VIROLOGICA SINICA 2016, 31 (4): 357–361 DOI: 10.1007/s12250-016-3821-6

INSIGHT

Accidental discovery and isolation of in and the relentless epidemiologist behind the investigations

Hedi Zhou1, Bryan Eaton2, Zhihong Hu1, Basil Arif3*

1. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China 2. 6 Belvedere Terrace, Ocean Grove, Victoria 3226, Australia 3. Scientist Emeritus, Laboratory for Molecular Virology, GLFC, Sault Ste. Marie P6A 2E5, Canada

INTRODUCTION 2016). These images were the im- tory on tropical diseases and named petus for concerted efforts to study viral the Uganda Virus Research Institute Zika virus (ZIKV) has captured the tropism and to put into motion efforts (UVRI) with a much broader man- attention of the world because of its to combat its spread. The World Health date. At the center of all that research Organization has deemed ZIKV a potential to infect neural cells and its is Zika Forest, believed to be owned “public health emergency of interna- teratogenic effects on foetuses and by UVRI (Figure 1). the new born. The virus seems to tional concern” (Cohn J, 2016). The have various modes of transmission purpose of this manuscript is to re- and has been the subject of many re- view the very early research that led views in the literature (example, to the discovery and partial charac- Musso and Gubler, 2016, Wang et terization of the virus along with some al., 2016). ZIKV was first isolated in current thoughts and add few per- 1947 but remained almost innocu- sonal anecdotes about the scientist who ous causing few and sporadic mild discovered it. infections until 60 years later when Figure 1. The Uganda Virus Research THE FIRST ISOLATION OF ZIKV an outbreak occurred in YAP State Institute initially established by the in the Federal State of Micronesia in Rockefeller Foundation in 1936. 2007 infecting nearly 75% of the ZIKV (Family Flaviviridae, Genus population (Duffy et al., 2009; Ai et Flavivirus) was first isolated from Because of the prevalence of Yel- al., 2016). A few years later (2013– and named after Zika forest located low Fever Virus (YFV) in the En- 2014), there was an epidemic in the parallel to the road from Kampala to tebbe peninsula, Zika forest was islands of French Polynesia located Entebbe and along the edge of a long chosen as the site to isolate strains of about half way between Mexico and arm of in Uganda. Ini- the virus. Rhesus monkeys, used as Australia. Almost concomitantly, tially, two strains of the virus were baits for mosquitoes, were placed on minor outbreaks occurred in other isolated, one from a sentinel rhesus isolated Pacific islands such as Cook macaque in 1947 and the second from platforms and secured on tree cano- Islands, Samoa, Fiji and New Cale- Aedes africanus mosquitoes in 1948 pies in different locations of the forest. donia. It is truly remarkable how the (Dick et al., 1952). At that time, The first isolate of ZIKV occurred virus has spread within a short time there was considerable interest in following infection of one of the to these seemingly isolated islands. in Africa and in 1936, sentinel rhesus monkeys (number However, nothing brought ZIKV to the Rockefeller Foundation estab- 766), which fell ill in April, 1947. the attention of the world more than lished the Yellow Fever Research The animal developed a fever of 40 the horrific images of newborn ba- Institute (YFRI) in Entebbe with a °C and serum withdrawn during the bies with microcephaly in north east- mandate to conduct research on this acute stage caused mortality in Swiss ern Brazil (Adibi et al., 2016, Rubin virus among others. Later, the insti- albino mice following intracerebral et al., 2016; Schuler-Faccini et al., tute evolved into a top-notch labora- injection. Interestingly, adult mice

© Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2016 AUGUST 2016 VOLUME 31 ISSUE 4 357 Discovery of Zika virus by George W. Dick were refractory to the virus when in- pigs (Dick, 1952). However, anti- Zika Viruses, Chikungunya (CHIKV), oculated intraperitoneally. A Seitz bodies have been detected in large Semliki Forest, O’nyong’nyong, Ka- E.K. filterable transmissible agent animals such as elephants, lions, dam and Entebbe bat viruses (Dick, was isolated from the brains of in- zebras, some rodents, kangaroos and 1953). fected mice and serum withdrawn others (Darwish, 1983; Henderson et from the same monkey at the con- al., 1968). It was later demonstrated EPIDEMIOLOGY AND DISEASE valescent stage completely neutral- that while rhesus 758 did not show ized the transmissible agent. Thus, overt signs of infection, it neverthe- Soon after the first isolation in 1947, ZIKV was born and named after the less developed neutralizing antibo- the virus was reported to have infected forest where it was isolated (Dick et dies against both strains of ZIKV. three people in Nigeria in 1954 al., 1952). The monkey did not de- All cross neutralization tests showed (McNamara, 1954). The virus was velop a serious illness other than conclusively that ZIKV was not re- isolated from one patient and the pyrexia. In 1948 a second ZIKV was lated to YFV, Dengue or to Theiler’s other two had an upsurge in serum isolated by an entirely different murine encephalomyelitis virus. We antibodies. However, the first well method. In attempts to isolate strains now know that ZIKV appears to be documented case of ZIKV in man of YFV from mosquitoes, batches of associated with an autoimmue dis- was reported by Simpson (1964) Aedes africanus were ground in a ease, the Guillain-Barré syndrome who eloquently described his own serum/saline solution, clarified and where the body’s own immunity be- infection by the virus. The 28 year filtered through Seitz E.K. filter gins to attack the peripheral nervous old Dr. David Simpson contracted pads. A group of mice was inocu- system and could lead to motor im- the illness while working at the En- lated intracerebrally with unfiltered pairment and paralysis (Hughes and tebbe laboratory, Uganda. He took and filtered suspensions. Evidence Cornblath, 2005), a fact clearly noted his own serum during the acute fe- showed that a second strain of ZIKV by Dick et al. (1952) in his original brile phase and inoculated two litters (E/1) was isolated from infected publication. of newborn mice intracerebrally. All mosquitoes (Dick et al., 1952). A The UVRI built a 35.5 metre tower the mice died. He then isolated the rhesus monkey 758 was inoculated in the forest to isolate mosquitoes virus from the brains and showed subcutaneously with E/1 strain but from different levels and, the tower that it was completely neutralized by did not exhibit any abnormalities is still standing. The tower has seven anti-ZIKV antiserum. other than a mild elevation in tem- dilapidated platforms set perilously While interesting and important, perature. Serum was taken during over a swamp full of mosquitoes and these early sporadic and isolated the “acute” phase and injected into crocodiles but has been used by sci- cases of ZIKV infection did not re- the brains of six mice demonstrated entists for generations to isolate veal anything extraordinary about the virus had multiplied in rhesus some of the world’s most nasty the nature of the virus and the dis- 758. Eventually, the monkey de- viruses (http://www.theguardian. ease it causes. It was not until 2007 veloped neutralizing antibodies com/global-development/2016/ (60 years after the first isolate) that, against ZIKV. Very interestingly, feb/13/zika-uganda-world-most-dan- now the well-known outbreak oc- early passages derived from the se- gerous-viruses-malaria). Investi- curred in YAP State of Micronesia rum of rhesus 758 caused motor gators went on to isolate no less than when nearly 75% of the small popu- impairment and paralysis in some 12 strains of ZIKV (Haddow et al., lation contracted the virus (Duffy et mice, some of which recovered but 1964; Haddow and Ssenkubuge al., 2009; Ai et al., 2016). This was infection was usually followed by 1965) and many of the isolates were followed by a major outbreak in death. In contrast, acute serum from from mosquitoes captured after sun- French Polynesia such as Tahiti and rhesus 766 produced 100% mortality set and from above the forest ca- Moorea in 2013–2014. Other out- in mice (Dick et al., 1952). The nopy. This was considered an impor- breaks continued to appear in many authors suggested that paralysis was tant epidemiological finding since Pacific and South Pacific islands (Ai an example of Theiler’s encepha- convection currents are quite active et al., 2016). To date, more than 50 lomyelitis. Now we know this to be at that time of the day and could countries harbour the virus. However, an error since Theiler’s encepha- carry mosquitoes to far locations none of these outbreaks made a ma- lomyelitis virus is a picornavirus and (Haddow et al., 1964). It is note- jor impact until the world was shocked not related to flaviviruses or togavi- worthy that among many viruses by the appearance of the virus in ruses. isolated by investigators at the UVRI north eastern Brazil with mothers In contrast to the bias for nervous are Yellow Fever, Rift Valley Fever, giving birth to babies with micro- tissue in mice, ZIKV was not neuro- Bwamba Fever, West Nile, Mengo, cephaly (Heymann et al., 2016; tropic in monkeys, rabbits and guinea Bunyamwera, Ntaya, Uganda S, Broutet, 2016; Ventura, 2016a, 2016b).

