Abstract: Virus outbreaks have been found to be related to the concentration of a new class of genomic peptides, Replikins^1^. The eight genes of H5N1 influenza virus were analyzed for the distribution of Replikin Counts (number Replikins /100 amino acids) in 2,441 sequences from birds and humans. An increase (p&#x3c;0.001) occurred from 2004 to August 2011 in one gene, pB1

Elenore S. Bogoch

Samuel Bogoch

English

Nature Precedings

  1 
Marked Rise in Replikin Counts in H5N1 Influenza Virus Localized to Lethality Gene p B1. 
 
Samuel Bogoch* and Elenore S. Bogoch** 
 
Abstract: Virus outbreaks have been found to be related to the concentration of a new class of 
genomic peptides, Replikins1. The eight genes of H5N1 influenza virus were analyzed for the 
distribution of Replikin Counts (number Replikins /100 amino acids) in 2,441 sequences from 
birds and humans. An increase (p<0.001) occured from 2004 to August 2011 in one gene, p B1.      
Replikins         Mean, black 
Count          SD,  red 
     
     [ NS1]  [Matrix ]  [ p  A ]  [Hemag]  [ p B1 ]   [Neuram]  [p B2]  [Nucleo] 
[Birds/Human] [B/H][B /H]   [B     /  H ]  [ B  / H]   [B   /  H   ]  [B /H ]   [B   / H  ] 
 
Legend for Figure:   Eight Groups of Replikin Counts, one Group [   ] for each gene:  
          [NS1],   [Matrix],         [pA],   [Hemagglutinin],  [ p B1],  [Neuraminidase], [p B2],[Nucleoprotein]. 
Two Subgroups are shown for each gene:  Counts on virus isolates from birds [B],   
                                                     Counts on virus isolates from humans [H].  
Each Subgroup contains 8 annual Replikin Counts, one for each year available from 2004 to 2011 
                                                                          (no data on PubMed in some years).   
Total Number of sequences analyzed for 
Replikins= 2,441 
Number of sequences in each subgroup:  
    [10 /7] [277 / 466] [153/ 62]   [209/ 425] 
 
     
 
 
[191/ 316 ]      [194 /  322 ]   [140 /75 ]  [142 / 212] 
 
In H1N1, the Replikin Count analyses of the hemagglutinin (‘Infectivity’) gene predicted the 
2009 pandemic one year in advance. This analysis then was repeated every few days to monitor 
‘real-time’ the course of the pandemic1. In H5N1, the potential for high lethality of H5N1 was 
predicted for Indonesia in 2006 by a rising H5N1 Replikin Count, then realized in 2007.  In 
H5N1 currently, the continuing increase in the p B1 Replikin Count to record highs in August, 
2011, and the current outbreaks of high lethality H5N1 in Egypt and Asia, are therefore of 
concern. The increasing Replikins in H1N1 hemagglutinin, together with the localization of the 
H5N1 Replikin Count increase to p B1, has led to the development of a completely synthetic 
Replikins pan-influenza vaccine effective against H5N12. 
0 
5 
10 
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20 
25 
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  2 
 
*Senior Scholar, Boston University School of Medicine 
** Director, Foundation for Research on the Nervous System 
 
Introduction  
  
Replikins are the only conserved  structures of infectious organisms described to date 
which correlate quantitatively and temporally with epidemic outbreaks, course, and 
lethality, and permit early or advance warning of such outbreaks. Replikins are genomic 
structures defined by the algorithm: peptides 7 to 50 amino acids long, containing two or 
more lysines, six to ten amino acids apart, at least one histidine, and a lysine 
concentration of 6% or more3. Replikins have been counted and their spread over the 
gene’s surface has been visualized in the hemagglutinin gene by X-ray diffraction in pre-
pandemic and pandemic periods1. 
  
Methods  All  H5N1  genomic  sequences  in  Pubmed were  analyzed  by  software  based on the 
authors’ algorithm4. Replikin peptides were first identified and then counted in each 
genomic sequence (Replikin Count = number of replikins per 100 amino acids). For each 
group of specimens’ replikins, the mean and standard deviation of the mean (SD) were 
calculated and compared between 2004 and August 2011. The terms ‘increase’ and 
‘decrease’ of Replikin Counts were used only when the p level was less than 0.001. 
Counts in H1N1, H5N1 and other influenza strains were each monitored separately. All 
sequence data submitted to Pubmed by laboratories throughout the world relevant to the 
keyword subjects of the inquiry were analysed. The number of sequences published on 
Pubmed for each inquiry are indicated in the Figure. Some years had no sequences 
published for some genes. During outbreaks, Replikin Counts were compared to Counts 
for the same strain in non-outbreak time periods. Replikin genes were isolated in silico by 
scanning and identifying those areas of the virus genome which had the highest 
concentration of Replikins. When a change was found to be associated with high 
infectivity (morbidity over the time period, CDC and WHO epidemiological data), the 
high Count area was named Replikin Infectivity Gene; Infectivity Genes were found in 
the hemagglutinin area. Similarly, areas in the genome were sought which were 
associated with high Replikin Counts and high mortality rates; the high Count area then 
was named Replikin Lethality Gene. In the present study, the Replikin Lethalty Gene was 
found specifically in the p B1 area. 
 
