Ann M. Powers Centers for Disease Control and Prevention, Fort Collins, CO, United States
The first half of the 20th century was the golden age for arboviral discovery. Beginning in 1913–16, the International Health Commission and the Yellow Fever Commission were founded to promote knowledge of scientific medicine and eradicate yellow fever. A number of national and international agencies, such as the Rockefeller Foundation (RF), were involved in this effort through the 1930s in laboratories developed and expanded around the world (one such laboratory was the Yellow Fever Research Institute in Entebbe, Uganda). With the closing of the RF yellow fever program in the late 1930s, the scientists involved with this work turned their efforts to further virus discovery, characterization, and classification. Their broader work with arthropod-borne viruses (arboviruses) began in earnest in 1951 with the establishment of a program to determine “what may be out there.” RF scientists, among others, working in collaboration with scientists at labs such as the Entebbe virus laboratory (then called the East African Virus Research Institute and later the Uganda Virus Research Institute) isolated and characterized numerous arboviruses. It was due to the efforts of programs such as these that viruses including chikungunya (CHIKV) and Zika (ZIKAV) were first isolated and initially described. Mosquito and animal studies were undertaken to characterize zoonotic cycles and efforts made to determine the risk to humans from these agents. Some pathogens, such as CHIKV, were identified during outbreaks while others, including ZIKAV, were discovered during earlier studies but were not shown to be the cause of human disease for years or even decades. It was the outstanding work of these pioneering field and lab researchers at this global network of institutes that set the foundation for much of the current arboviral field work. These investigators not only found dozens of novel arboviruses but performed extensive viral and serological characterization that provided an understanding of the basic relationships among all these agents that would be used for decades to come and aid in the identification of novel emergence events.
Arbovirus; Chikungunya; Discovery; Dengue-like illness; Emergence; Zika
The first half of the 20th century was the golden age for arboviral discovery. Beginning in 1913–16, the International Health Commission and the Yellow Fever Commission were founded to promote knowledge of scientific medicine and eradicate yellow fever (Downs, 1982). A number of national and international agencies, such as the Rockefeller Foundation (RF), were involved in this effort through the 1930s in laboratories developed and expanded around the world (one such laboratory was the Yellow Fever Research Institute in Entebbe, Uganda; (Sempala, 2002)). With the closing of the RF yellow fever program in the late 1930s, the scientists involved with this work turned their efforts to further virus discovery, characterization, and classification. Their broader work with arthropod-borne viruses (arboviruses) began in earnest in 1951 with the establishment of a program to determine “what may be out there.” RF scientists, among others, working in collaboration with scientists at labs such as the Entebbe virus laboratory (then called the East African Virus Research Institute and later the Uganda Virus Research Institute; (Sempala, 2002)) isolated and characterized numerous arboviruses. It was due to the efforts of programs such as these that viruses including chikungunya (CHIKV) and Zika (ZIKAV) were first isolated and initially described. Mosquito and animal studies were undertaken to characterize zoonotic cycles and efforts made to determine the risk to humans from these agents. Some pathogens, such as CHIKV, were identified during outbreaks while others, including ZIKAV, were discovered during earlier studies but were not shown to be the cause of human disease for years or even decades. It was the outstanding work of these pioneering field and lab researchers at this global network of institutes that set the foundation for much of the current arboviral field work. These investigators not only found dozens of novel arboviruses but performed extensive viral and serological characterization that provided an understanding of the basic relationships among all these agents (Casals, 1961) that would be used for decades to come and aid in the identification of novel emergence events.
As was the case in the early days of arboviral discovery, the original discovery of CHIKV occurred during an outbreak of a suspect vector-borne disease. Discovery of CHIKV was a result of studying an epidemic of what was originally thought to be dengue by a hospital clinician. Certainly, dengue had been known for decades in East Africa (e.g., Tanzania, Madagascar, Ethiopia, and Somalia) (Godding, 1890; McCarthy and Wilson, 1948), but epidemics of this disease were not common in this region and, therefore, of interest for scientific study.
The outbreak occurred on the Makonde Plateau (~ 2000 feet above sea level, 45 miles across, and 80 miles from the coast (Robinson, 1956)) in the Newala District of what was then known as Tanganyika (modern Tanzania). While allowing for normal clinical variation, the cases were reported to have disease clinically indistinguishable from dengue (Robinson, 1955). Nobody in the affected area could recall such an outbreak ever occurring before and no record of a similar outbreak was reported in the annual reports of the District Health Commission from 1930 forward (Lumsden, 1955). The distinguishing trait of the illness was the extreme joint pain that led to the name of “chikungunya” (“that which bends up or contorts” in the local dialect) being given to the disease by the affected population.
