Veepox: A Recombinant Chimera BioweaponBY ERIC LUELLEN

VIRUSES: MOLECULAR MACHINES PERSISTING AT THE BOUNDARIES OF LIFE (BIOS E-157)HARVARD UNIVERSITY, DECEMBER 2015

What we are going to talk about?

Why is veepox important? (2 min) Genesis informs public health policy decisions Epidemiological monopoly Novel genetic-engineering method Triggers a biological arms race

Smallpox – Variola major (2 min) Venezuelan equine encephalomyelitis (VEE) (2 min) Recombination – The Splicing (2 min) Epidemiological Impact: Dark Winter & Dark Winter 2.0 (2 min) Questions & Answers

Qualifiers

Amalgamated Information By definition, the creation of most bioweapons is an area of “dark” biology; therefore, most of what we think we know is based on a “convergence of evidence;”

Tip of the Classified Iceberg - Much of what is known is classified by the governments that created, or discovered, it; therefore, public scientific knowledge is disparate – combined from bits and pieces from varied sources; and,

Some Sources Biased - Much of what we think we know about veepox comes from the testimony of defectors from the Soviet/Russian bioweapon program; because it was necessary for them to appear valuable, we must note they had their own agendas – however, several independently corroborated each other over many years, as did scientific publications.

Theoretical – Veepox was tested on monkey animal models, according to the former Deputy Director of the Soviet bioweapons program; however, he has not discussed the results in public. How the spliced “double virus” would behave is then predicted largely from what we know about how they behave separately.

Why is veepox important? The Take Aways

1. Genesis Informs Public Health Policy Decisions – The presumption that smallpox eradication was universally humanitarian was erroneous; the goal of large-scale smallpox weaponization came about as a “next strategic move” by a principal advocate (USSR) of eradication because it greatly increased smallpox’s strategic value as a bioweapon. It represents a divisive and intentional misuse of public health for geopolitical and strategic advantage. It can inform the design of public health programs and vaccine research to prevent or inhibit misuse.

2. Epidemiological Monopoly – One country, as of the early 1990s, possessed enough weaponized smallpox to infect 1.841 billion people and the capacity to make 4-5x that amount every year. Vaccine research largely stopped for decades, mandatory inoculations stopped entirely, exposing most of the global population to at-risk status. From an epidemiological perspective, it is the only incurable pathogen possessed by one decision maker capable of global infection.

3. Novel Genetic Engineering Method – One, researchers who created veepox determined that foreign DNA material can be injected between genomes in multiple pieces as a method of recombination, especially in large viruses (186,102 bp here). Two, researchers were able to use one virus as a vector for another, in this case with different routes of pathogenesis (neuroinvasion) giving recipients two independent diseases at once.

4. Triggers a Biological Arms Race – The creation of chimera permutations necessitates a biological research response to counter the original research, increased inspection and control of vaccine research, and a rationale for investing more in recombinant pathogen research to stay “one step ahead” of those with malevolent or selfish intents.

Genesis of veepox … weaponized smallpox A geopolitical & historical context

Weaponized smallpox constitutes a “weapon of mass destruction” capable of incapacitating nearly 100% of those exposed and killing at least 30% without harming territory or equipment

British weaponized smallpox -1950 (vaccinia); Soviet smallpox weaponization laboratory opened in 1947; US unaware until 1989 (http://cns.miis.edu/opapers/op9/op9.pdf)

In 1969, at the US offense bioweapons program’s end, Dr. Bill Patrick, its Director, estimated 1 gram infected 100 people

Vozrozhdeniya Island accident in 1971, 400 grams test exploded, researcher on a ship deck 15 km away became infected and transmitted to all children after returning home; all died – safe zone est. at 40 km (others in 1970 & 1979)

From the late 1970s to early 1990s, the Soviet Union maintained an arsenal of weaponized smallpox of 20 tons, with the capacity to manufacturer 80-100 tons more per year

How many grams are in 20 tons? Enough to infect 1.814 billion people with smallpox – and veepox is worse