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Originally, it was not clear if ZIKV distribution and paucity of human al., 2007). It is very tempting to targeted developing foetal brain cells cases suddenly appears in 2007 as speculate that a mutation(s) in ZIKV or even infects other neural cells. To the cause of a human epidemic in E protein might have contributed to resolve this dilemma, investigators Yap State of Micronesia. Since then, the geographic explosion of virus induced pluripotent stem cells to the virus continued to spread to other since 2007. Recently, phylogenetic form human neural progenitor cells, parts of the world until its alarming analyses have shed much light on the hNPCs (Tang et al., 2016) and found manifestation in Brazil. The ques- movement and migration of ZIKV that the original ZIKV strain iso- tion is what happened to cause a among different countries. Initially, lated from rhesus 766 efficiently in- relatively harmless virus suddenly two phylogenetic lineages, the Asian fected hNPCs. They concluded that appear in such a malevolent capacity. and the African were identified the virus targets these cells directly. The answer has not yet been eluci- based on the availability of rather The investigators conducted a global dated and, most likely several factors limited genomic sequences (Had- transcriptome analysis and found such as viral, vectorial, environmental, dow et al., 2012). More recently, that upon virus infection, there was a etc. must have played significant roles phylogenetic analyses based on all significant number of differentially in its resurgence. If one looks at the available viral sequences including expressed genes with up-regulation epidemiologic history of another the open reading frames (ORFs) en- of a large number of genes involved mosquito-borne virus, Chikungunya coding the envelope E protein and in transcription, protein transport (CHIKV, Alphavirus, Togaviridae) the non-structural protein NS5 re- and catabolic processes. Concomi- also isolated at the UVRI, we see solved three lineages identified as tantly, there was a significant down- that it was innocuous until 2005 the African 1, African 2 and Asian/ regulation of genes related to cell American (Shen et al., 2016). The cycle pathway (Tang et al., 2016). when it started an epidemic in Re- latter study elucidated the central This was the first evidence that ZIKV union Island (Chastel, 2005; En- role of two Western African coun- targeted neural cells. serink, 2006; Ligon, 2006). The is- tries, Senegal and Cote d’Ivoire in With all the above solid evidence land is located in the Indian Ocean the evolution of ZIKV and the vi- plus numerous seminal observations, just east of Madagascar and south of ruses that caused the outbreaks in it was still not possible to conclude Mauritius. Within two years starting that ZIKV is the causative agent of in 2005, the virus caused an epidemic Asia and the Pacific islands were microcephaly and/or Guillain-Barré that infected more than a third of the born in Western Africa. syndrome. However, evidence has population in Reunion Island with been mounting at a lightning speed 260 deaths (Simon et al., 2007). Prior WHAT IS NEXT? and after thorough and careful exami- to this, CHIKV was not known to nation of all the existing evidence, cause death in humans, then was Prevention measures to stop the spread the Centers for Disease Control and transformed from nearly harmless to of ZIKV are no different than any Prevention in the US has clearly con- deadly. In comparison to the east others in the control of mosquito- cluded that ZIKV is the causative agent African strain, the virus from Re- borne viral disease. They include the of microcephaly (http://www.cdc. union Island had a single mutation of obvious control of mosquitoes in gov/media/releases/2016/s0413-zika- A226V in the envelope E1 protein their breeding ponds and the use of microcephaly.html). They also indi- (Tsetsarkin et al., 2007). Detailed personal insect repellents. We also cated that this could be the tip of the analyses showed that this single know that it is transmitted sexually iceberg in terms of damaging effects mutation influenced vector fitness so strict precautions in this regard on the brain with other neurological and viral infectivity. CHIKV is nor- will have to be taken (Cooke, 2016). manifestation. Having established mally harboured and transmitted by Anti-viral drugs would be the choice the cause and effect relationship Aedes aegypti but this one mutation option since the virus is spreading at between ZIKV and foetal brains, the allowed the virus to be carried by such a rapid rate and with adverse CDC believes that their research and multiply in the Asian tiger mos- consequences. Many antiviral drugs should drive further efforts to con- quito, Aedes albopictus, which is have proven to be quite effective but trol the spread of the disease. known for its anthropophylic bias they need to affect virus replication and exists in many countries in Europe negatively without major toxicity to FROM DORMANCY TO and North America. Certainly, this the cell. Many flavivirus drug tar- EPIDEMIC adaptive evolution allowed the virus gets have been identified and many to rapidly expand its geographic dis- appear to be promising (Sampath It is, indeed, enigmatic that a virus, tribution. As of 2007, at least 1.4 and Radmanabhan, 2009). Molecu- which was innocuous for 60 years, million people have been infected lar, structural and biochemical chara- as exhibited by its limited geographic with CHIKV (Ravi 2006; Pialoux et cterization of many vital viral pro-

