, and its precision is 20.3801, as shown in Table 2. Four decimal places are retained because this situation is not involved in the mathematical model. If the outbreak time is not completely correlated with the superantigen, the correlation coefficient of R is less than 1. Even if R = 0.9999, the mathematical model will not hold. Only when R = 1.0000 can the mathematical model be true.

[0074] Compared to monkeypox, superantigen D1398 has a precision of 17.6876 in smallpox, which is much lower than the 20.3801 in cowpox. The high precision antibodies produced in cowpox can easily fight against the least precise superantigen in smallpox. Monkeypox superantigen D1381 has a precision of 19.9325, which is lower than the 20.3801 in cowpox. Before May 7, 2022, it was endemic monkeypox and should be protected by cowpox, but not as effective as smallpox because it has a higher precision. The lower the precision of the superantigen, the easier it is to be neutralized by antibodies with higher precision.

[0075] 2. The smallpox epidemic on February 16, 1972 was caused by the second-ranked superantigen E60, which consists of 39 amino acids, 1W.

[0076] As the evolution law of superantigens discovered by the present invention, the secondary superantigen is not shorter than the primary superantigen, but usually longer. Smallpox is an ancient epidemic. In order to determine the primary superantigen, at least 1W must be contained in the epidemic. In the case of smallpox, the 1W secondary superantigen E60 is the same length as the primary superantigen D1398. In this case, the secondary superantigen must improve its precision, otherwise, antibodies with higher precision will neutralize the secondary superantigens with lower precision. The accuracy of E60 is 28.0381279. Seven decimal places are retained because this situation is involved in the mathematical model, z = 13.762x^2-109.376x-63.290y+221.197. In this case, x = 25.2286969-28.0381279 = -2.8094310, y = 1, z = 573.8134503. The time interval from February 16, 1972 to the reference date December 12, 2019 is 573.862 months. The prediction error is only 0.0485497 months or 1.4079413 days later, while the time span is 47 years and 573.93 months ago. If the reference date is 0:00 Beijing time on December 12, 2012, the predicted outbreak time of smallpox is 2:47 am on February 17, 1972 in Central European Time. The expected outbreak time is 2 hours later than the actual historical time of February 16, 1972. This mathematical model should be correct.

[0077] Interestingly, the second most common superantigen in cowpox is I1615, which is 70 amino acids long and has 4W tryptophans. Its precision is 28.7655. In this case, x = 28.7655-25.2286969 = 3.536803, y = 4, z = 13.762x^2-109.376x-63.290y+221.197 = -246.655846. Since the z value between the predicted outbreak date and the reference strain outbreak date is negative, -246.655846 months, this is a negative interval month, which is impossible in theory and cannot be achieved in practice. Therefore, cowpox will never break out in humans. Compared with the second-best superantigen E60, which has an accuracy of 28.0381279 in smallpox and 25.8841380 in epidemic monkeypox, the second-best superantigen I1615 has a higher accuracy of 28.7655 in cowpox, which has never caused a human outbreak. The cowpox vaccine should be able to effectively protect humans from smallpox and epidemic monkeypox.

[0078] Table 3. Molecular weight and standard deviation of coronavirus superantigens D614, N148, I358 and other potential mutant antigens

[0079]

[0080] Similarly, the accuracy of the bat coronavirus D614 antigen is 27.57949, which is 2.35079 higher than the accuracy of the reference antigen 25.23. Substituting x = 2.35079 into the formula

[0081] z=13.762x^2-109.376x-63.290y+221.197=-23.1613937.

[0082] It will never infect humans. Success with a vaccinia vaccine would suggest that bat coronaviruses may be candidates for developing a vaccine.

[0083] 3. The monkeypox outbreak on May 7, 2022 was caused by the second-ranked superantigen monkeypox E60, which is 75 amino acids long, 2W.

[0084] Monkeypox was an endemic infectious disease before May 7, 2022. In order to determine the chief superantigen, at least 1W must be present in the outbreak. In the case of epidemic monkeypox, the secondary superantigen monkeypox E60, which is 2W in length and 75 amino acids, is the second shortest superantigen after the chief superantigen D1398. In this case, the secondary superantigen must improve its precision, otherwise, the antibodies with higher precision will neutralize the lower precision secondary superantigen. The precision of monkeypox E60 is 25.8841380, which is higher than the precision of the chief superantigen D1398, which is 19.9325. Seven decimal places are retained because this situation is involved in the mathematical model, z = 13.762x^2-109.376x-63.290y+221.197. In this case, x = 25.8841380-25.2286969 = 0.655441, y = 2, z = 28.8396710. The time interval from May 7, 2022 to the reference date of December 12, 2019 is 28.839 months. The prediction is only 0.0006710 months after the theoretical outbreak start time. In May 2022, there are 31 days. The prediction is only 0.020801 days after the theoretical outbreak start time, 0.499224 hours later. Since the actual outbreak time is not accurate to the hour, but only to the day. The theoretical outbreak time is from 0:00 to 24:00 on May 7, 2022, and the predicted outbreak time is 0.499224 hours on May 7, 2022, which is within the theoretical outbreak time. This mathematical model should be true.