Smallpox (Variola major) Fact Sheet An enveloped ds DNA virus (varius: spotted)

Pathogenesis & Virulence1. Cough droplets infect throat 2. Fever in 2-4 days & contagious3. Exanthema then forehead rash4. Raised rash on arms, legs most5. Herd immunity at 80-85%6. R0 = 5-10

Replication Immune Evasion1. SPICE is soluble complement

regulator that deactivates the human complement system

2. CKBP-II blocks signaling pathways to inflammatory response

1. 186,102 bps (large) encode 187 proteins

2. Cytoplasmic replication3. Unique viral-specific DNA-

dependent RNA polymeraseSources: http://www.pnas.org/content/101/31/11178.figures-only

Venezuelan equine encephalomyelitis Non-segmented, (+) ssRNA zoonotic arbovirus

(a virus complex of six serotypes)

Pathogenesis & Virulence1. Bite by Melanoconion genus

mosquito (10)2. Horses and rodents act as

amplifying reservoirs3. Bi-phasic 4-8 day flu symptoms4. R0 = 11

Replication1. Attach via 240 E1 & E2 glycoproteins

(unknown receptor – vert/invert.)2. Endocytosis, pH drop, conformational

change, +/- template synthesis3. 11,700 nucleotides

Immune Evasion1. Inhibits global inhabitation of

transcription2. Involves nsP2 protein but

otherwise, is poorly understood

Veepox - a recombinant injection First attempt decreased virulence

1. Sources: http://www.proteinmodelportal.org/?pid=modelDetail&provider=SWISSMODEL&template=2j87D&pmpuid=1000778925895&range_from=1&range_to=177&ref_ac=Q0NCP2&mapped_ac=Q0NCP2&zid=async

2. http://www.sciencedirect.com/science/article/pii/S00426822999964843. http://www.biomedcentral.com/content/pdf/1472-6807-6-22.pdf

After 1988, Soviets re-assembled VEE and inserted into thymidine kinase (TK) gene of variola using a plasmid (pieces of circular DNA used to insert into cells) but it reduced virulence – putatively because it interfered with action of the gene (vaccinia and variola TK genes are 97.2% the same)

KT is a phosphotransferase (enzyme) that catalyzes a reaction during the synthesis of DNA. The version in variola is highly similar to TK2, one of two versions found in humans.

Veepox - a recombinant double splice Second attempt increased virulence

1. Sources: http://www.proteinmodelportal.org/?pid=modelDetail&provider=SWISSMODEL&template=2j87D&pmpuid=1000778925895&range_from=1&range_to=177&ref_ac=Q0NCP2&mapped_ac=Q0NCP2&zid=async

2. http://www.sciencedirect.com/science/article/pii/S00426822999964843. http://www.biomedcentral.com/content/pdf/1472-6807-6-22.pdf

Between 1988 andn1991, Soviet scientists found that by inserting the re-assembled VEE genome into two locations “immediately after the KT gene” both viruses were stable and expressive.

Speculation from similar work done with rabies, is the splices may have been placed downstream of the vaccinia P7.5 and P11 promoters and flanking by TK sequences.

It’s unknown in the public domain if two copies were spliced in or two pieces of one copy, nor has the exact second location been disclosed. Similarly, while tested on animal models (monkeys), it’s unknown if the two viruses worked together. Most likely they “piggy-backed” on pathogenesis and caused smallpox plus VEE.

