47. Genomic Replikin Counts™ of Infectious Salmon Anemia Virus (ISAV) in Canada
Exceed the Counts in Lethal Outbreaks in Norway, Chile, and Scotland March 16, 2012
(From Nature Precedings: doi:10.1038/npre.2012.6991.1:15 March 2012)
Real-Time Tracking of the Evolution of the ISAV Genome and the Resultant Replikins Solid Phase ISAV Vaccine Make ISAV Pandemic Prevention Possible.
Samuel Bogoch and Elenore S. Bogoch. Boston University School of Medicine,
Foundation for Research on the Nervous System, Bioradar UK Ltd and Replikins,Ltd.
36 The Fenway, Boston, MA 02215 Nature Precedings : doi:10.1038/npre.2012.6991.1:15 March 2012
Advance warning of pathogen outbreaks, such as those by influenza and ISAV, both members of the orthomyxovirus family, has not been possible
heretofore. A new class of genomic peptides associated with rapid replication was discovered and named Replikins. Software was designed to analyze
Replikins quantitatively. Replikin concentration changes were measured annually prior to, and "real time" every few days during, the 2009 H1N1
influenza pandemic. Replikins were seen by both linear sequence representation and three-dimensional X-ray diffraction, and found to increase in
concentration from Replikin Counts of 3.2 to 5.5 to 10.1 and to expand on the virus hemagglutinin surface prior to and during the H1N1 pandemic.
A highly significant increased concentration of virus replikins was found to correlate with influenza virus outbreaks a) retrospectively in three
pandemics from 1918 to 1999 (14,227 sequences)(p<0.001), and b) prospectively before the H1N1 2009 pandemic in the hemagglutinin gene, 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. Rising
replikin concentration in H1N1 from 2006 to 2008, predicted one year in advance the H1N1 outbreak of 2009; and in H5N1, predicted the lethal outbreaks
of H5N1 1997-2010 (1-7). We then found (8) that the area in the genome of the highest concentration of Replikins, and the country in which this peak
exists in scout viruses, permitted in the past five years seven consecutive accurate predictions of the geographic localization of coming influenza
outbreaks, including those now realized in Mexico for H1N1, and in Cambodia for H5N1. Real-time Replikin analysis of the evolution of the virus genome
identified both mutations and structural reorganization of the hemagglutinin and p B1 genes over as much as two years before each outbreak. This
information, together with the specific Replikin sequences so obtained, permitted solid-phase synthesis of Replikin vaccines in seven days, which
blocked H5N1 in chickens (11). The information also now provides up to two years of time to thoroughly test and distribute vaccines to high risk
animals and humans in the countries identified; thus for the first time, a quantitative genomic Replikins method is available to both predict initial
outbreaks and to prevent the development of a pandemic. This new technology is now also being applied to outbreaks of another member of the orthomyxovirus
family, Infectious Salmon Anemia Virus (ISAV).
Software based on the authors' algorithm (1) first identified and then counted the replikin peptides in each genomic sequence
(Replikin Count™ = number of replikins per 100 amino acids). For each annual group of sequences, the Replikin Count™
mean and standard deviation of the mean (SD) were calculated and compared with other years. Highly statistically significant increases
and decreases were examined, for example by strain, host, country, history, year, month or week; by substitution, morbidity, and lethality.
The terms 'increase' and 'decrease' of Replikin Counts™ were used only when the p level was less than 0.001. All Counts for all
sequences on Pubmed were each monitored separately, from 1997 to 2012, for all countries reporting to Pubmed. Statistical analyses of
rate of change, trend, pattern, and growth models in the evolution of each virus strain were initiated. Replikin genes were isolated in
silico by scanning and identifying those areas of the virus genome which had the highest concentration of replikins. The Count was compared
with the Count in other countries in the same year, and with the occurrence of outbreaks (Figure). Replikin peptides were visualized by two
means: a) by linear display of sequences of contiguous numbered amino acids in the primary structure and b) by X-ray diffraction analysis of
the 3-dimensional folded structure, which showed the increased coverage by Replikins of the HA gene surface in H1N1 real-time as the Replikin
Count™ increased from 3.2 to 5.5 to 10.1 from before and during the course of the H1N1 pandemic of 2009 (1).