www.virosin.org AUGUST 2016 VOLUME 31 ISSUE 4 359 Discovery of Zika virus by George W. Dick teins is a prerequisite to identifying mouse models may help explain viru- small molecules to target essential lence of ZIKV to humans and may steps in virus replication. The flavi- aid in the design of intelligent strate- virus RNA dependent RNA poly- gies to generate effective drugs and merase (RdRp) may turn out to be an vaccine. excellent target. Random screening A novel and audacious genetic of nucleoside analogues identified control strategy was developed at several adenine derivatives that were Oxford Insect Technologies. By in- effective inhibitors of RdRp with troducing fatal genes into male Ae- negligible cell toxicity (Angusti et des aegypti mosquitoes, they geneti- al., 2008). Virus entry into cells is cally modified the insect to produce another target that can be exploited progeny that get killed before reach- to prevent virus replication. Such a ing adulthood (Oxitec mosquitoes Figure 2. Prof. George W. Dick (Aug. target was an excellent choice in known officially as OX513A, Ox- fighting HIV-1 infection (Citterio 14, 1914–July 3, 1997), the Epi- itec.com). This depends on the fact demiologist/Virologist who first and Rusconi, 2007; Munch et al., that the main purpose in life of a 2007). isolated ZIKV. (Courtesy of Prof. male mosquito is to find a female to George W. Dick) A unique anti-flavivirus inhibitor mate and produce progeny. Evi- was discovered when high through- dently, this is a different model from the British Royal Army Medical put screening of nearly 240,000 other genetic strategies. Saturating a Corps and was sent to East Africa to compounds for small molecular in- dense urban environment with be in charge of the army’s pathol- hibitors of the non-structural protein Oxitec mosquitoes should, theoreti- 5 (NS5) of DENV. NS5 is the RNA ogy services. After the Second World cally, drastically reduce the progeny War, he became a Fellow of the Rocke- capping enzyme (Stahla-Beek et al., from wild-type Aedes aegypti. The 2012) in flaviviruses. The investi- feller Foundation with appointments approach is unique because it is self- at the John’s Hopkins University gators identified a number of com- limiting with no residue left in the pounds with powerful capacity to in- (JHU) in Baltimore, USA and a Fel- environment. Essential to note that low of the School of Hygiene and hibit GTP binding and Guanylyl- Brazil has an enormous water sys- trasferase activity of NS5. These Public Health of the JHU but re- tem in the Amazon ideal for mos- mained in Uganda working at the compounds exhibited profound anti- quito breeding where Oxitec mos- DENV activity and also had a par- YFRI. For about five years he fo- quitoes may not be a viable control cused on the natural history of yel- tially negative effect on the replica- option. tion of YFV and West Nile virus low fever virus. In 1950 following One cannot help but mention the his return to England for a position (Stahla-Beek et al., 2012). Naturally, summer Olympics scheduled in Brazil the development of an anti-ZIKV at the Medical Research Council, in August with the expected influx vaccine much like the one devel- George Dick then accepted a pro- of hundreds of thousands of people. oped against yellow fever virus would fessorship and became the Chair- No doubt, a large number will be in- be a very good long term mitigation man of the Department of Microbiol- fected with ZIKV and will transport measure. One avenue that promises ogy at Queen’s University of Bel- it back to their home countries. We success has been reported recently fast in 1955. In addition to being a might expect to see another spike in (Shan et al., 2016) where the inves- brilliant investigator, Prof. Dick was the ZIKV infection rate in the world. tigators generated an infectious RNA distinguished as a mentor and edu- Indeed, at these Olympics, ZIKV derived from a cDNA clone. A full cator of a number of young scien- could be the most decorated athlete. length transcript of the cDNA clone tists who went on to develop careers was infectious to Vero cells and gene- of their own in microbiology and rated a virus infectious to mice by THE SCIENTIST virology. Two of the authors (B. the intracerebral route and also rep- Eaton and B. Arif) were fortunate to licated in Aedes aegypti. The au- This manuscript is as much about the be two of three students studying thors went on to generate a lucife- discovery of ZIKV as it is about the honours microbiology (1963–1967) rase-marked virus for use in drug famous Virologist/Epidemiologist, at that time at the Queen’s University screening and other downstream op- George W. Dick (Figure 2), who dis- of Belfast and received much atten- tions (Shan et al., 2016). They indi- covered it in 1947 while working as tion and personalized training from cated that this system of reverse ge- a research scientist at YFRI in En- Prof. Dick and all the professors at netics along with the mosquito and tebbe, Uganda. In 1939, he joined the department. Indeed, Prof. Dick’s