Viral idiosyncrasies

Replicates in the cytoplasm instead of nucleus Morbidity varies widely by age – while mean is

30% -- ages 30-49 is actually 54% and > 50 is closer to 50%

Virus and vaccine are 96% the same differing by only 37 proteins

Vaccine complement control protein differs from virus in only 11 amino-acids residuals – believed to be the key why variola major is highly virulent and the vaccine is not infectious

Encephalitis from variola is more damaging then from VEE, hence giving great destructive value to variola using VEE as a vector for neuroinvasion

Venezuelan equine encephalomyelitisSmallpox (Variola major)

Maximum viral replication occurs in 8-10 hours, not days

Replicates highly in almost any host (e.g., 1 billion virions per mm culture)

Pathogenicity is distinct because it uses neuron transmission

Nasal epithelial lining to olfactory bulb within 24-48 hours via bi-polar neuron

Even when neuron is removed, still gets to brain within 36 hours via trigeminal nerve via teeth

More laboratory infections have occurred with VEE than any other pathogen – 150 and all by aerosol exposure

Why is veepox a good bioweapon?

To shorten the incubation period from ~ 17 days to 2-6 days To make pathogens “stickier,” strengthen binding between virions & hosts Accelerate virion propagation (many host types and two viruses) Further reduce infectious dose below already low 10-20 virions Increased mortality by multiple and diverse immune suppression methods Worsened the severity and diversity of symptoms – dozens of differential

diagnoses with early symptoms appearing flu-like Assays, cultures, and diagnostic tests ineffective or, at a minimum, slow to

interpret because of unrecognized gene expressions making diagnosis extra difficult and likely impossible outside of major research centers

Veepox Applied An epidemiological exercise

Dark Winter & Dark Winter 2.0

Dark Winter (Weaponized smallpox) Bioterrorism Exercise, Andrews Air Force Base, June 22-23, 2001

Assumptions1. 3 10 g releases of weaponized

smallpox in Phil., Atlanta, OK City 2. 3,000 people infected – Dec 13. 30% morbidity - R0 = 10 (mean of

1958-1973) - 56.75% of contacts4. 228 million Americans susceptible

The First 22 Days1. 16,000 cases, 14K in last 24 hours2. 30K second-generation cases3. US vaccine stockpile + depleted4. States deploy National Guard &

declare martial law – close borders5. Canada & Mexico close borders

Outcome & Projections

Sources: http://www.upmchealthsecurity.org/our-work/events/2001_dark-winter/Dark%20Winter%20Script.pdf http://cid.oxfordjournals.org/content/34/7/972.full

1. Four-generation outbreak – 3m mortality & 1m morbidity in US

2. DoD enforced quarantine3. 48% of Americans (+/- 5%)

demand nuclear retaliation (CNN/Gallup)

Assumptions for Dark Winter 2.0

The mortality rate for traditional smallpox is 30% The morbidity rate for vaccinia when it develops into encephalitis is 15-25% higher (CDC) The mortality rate for vaccinia when it develops into encephalitis is 25% - meaning a quarter

of survivors have long-term neurological issues (CDC) The morbidity rate for stand-alone VEE is low (1-5%) depending on age of infected Because the smallpox and VEE genomes are complete and “piggy-back” on each other, and

because VEE travels through neural pathways to the olfactory bulb within 24 hours, we assume 100% of those infected will use VEE as a vector to deliver smallpox virions to the brain in first 24 hours

We, therefore, assume the mortality rate of veepox is additive and a range – 30% for traditional smallpox, 15-25% for smallpox encephalitis, and 8% for traditional VEE – a combined veepox mortality range of 46-60%; we assume a conservative 55% in this model

Sources: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5204a1.html

Dark Winter 2.0 (Veepox) Bioterrorism Exercise, Cambridge, MA December 3, 2015

Assumptions1. 3 10 g releases of weaponized

veepox in Phil., Atlanta, OK City 2. 3,000 people infected – Dec 13. 55% morbidity - R0 = 18 (100%

transmission) – 25% chronic neuro4. 322.3 million susceptible (US)

Day 22 Numbers – Morb/Mort1. Borders closed within ~ 10 days2. 27,000 cases, 14,850 dead by

Xmas3. 54K 2nd gen cases - 29.7K/13.5K4. 972K 3rd gen cases – 534K/243K5. Neither vaccine nor cure available

Outcome & Projections

Sources: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5204a1.htm http://wwwnc.cdc.gov/eid/article/7/6/01-0607-techapp1.pdf