Results and Discussion
The Replikin Counts® of Infectious Salmon Anemia Virus (ISAV) lethal Replikin genes have increased since 2005 (p<0.001) to mean Counts
greater in Canadian salmon than those found during highly lethal ISAV outbreaks in Norway, Chile, and Scotland (Figure). The Replikin Count™
equals the number of Replikins per 100 genomic amino acids specified. In addition to the significance of the mean annual Count and the standard
deviation of the mean, as discussed in the Legend to the Figure above, the peak individual specimen Replikin Counts reached (not shown) were also
instructive as markers of the progress of the evolution of the virus. The peak individual Counts observed to date in all Pubmed ISAV data are 5.6
in Chile, 7 in Norway, 9.4 in Scotland, and 22.6 in Canada. Replikin Count™ increases correlate with increased lethality in several classes
of infectious disease, in influenza viruses, in Taura Syndrome virus in shrimp, in other viruses, in bacteria, and in plasmodia (1-8). The Replikin
Count has been used to predict the relative lethality of shrimp Taura Syndrome Virus (TSV) isolates, the accuracy of the prediction confirmed in the
linear relationship observed in the laboratory. Replikins TSV Synthetic Vaccines have been shown in the laboratory to block TSV infection in shrimp
(12). Regional temporal cyclic expansion preceded by increased concentration of Replikins observed in other viruses (9) also may be occurring for
ISAV, as seen in the Figure.
The present data suggest that the marked decline in salmon yields in recent years in Canadian and U.S. waters (10,19), at least in part, may be
related to ISAV. ISAV is acknowledged to exist on Canada's Atlantic coast; but there has been some disagreement expressed about the presence of ISAV
in Pacific Canadian salmon farms in British Columbia, whether it has reached salmon in the Fraser River, and whether it is a lethal strain. Some
government ISAV data apparently has not yet been completely published (14,19). The situation in British Columbia could also impact the Alaska
salmon fisheries, should the virus be found spreading among wild stocks.
To counteract this risk of spreading, a solid phase synthetic ReplikinsISAV™Vaccine has been formulated, and is now available for trials,
based on ISAV Replikins genomic structures conserved back to 1997. The ISAV vaccine is similar in its basis to the oral Replikins TSV Vaccine, and
to the TransFlu™Vaccine which has been demonstrated to block H5N1 virus and its excretion by chickens (12). For the first time it may be possible
to counter virus reservoir formation where mutation can occur to more lethal strains (12). The ReplikinsISAV™Vaccine will be applied in the same
way to counter reservoir formation in Canada and elsewhere.
Lack of basic information and transparency hinder progress in solving the challenge of ISAV, as noted both in Canada (10,14,19) and earlier in
Chile (18). The absence of published data at particular times, some critical for interpretation, is evident in the Figure.
Salmon and other fish farms are an increasingly important source of food in many areas of the world. Outbreaks of Infectious Salmon Anemia Virus
(ISAV) are responsible for large losses in salmon farms. Lethal ISAV in Chile is reported to have resulted in over $2 billion dollars in losses in
2010. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus,
Isavirus. Some ISAV strains appear to be relatively 'benign' (17), sometimes referred to as salmon 'flu', and apparently produce no lethal disease.
But no quantitative methods have been available previously to distinguish genomic sequence structures which are benign from those which relate to
lethality, nor which indicate when apparently benign strains mutate to lethal ones. Consequently, salmon farmers in Chile who have salmon pens
infected with any ISA strain other than HPR0 (although its allegedly benign disposition may be changed by rapid replication and mutation), are
required to kill their fish, and they have no way to tell in advance if the strain they are dealing with is lethal or not. This also makes the
exclusion of lethal strains of ISAV in salmon eggs impossible when deciding which to use to stock new salmon aqua farm pens. The determination
of Replikin Counts™ now should help in defining the degree of lethality, as they have been shown to be able to do with Taura Syndrome Virus
in shrimp (11).
The high Replikin Counts™ of ISAV in Canada serve as evidence supporting ISAV as a cause of salmon loss currently, as well as a warning
of the risk of outbreaks to come. The risk may be substantial in view of the accuracy of the Replikin Count predictions in seven successive instances
of lethal outbreaks over the past five years by ISAV's related orthomyxovirus, influenza, in all of which not only the prediction of the strain-outbreak
(as discussed by the UN Food and Agriculture Association (3)), but also the geographic location of the outbreaks (8), were correctly predicted. Replikin
studies on ISAV are discussed in greater detail in a book in preparation, The Evolution of Lethal Replikins (7).
We thank Paul Molyneaux ("Swimming in Circles: Aquaculture and the End of Wild Oceans", Amazon Press), Dr. Kristi Miller of the Canadian Department
of Fisheries and Oceans, Nanaimo, B.C., and Dr. Victoria Pedersen and associates, Canadian Food Inspection Agency, for discussions of the ecological
status of salmon in Canada and worldwide.