Results and Discussion 
 
In contrast to the rising Replikin Counts in one gene, p B1, the constancy in the other 
seven out of eight of the influenza genes of both the mean and the standard deviation of 
the Replikin Counts, over a seven and one half year period, is notable. Confidence is 
increased in the reliability of the software methods used by the presence of this constancy 
over years. Further, controls are thereby provided for the evaluation of changes in that 
constancy when they occur. An independent statistical analysis for H1N11 of the 
significance of the changes in Replikin Counts is relevant as precedent to evaluating both 
  3 
the constancy and changes observed in H5N1 in the present study. Rising replikin 
concentration in H1N1 from 2006 to 2008, predicted one year in advance the H1N1 
outbreak of 2009. A highly significant increased concentration of virus replikins was 
found prospectively before the H1N1 2009 pandemic1 (12,806 sequences) (in the 
hemagglutinin gene (N=8,046), p values by t-test = 1/10130, by linear regression = 1/1024 
and 1/1029, by Spearman correlation < 2/1016, by Wilcoxon rank sum<1/1016, by multiple 
regression adjusting for correlation between consecutive years = 2/1022.  
 
Mean Replikin Counts in six of the eight genes of H5N1 are seen in the Figure to be 
under 5, as in earlier evidence found for H1N1 genes in non-outbreak ‘resting’ periods1. 
In all genes with mean Replikin Counts under 5, the Figure shows that the SD is small, 
reflecting the restricted range of the virus population1. Although the hemagglutinin gene, 
showed a recent significant increase in the means in birds (non-significant in humans), 
the standard deviation of the mean (SD) increases shown in the Figure indicate that ‘scout 
viruses’1 with increased Replikin Counts have appeared among the virus population, 
possibly signalling a coming increase in H5N1 infectivity.  
 
Surprisingly, while a major reservoir and vector for H5N1 is thought to be in birds, as 
seen in the Figure, the ‘preferred’ host in terms of recent increases in Replikin Count 
appears to be humans. 
 
H5N1 Outbreaks and Lethality  
  
In H5N1, increased Replikin Counts predicted the lethal outbreaks of H5N1 between 
1997 and  2010. H5N1 is very different from H1N1 in terms of its infectivity and 
lethality. H5N1’s infectivity is lower and lethality higher than H1N11. During the worst 
recorded period of H1N1, the 1918 pandemic, the mortality rate was estimated to be only 
2.5%, whereas the human mortality rate for H5N1 has been reported by WHO to be as 
high as 82%5. From specimens of 1957, when H5N1 sequences were first reported, until 
1996, the mean Replikin Count did not exceed 41.  A slight but statistically significant 
increase in H5N1 Lethality Gene Replikin Count occurred in 1996, in advance of the 
outbreak in Hong Kong in 1997. In 1996, an increase in Replikin Count preceded the 
2007 Hong Kong outbreak in which 30 human cases occurred with a mortality rate of 
27%. The Count increased between 2005 and 2008 (Figure) with subsequent animal and 
human outbreaks in Asia and Egypt in 2007-2010 (WHO)6.   
 
Prediction of Geographic Location of H5N1 (Avian flu) Outbreak in Indonesia  
 
Instead of comparing neighboring genes, neighboring countries were compared for the 
Replikin Counts of H5N1 scout infections in humans over several years. In the replikin 
prediction of 2005-2006, Indonesia was predicted to be the country that would be worst 
affected in terms of increased human mortality rate7. Following the replikin prediction, 
277 human H5N1 cases were reported and the human mortality rate increased in 
Indonesia from 40% to 82% (WHO)5.   
  
 
  4 
Concurrent H5N1 and H1N1 Build-Up in 2011  
 
H5N1.  
Because of the increase in H5N1’s Replikin Count in birds from 2002 to 2008 (Figure), 
and the increased Counts in H5N1’s proposed precursor H9N2 in chickens8.9, the authors 
issued a warning in January 2009 that H5N1 outbreaks would surge10. By January of 
2010, H5N1 outbreaks occurred in birds and chickens in 63 countries12. Human cases 
appeared monthly, most prominently in Egypt, where there were 106 WHO confirmed 
human cases and 32 deaths (mortality rate 30.2%) as of March 16, 2010. Also in March, 
2011, the case-fatality rate was reported by CIDRAP to be 34%, versus 60% for other 
countries with human cases. Now, as of June and July, 2011 respectively, the cumulative 
mortality rate is reported by WHO to have risen since 2006 to 34.7%5; and the current 
mortality rate reported by the Egyptian government is 38.7%6. Globally, as of 2010, the 
H5N1 hemagglutinin Infectivity Gene Replikin Count had reached its highest level in 
humans since 1998, 4.6+/-4.31.   
  
