Eric Luellen's presentation at Harvard University virology class on December 3, 2015 about veepox, the weaponization of smallpox via recombination with Venezuelan equine encephalomyelitis (VEEV); Dark Winter, a model for extrapolating the impact of weaponized smallpox; and, Dark Winter 2.0, one example of applying veepox to that model.

1. Veepox:
A Recombinant Chimera Bioweapon
BY ERIC LUELLEN
VIRUSES: MOLECULAR MACHINES PERSISTING AT THE BOUNDARIES OF LIFE (BIOS E-157)
HARVARD UNIVERSITY, DECEMBER 2015

2. 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

3. 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.

4. 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.

5. 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

6. Smallpox (Variola major) Fact Sheet
An enveloped ds DNA virus (varius: spotted)
Pathogenesis & Virulence
1. Cough droplets infect throat
2. Fever in 2-4 days & contagious
3. Exanthema then forehead rash
4. Raised rash on arms, legs most
5. Herd immunity at 80-85%
6. R0 = 5-10
Replication Immune Evasion
1. 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 replication
3. Unique viral-specific DNA-
dependent RNA polymerase
Sources: http://www.pnas.org/content/101/31/11178.figures-only

7. Venezuelan equine encephalomyelitis
Non-segmented, (+) ssRNA zoonotic arbovirus
(a virus complex of six serotypes)
Pathogenesis & Virulence
1. Bite by Melanoconion genus
mosquito (10)
2. Horses and rodents act as amplifying
reservoirs
3. Bi-phasic 4-8 day flu symptoms
4. R0 = 11
Replication
1. Attach via 240 E1 & E2 glycoproteins
(unknown receptor – vert/invert.)
2. Endocytosis, pH drop, conformational
change, +/- template synthesis
3. 11,700 nucleotides
Immune Evasion
1. Inhibits global inhabitation of
transcription
2. Involves nsP2 protein but
otherwise, is poorly understood

8. 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/S0042682299996484
3. 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.

9. 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/S0042682299996484
3. 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.

10. 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

11. 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

12. Veepox Applied
An epidemiological exercise
Dark Winter & Dark Winter 2.0

13. Dark Winter (Weaponized smallpox)
Bioterrorism Exercise, Andrews Air Force Base,
June 22-23, 2001
Assumptions
1. 3 10 g releases of weaponized
smallpox in Phil., Atlanta, OK City
2. 3,000 people infected – Dec 1
3. 30% morbidity - R0 = 10 (mean of
1958-1973) - 56.75% of contacts
4. 228 million Americans susceptible
The First 22 Days
1. 16,000 cases, 14K in last 24 hours
2. 30K second-generation cases
3. US vaccine stockpile + depleted
4. States deploy National Guard & declare
martial law – close borders
5. 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 quarantine
3. 48% of Americans (+/- 5%) demand
nuclear retaliation (CNN/Gallup)

14. 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

15. Dark Winter 2.0 (Veepox)
Bioterrorism Exercise, Cambridge, MA
December 3, 2015
Assumptions
1. 3 10 g releases of weaponized
veepox in Phil., Atlanta, OK City
2. 3,000 people infected – Dec 1
3. 55% morbidity - R0 = 18 (100%
transmission) – 25% chronic neuro
4. 322.3 million susceptible (US)
Day 22 Numbers – Morb/Mort
1. Borders closed within ~ 10 days
2. 27,000 cases, 14,850 dead by Xmas
3. 54K 2nd gen cases - 29.7K/13.5K
4. 972K 3rd gen cases – 534K/243K
5. 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 cases
2. 9.6 million fatalities
3. 4.37 million survivors with long-term
neurological issues
4. Excludes foreign transmissions
5. Elapsed time = 68 days

16. 41
54
64
133
66
42
302
27
41
By December 22, 2015 = 27,000 Cases
22
66
30
334
25
66
46
20
2,000
3,333
18,664
66
359
334
668
By Cumulative Reported Veepox Cases (Day 22)
400
Veepox cases

17. 64
133
66
42
302
41
By February 6, 2016 = 17.5 million cases, 9.6 million deaths,
4.36 million neuro deficits
3,333
66
359
334
By Cumulative Reported Veepox Cases (Day 68)
35,000
11,550
5,863
26,250
8,663
4,397
Cases Reported
Fatalities
Chronic neuro deficits
41,563
13,716
6,962
86,406
28,514
14,473
72,188
23,822
12,091
195,781
64,608
32,793
12,096,875
3,991,969
Pop. 3.878m
No survivors
432,031
142,570
72,365
27,334
9,023
4,580
27,334
9,023
4,580
17,500
5,775
2,931
232,969
76,880
39,022
2,160,156
712,852
361,826
7% Pop. Dec.
42,656
14,077
7,145
19,688
6,497
3,298
42,656
14,077
7,145
216,563
71,466
36,274
16,406
5,414
2,748
1.8% Pop. Dec.
259,219
85,542
43,419
29,531
9,745
4,946
13,125
4,331
2,198
42,656
14,077
7,145
1,296,094
427,711
217,096
3.3% Pop. Dec.
24,063
7,941
4,030
26,250
8,663
4,397

18. Mass Casualty Capacity
Exceeded in 2-4 weeks by 200% to 6,400%
Oklahoma
Beds available: 11,011
Beds needed (Day 22): 18,664
Georgia 6,400%
Beds available: 22,125
Beds needed (Day 22): 3,333
Beds needed* (Day 68): 1,447,304
* Total cases - deaths
Pennsylvania 2,222%
Sources: https://www.ahd.com/state_statistics.html
Beds available: 37,384
Beds needed (Day 22): 2,000
Beds needed* (Day 68): 868,383
* Total cases - deaths
New York 202%
Beds available: 57,504
Beds needed (Day 22): 400
Beds needed* (Day 68): 173,677
* Total cases - deaths
Alabama 866%
Beds available: 16,166
Beds needed (Day 22): 18,664
Beds needed* (Day 68): 156,089
* Total cases - deaths
Kansas 4,276%
Beds available: 6,614
Beds needed (Day 22): 668
Beds needed* (Day 68): 289,461
* Total cases - deaths

19. Further Reading
The genesis & epilogue of Earth’s largest bioweapons
program
Biohazard
Ken Alibek
The Soviet Biological Weapons Program
A History
Milton Leitenberg
Raymond A. Zilinskas

20. 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.

21. Questions & Answers