- Bogoch, Samuel and Bogoch, Elenore. Prediction of specific virus outbreaks made from the increased concentration of a new class of virus genomic peptides, replikins. Nature Precedings http://dx.doi.org/10.1038/npre.2011.6279.1 (2011)
- Bogoch, Samuel and Bogoch, Elenore. Marked Rise in Replikin Counts in H5N1 Influenza Virus Localized to Lethality Gene p B1. Nature Precedings http://dx.doi.org/10.1038/npre.2011.6420.1 (2011)
- UN Food and Agriculture Organization (FAO) discussion of Replikins, DVM Newsmagazine, Sept. 8, 2011. Reproduced in Replikins Report #42 , <Replikins.com>.
- H1N1 Influenza Virus with Highest Replikin Count™ Since the 1918 Pandemic Identified in the U.S. and Austria (April 7, 2008). Replikins Report #18, <Replikins.com>.
- Replikins Provided Advance Warning of Mexican H1N1 "Swine Flu" Virus Outbreak (April 25, 2009). Replikins Report #26, <Replikins.com>.
- Indonesia Reports Experiencing Human H5N1 Mortality Increase, as Predicted Last Year by Replikins' FluForecast® Quantitative Virus Analysis (June 8, 2007). Replikins Report #12, <Replikins.com>.
- Bogoch S and Bogoch ES. The Evolution of Lethal Replikins. In preparation, 2012.
- Bogoch S and Bogoch ES. Bogoch Replikins Pandemic Prevention: Increase of Strain-Specific Influenza Genomic Replikin Counts™ , Having Predicted Outbreaks and their Location Seven Times Consecutively,Up to Two Years in Advance, Provides Time for Prevention of Pandemics. Nature Precedings : doi:10.1038/npre.2012.6952.1 : Posted 1 Mar 2012.
- Replikins Report #19<Replikins.com>. Cycles of West Nile Virus replikin increases precede increases in the number of human cases (May 1, 2008).
- Columbia Basin Fish and Wildlife News Bulletin. Researchers Say Lethal Marine Influenza Virus Found In Wild Salmon Off British Columbia Coast .Posted on Friday, October 21, 2011; Alaska Native News Staff, Journalist team. ISAV Canada Covers Up the Presence of Salmon Virus on the West Coast 12,1,2011; Morton, Alexandra and Routledge,Rick.Lethal Atlantic Virus Found in Pacific Salmon.Simon Fraser U.,Media Release,10,17, 2011; www.cbc.ca/news/canada/british-columbia/story/2011/12/17/ bc-cohen-commission-salmon.html
- Samuel Bogoch and Elenore S. Bogoch. New Synthetic Replikins Aquaculture Vaccines Against TSV and WSSV Viruses in shrimp, and ISAV in salmon. Aquaculture America 2012 Meeting, Abstract Number 526, March 1, 2012.
- Jackwood MW, Bogoch,S., Bogoch ES, Hilt, D and Williams SW. Efficacy of a Replikin peptide Vaccine Against Low Pathogenicity Avian Influenza H5 Virus. Avian Diseases 53(4):613-617, 2009.
- Kibenge FSB et al. Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences. Virol J. 6: 88, 2009. doi: 10.1186/1743-422x-6-88.
- Kibenge, MT et al. Infectious Salmon Anemia in juvenile Onchorhychus species from the North West Pacific Ocean. In alexandramorton.typepad.com. Nov. 14, 2011.
- Report #37 <replikins.com>. Current Severe Outbreaks of Foot and Mouth Disease Predicted by Replikins' BioRadar™ One Year in Advance - June 2009. July 16, 2010
- Report #39 <replikins.com>. Rising Replikin Counts in E. Coli in Germany Since 2005 Preceded Current E. Coli Outbreak, June 20, 2011
- Christiansen, D. et al. A low-pathogenic variant of infectious salmon anemia virus (ISAV-HPRO) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L.) in the Faroe Islands. Journal of general virology 92 (4), 909-918, 2011
- Harbor Watch.Cooke Aquaculture Chile Operations see More ISA Infections. Responsible Aquaculture. <Wordpress.com/2012/01/09>
- Hon. Bruce Cohen, Commissioner. Commission of Inquiry into the Decline of Sockeye Salmon in the Fraser River. http://www.cohencommission.ca/en/ContactUs.php