A number of villages in the region reported similar outbreaks beginning in mid-1952 (Robinson, 1955). The outbreaks were reported to spread very fast and affect the majority of individuals in any given village. The onset of the disease was reported to be rapid; patients reported the pain to be virtually incapacitating within just minutes to hours with a rapid-onset high fever (102–105°F) occurring 3–9 days prior to the joint pain. The fever initially lasted 1–6 days with several individuals having a recurrence of a lower fever 1–3 days after the initial fever. The joint pain that followed the fever was described as “frightening” and exacerbated by movement. In this epidemic, the large joints were primarily affected and were occasionally accompanied by backache. Most cases (80%) also developed a maculopapular rash (within the first 10 days of illness) predominantly on the truck. While the rash was found to be “irritating,” it usually faded within just days. While the fever and rash resolved during the acute period, in some patients, the joint pain continued for up to 4 months. Significantly, there were so many affected individuals that the normal agriculture practices of the region were being adversely impacted. A range of other symptoms were reported to occur occasionally (tachycardia, vomiting, abdominal pain, residual swelling of the ankles, etc.) but notably, there were no respiratory or CNS abnormalities, no lymphadenopathy, and no mortality. All ages and ethnicities were affected but children did appear to recover more quickly. Also, importantly, the lack of adenopathy, lack of retro-orbital pain, and the persistence of the severe joint pain distinguished this epidemic from those preceding it in East Africa.
After initial reporting of this epidemic to the regional Virus Research Institute in Entebbe, personnel and resources were dispatched to the area to perform further investigations (Lumsden, 1955; Ross, 1956). The goals of this work included describing the epidemiology of the outbreak and virus isolation attempts. The description of the affected area was of initial interest because cases were primarily focused in the plateau areas (incidence rates of 13%–95%) with fewer cases reported in the neighboring lowland areas (incidence of 0%–37%) separated sharply by high escarpments (Lumsden, 1955). The period of the epidemic in any particular locality was brief lasting an average of 2.25 months before subsiding. The last known case of this outbreak occurred in May of 1953 (Robinson, 1956). While epidemic cases were primarily found on the plateau center, there was speculation that the virus actually originated in a lowlands valley (where men from the central plateau would travel from after working in agriculture). These individuals were infected in a region of endemic activity and transported the agent to a completely susceptible population where the agent spread rapidly with the abundant Aedes aegypti populations closely associated with the humans (Lumsden, 1955). The lower number of cases in the lowlands might support this hypothesis, but lack of knowledge of the agent prevented further assessment. Interestingly, the same symptoms were being found on a nearby plateau and adjacent coastal regions of Portuguese East Africa (Robinson, 1955). This outbreak differed by inclusion of the coastal areas, which did not occur in Newala, suggesting ecological differences may have been present that impacted disease development.
Because the illness resembled dengue, the supposition was that transmission of the infecting agent was likely via blood-sucking arthropods (of which there was noted to be a large abundance and variety of on the plateau). To investigate if this was indeed the case, a range of insect catches were performed including knockdown collections inside huts, baited catches (human or bird) inside huts, outdoor baited collections, mosquito larval collections, castor-oil paper collections, and search of hut floors for ticks. From these collections, insects ranging from redjuvid bugs, sandflies, bed-bugs, mosquitoes (both anophelines and culicines), flies, and ticks were identified.
The hypothesis was that insects closely associated with huts of case patients were likely responsible for transmitting the agent and, therefore, higher incidence rates would be linked to greater abundance of the transmitting arthropods in the huts. The lack of anophelines on the plateau regions and an abundance of this genera in one lowland area with no cases suggested anophelines were not the epidemic vectors. Generally, Ae. aegypti and Culex species were found most abundantly on the plateau. Further analysis of the mosquito data showed that while Cx. species numbers were high on the plateau, they were high in both high- and low-disease incidence localities and/or huts as well as being abundant in lowlands areas. In contrast, Ae. aegypti had a high prevalence in plateau areas and huts with high-disease incidence. Additionally, the Ae. aegypti were found to feed only on humans while the Cx. sp. fed on birds as well. Significantly, areas with high infection rates were associated with huts where water (not readily available on the porous-soiled plateau) was stored in clay jars (frequently containing mosquito larvae) in the huts.