H1N1. 
The H1N1 Infectivity Gene Replikin Count, never returned to pre-pandemic levels in 
2010; from the outbreak in April 2009 its peak Replikin Count persisted at approximately 
10 through 2010, then peaked globally in January 2011 at 13.5+/-4.2. In Mexico, 3/21 
specimens in the first four months of 2011 had record high levels of 16.71. The failure of 
the Replikin Infectivity gene to return to pre-2009 outbreak levels two years later is in 
marked contrast to the SARS Replikin Count which promptly returned to pre-outbreak 
Replikin levels in approximately 9 months1 to signal the end of the SARS outbreak. The 
present data therefore strongly suggests that the H1N1 pandemic of 2009 is continuing to 
develop. While there were insufficient H1N1 p B1 sequences submitted to PubMed from 
Mexico as of August, 2011, the global H1N1 Lethality Gene Replikin Count level in 
humans, which had decreased at the end of 2010 to its pre-pandemic level of 2.0+/-0.2, 
was again increased to 5.6+/-4.9 as of August 2011, higher than the range that it was in 
before the 2009 H1N1 pandemic1.  
 
Conclusions 
 
The elevation of the concentration of both the H1N1 Infectivity and Lethality Genes, is 
invariably (in 18/18 predictions) followed by clinical outbreaks1,3. The concurrent 
combined activation of both the Infectivity and Lethality Genes of H5N1 as well in 2011 
therefore is of concern1. The 2011 outbreaks have begun for both H1N1 and H5N1. 
Again as for H1N1 in 2008, in Mexico, initial ‘scout‘ virus outbreaks of H1N1 have 
occurred in 56 cases with Replikin Counts of the Infectivity Gene up to a record 16.7 and 
a human mortality rate of 10.7%13,14. Outbreaks of H5N1 in Egypt have begun with a 
current cumulative mortality rate of from 34.7% (3,4) to 37.8%5,6,12. A structural build up 
of virus Replikins, in both H1N1 and H5N1, consistent with that observed in advance of 
the last influenza pandemic of 20091, therefore appears to be in progress towards another 
pandemic of one or both strains.  
 
 
  5 
Acknowledgements  
  
We are grateful to Pubmed, and to its contributors, whose data were used extensively in 
these studies.  Dr. S. Winston Bogoch, with the authors, designed the software used in 
this study1. Anne Bogoch Borsanyi was responsible for the preparation of research 
protocols and contributed to the writing of this manuscript. We are grateful to Professor 
Mark Jackwood, University of Georgia, for the independent testing of synthetic replikins 
TransFluTM vaccine against H5N1 in chickens2. We are grateful to the United Kingdom 
Department of Trade and Investment for sponsoring academic seminars to introduce the 
technology of the Replikins Bioradar Global Surveillance SystemTM 15 .  
 
References 
 
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A new way to predict outbreaks: replikin peptide concentration in H5N1 influenza virus genome as a marker for lethal outbreaks.

Efficacy of a Replikin peptide Vaccine Against Low Pathogenicity Avian Influenza H5 Virus. Avian Diseases 53(4):613-617,

FluForecast TM and Bioradar TM from Replikins, Ltd., London Bioscience Innovation Centre, 2 Royal College Street,

For Infectious Disease Research And Policy) A year's experience with the 2009 H1N1 influenza virus has underscored the endless unpredictability of flu.

For Infectious Disease Research And Policy). FAO says H5N1 outbreaks are up this year

Health Organization Alert. Archive Number 20110425.1292.

Health Organization). Cumulative Number of Confirmed Human Cases of Avian Influenza A (H5N1) Reported to WHO. 16

New H1N1 Genomic Data Shows Decrease in Replikin Count* of Lethality Gene; Replikin Count of Infectivity Gene Remains High. .<replikins.com> Report Number 31,September 30,

Prediction of specific virus outbreaks made from the increased concentration of a new class of virus genomic peptides, replikins. Available from Nature Precedings

Replikins: the chemistry of rapid replication. Begell House,

Rising H9N2 Influenza Replikin Count Has Doubled That of H5N1.

Marked Rise in Replikin Counts in H5N1 Influenza Virus Localized to Lethality Gene p B1.

AbstractAbstract: Virus outbreaks have been found to be related to the concentration of a new class of genomic peptides, Replikins^1^. The eight genes of H5N1 influenza virus were analyzed for the distribution of Replikin Counts (number Replikins /100 amino acids) in 2,441 sequences from birds and humans. An increase (p&lt;0.001) occurred from 2004 to August 2011 in one gene, pB1.</jats:p

Elenore Bogoch

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http://precedings.nature.com/documents/6420/version/1

Marked Rise in Replikin Counts in H5N1 Influenza Virus Localized to Lethality Gene p B1.

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