[0085] 4. Four superantigens

[0086] The SD of superantigen SARS II F718 is 20.69, which is even smaller than 25.23. Why did this "rough" antigen not mutate? The possible reason is the distribution of amino acids, because it does not contain any of the largest amino acids, W. If any superantigen does not contain "W", it may not show a tendency to mutate first.

[0087] Tryptophan does have its own biochemical functions, one of which is that it can be translated from a "stop codon." It has been reported that thymine guanine adenine (TGA stop codon) is a codon for tryptophan in spirals, but a stop signal in other species.

[0088] Some scientists may be skeptical about this because empirical evidence shows that the correlation is close to 1, but cannot reach 1. If you ignore the epidemic cases with a correlation coefficient of 1.0000000, you may miss the way to discover the truth, and there is no reason to ignore it. To be cautious, the present inventor calculated more than 3 times, with a decimal point accurate to 7, and found that R is really 1.0000000. In theory, the month with a decimal point accurate to 7 can be accurate to the second, and the mathematical model can predict the seconds of the disease outbreak, but clinicians cannot make the same accurate diagnosis.

[0089] 5. Conclusion

[0090] The present invention has found through long-term research that the improvement of superantigen precision and the amount of tryptophan in it are the determining factors for the outbreak date of smallpox and monkeypox. The method of the present invention can predict the time when pox viruses such as smallpox, monkeypox and severe acute respiratory syndrome II may be widely prevalent, providing a theoretical basis for timely intervention and prevention.

[0091] According to the theoretical basis and mathematical model of the present invention, the strains with a negative time interval from the reference strain are calculated to be non-epidemic strains. Among the epidemic strains, if the chief superantigen contains tryptophan, the pathogen strain is likely to cause an epidemic in a larger area. If it does not contain tryptophan, it will generally only cause local epidemics in a small area.

[0092] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for identifying a non-epidemic strain of a pathogen, characterized in that: include: A. Detect the antigenic amino acid sequence of the pathogen strain; B. Determine the difference in antigenic precision between the pathogen strain and the reference strain based on the antigenic amino acid sequence of the pathogen strain; C. determining the epidemic time interval between the pathogen strain and the reference strain based on the antigen precision gap; D. If the epidemic time interval is a negative value, the pathogen strain is a non-epidemic strain.

2. The method for identifying a non-epidemic strain of a pathogen according to claim 1, characterized in that: In step B, the calculation of the antigen precision gap includes: firstly determining the antigen precision of the pathogen strain and the reference strain respectively, and then determining the antigen precision gap.

3. The method for identifying a non-epidemic strain of a pathogen according to claim 2, characterized in that: In step B, the calculation of the antigen precision includes: determining the superantigen, calculating the standard deviation of the amino acid molecular weight of the superantigen, and obtaining the antigen precision.

4. The method for identifying a non-epidemic strain of a pathogen according to claim 3, characterized in that: The method for determining the superantigen includes: cutting the antigen amino acid sequence with glycine as a node to obtain a plurality of longest amino acid peptide chains, which are the superantigen.

5. The method for identifying a non-epidemic strain of a pathogen according to claim 3, characterized in that: Among the superantigens, the shortest amino acid peptide chain is the leading superantigen, and the longest amino acid peptide chain is the trailing superantigen.

6. The method for identifying a non-epidemic strain of a pathogen according to claim 3, characterized in that: The method for determining the superantigen determinant cluster includes: taking glycine as a node, cutting the antigen amino acid sequence to obtain the longest 4 amino acid peptide chains, which are the superantigens; among the superantigens, the shortest amino acid peptide chain is the first superantigen, the second shortest amino acid peptide chain is the second superantigen, the third shortest amino acid peptide chain is the third superantigen, and the longest amino acid peptide chain is the fourth superantigen.

7. The method for identifying a non-epidemic strain of a pathogen according to claim 1, characterized in that: The pathogen strains include poxvirus strains.

8. The method for identifying a non-epidemic strain of a pathogen according to claim 7, characterized in that: The reference strains include one or more of smallpox, cowpox, and monkeypox.

9. The method for identifying a non-epidemic strain of a pathogen according to claim 7, characterized in that: In step C, the popular time interval calculation includes regression equation calculation, and the regression equation includes: z=13.762x^2-109.376x-63.290y+221.197 Wherein, z represents the epidemic time interval, x represents the difference in antigen precision, and y represents the amount of tryptophan contained in the superantigen of the pathogen strain.

10. The method for identifying a non-epidemic strain of a pathogen according to claim 9, characterized in that: The correlation coefficient of the regression equation is R=1.000000.

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