1. 17.5M 4th gen cases2. 9.6 million fatalities 3. 4.37 million survivors with long-

term neurological issues 4. Excludes foreign transmissions5. Elapsed time = 68 days

41

54

64133

6642

302

27

41

By December 22, 2015 = 27,000 Cases

22

66

30

33425664620

2,000

3,333

18,664

66

359334

668

By Cumulative Reported Veepox Cases (Day 22)

400

Veepox cases

64133

6642

302

41

By February 6, 2016 = 17.5 million cases, 9.6 million deaths, 4.36 million neuro deficits

3,333

66

359334

By Cumulative Reported Veepox Cases (Day 68)

35,00011,5505,863

26,2508,6634,397

Cases ReportedFatalitiesChronic neuro deficits

41,56313,7166,962

86,40628,51414,473

72,18823,82212,091

195,78164,60832,793

12,096,8753,991,969Pop. 3.878mNo survivors

432,031142,57072,365

27,3349,0234,580

27,3349,0234,580

17,5005,7752,931

232,96976,88039,022

2,160,156712,852361,8267% Pop. Dec.

42,65614,0777,145

19,6886,4973,298

42,65614,0777,145

216,56371,46636,274

16,4065,4142,7481.8% Pop. Dec.

259,21985,54243,419

29,5319,7454,946

13,1254,3312,198 42,656

14,0777,145

1,296,094427,711217,0963.3% Pop. Dec.

24,0637,9414,030

26,2508,6634,397

Mass Casualty Capacity Exceeded in 2-4 weeks by 200% to 6,400%

OklahomaBeds available: 11,011Beds needed (Day 22): 18,664

Georgia 6,400%Beds available: 22,125Beds needed (Day 22): 3,333Beds needed* (Day 68): 1,447,304* Total cases - deaths

Pennsylvania 2,222%

Sources: https://www.ahd.com/state_statistics.html

Beds available: 37,384Beds needed (Day 22): 2,000Beds needed* (Day 68): 868,383* Total cases - deaths

New York 202%Beds available: 57,504Beds needed (Day 22): 400Beds needed* (Day 68): 173,677* Total cases - deaths

Alabama 866%Beds available: 16,166Beds needed (Day 22): 18,664Beds needed* (Day 68): 156,089* Total cases - deaths

Kansas 4,276%Beds available: 6,614Beds needed (Day 22): 668Beds needed* (Day 68): 289,461* Total cases - deaths

Further ReadingThe genesis & epilogue of Earth’s largest bioweapons program

BiohazardKen Alibek

The Soviet Biological Weapons ProgramA HistoryMilton LeitenbergRaymond A. Zilinskas

FAQs

Why would a country develop a bioweapon with high mortality when it would expose their own soldiers or civilian population?

The Soviets envisioned mass aerosol inoculations with vaccines being developed along side the bioweapons – largely unsuccessful though

What role in strategy did veepox play relative to nuclear armaments? Soviet policy makers were of two views: (a) that the proximity and posture of China was a greater threat than the US and was the primary target; and, (b) they were influenced by a 1981 US paper indicating that ~ 28% (60 million in 1981) of the US population would

survive a nuclear attack and it was too many to fight – BW were the “mop up” strategy Was this technology proliferated and if so, how?

Depending on who’s analysis is used, the technology at some level exists in 3-4 other countries; however, the technology was not proliferated in a classical sense. The knowledge was transferred via BW “training academies” for scientists hosted in USSR/Russia for a fee (e.g., Iran). The second proliferation method was diaspora. Biopreparat had 30,000 employees at 18 laboratories (some in the “stan countries”) who, by early 1990s were being paid in some cases $150 per month – other countries offered up to $10,000 per month to leave. There is now public accounting as to where all these scientists went – at least four senior scientists ended up in the United States.Source: Leitenberg, Milton and R. A. Zilinskas, The Soviet Biological Weapons Program: A History, Harvard University

Press, Cambridge, MA, 2012.

Questions & Answers