360 AUGUST 2016 VOLUME 31 ISSUE 4 VIROLOGICA SINICA Hedi Zhou et al. virology lectures, which were held in ence. Hyg, 46: 521–534. his office often extended from the One of us (BE) wishes to acknowl- Dick GW, Kitchen SF, Haddow AJ. 1952. Trans R Soc Trop Med Hyg, 46: 509–520. mandatory hour to 3 or more hours. edge Prof. Dick’s instrumental role Dick GWA. 1953. Trans R Soc Trop Med Hyg, He was an enthusiastic and inspiring in getting funding at a time when the 47: 13–43. professor and one of the few scien- British system provided allowances Duffy MR, Chen TH, Hancock W, et al. 2009. tists who instilled into us the pas- for university students pursuing their New Eng J Med, 360: 2536–2543. sion for the science of virology that first degree. As a student who had Enserink M. 2006. Science, 311: 1085. remained with us throughout our ca- Calvet G, Aguiar RS, Melo AS, et al. 2016. The already graduated with a BSc (hons) Lancet, 16: 653–660. reers. Herewith a humorous anec- in Chemistry, BE was not eligible Haddow AJ, Ssenkubuge Y. 1965. Trans R En- dote: in describing his experiences in for further support to acquire a BSc tomol Soc London, 117: 215–243. Africa he recalled how human vol- (hons) in Microbiology. Prof. Dick Haddow AJ, Williams MC, Woodall JP, et al. unteers captured mosquitoes with ensured the funding became availa- 1964. Bull Wld Hlth Org, 31: 57–69. test tubes while sitting lightly clad Haddow AD, Schuh AJ, Yasuda CY, et al. 2012. ble. PLoS Negl Trop Dis, 6: 1–7. on a tree branch in Zika forest. Interestingly, Dr. David Simpson Henderson BE, Hewitt LE, Luke M. 1968. Virus George Dick was also recognized was also a member of the Department Research Institute Annual Report. Nairobi: for his stellar work on poliomyelitis of Microbiology in Belfast when we East African Printer, pp48–51. in Mauritius, his research on Yellow were there and relayed many of his Heymann DL, Hodgson A, Sall AA, et al. 2016. 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