The collection of the arthropods was not only performed to see which species were present in abundance but also to serve as a source of material to attempt virus isolation in young mice as had been previously successful for yellow fever virus (Theiler, 1930). The mosquitoes and bedbugs were ground up and inoculated into young mice to attempt virus isolation. Of 83 pools of mice inoculated with wild mosquito homogenates, 49 resulted in mortality indicating the presence of an infectious agent (Ross, 1956). After passage of the material through a Seitz filter, the filtrates were still able to kill baby mice, produced no growth on a variety of bacterial culture media, were not susceptible to a number of antibiotics, and showed no visible microscopic particles; all these characteristics indicated that the agent was a virus.
When antiserum generated against one of the isolates was used in a neutralization assay, protection against nearly 10,000 50% lethal doses (LD50) was observed and the name “chikungunya virus” (CHIKV) was applied to the agent. This was the official recognition of CHIKV. Because an isolate was now available, antibody studies could be performed on paired acute and convalescent sera collected from individual patients affected during the epidemic to further understand the nature of the virus. In samples from all cases evaluated (n = 15), there was an increase in neutralization index between the acute and convalescent samples. The convalescent titers were so high, an endpoint was rarely able to be calculated (Ross, 1956). Subsequent studies performed after prolonged storage of additional human serum samples collected during the outbreak (frozen for ~ 2 years) demonstrated a comparable increase in neutralizing antibodies in the convalescent sera where 400 LD50 of CHIKV could be neutralized (Mason and Haddow, 1957).
The next steps in characterization of the agent included evaluating the relationship of this agent to other known regional viruses. One very early assessment indicated it might be related to Hawaiian dengue virus as this was one heterologous agent that did generate neutralization (Ross, 1956). However, this was only a one-way relationship with no neutralization noted when using chikungunya virus and serum from Hawaiian dengue infections. Additional work showed CHIKV to be related to Semliki Forest virus (SFV) by hemagglutination tests (Mason and Haddow, 1957), but extensive characterization of serological relationships would only come several years later when the virus was determined to be related to the Group A (alphaviruses) agents (Spence and Thomas, 1959).
With at least a preliminary understanding of this new disease and the agent causing it, regional arboviral research units sought to evaluate the geographic range of the agent and determine if the disease was as initially reported. At one such facility, the East African Virus Research Institute in Entebbe, Uganda, CHIKV was identified in both mosquitoes and man in 1956. Mosquitoes were being collected from a 65-foot-high platform in a tree near the edge of Zika Forest as part of a program involving 24-h captures (Weinbren et al., 1958). Homogenate from a pool of Ae. africanus taken from this platform and ground level collected in June 1956 was inoculated into mice and monkeys to characterize illness and attempt virus isolation. In addition to the mosquito pool homogenate, serum from the mosquito collectors who became sick were similarly injected into mice and used for neutralization testing (Weinbren, 1958a,b). These experiments showed that the human serum neutralized CHIKV but not Sindbis virus and that virus isolated from either the human or mosquito pool was neutralized by convalescent serum confirming a two-way serological relationship. Importantly, this work showed the agents from both the human subject and Ae. africanus were the same and reinforced the ideas that CHIKV was indeed mosquito-borne as well as having a distribution into Uganda (Weinbren, 1958a, b). Detections of CHIKV continued to occur during epidemics or as periodic cases recognized during subsequent years in other areas of East Africa including the Transvaal Province of South Africa (Gear and Reid, 1957), Northern Rhodesia (Rodger, 1961), Belgian Congo (Osterrieth and Blanes-Ridaura, 1960), Southern Rhodesia (McIntosh et al., 1963), and Uganda (Henderson et al., 1970; McCrae et al., 1971).
While searching for the virus in sick individuals was starting to reveal the range of the viral distribution, this was, of course, an incomplete picture and would only reveal active transmission. Serosurveys across various regions of Africa (beyond the known range of East Africa) were being performed to see how extensively the virus was distributed both geographically and by prevalence. Based on this work, it was revealed that the prevalence varied considerably based on location sampled. For example, in Uganda, high rates of CHIKV-specific antibodies were found in the western and northwestern parts of the country but virtually absent from the southern and eastern regions (Henderson et al., 1970). Perhaps not unexpectedly, the seroprevalence increased with increasing age. While testing of these sera for the related o’nyong nyong virus (ONNV) was not performed, some of this positivity could have been due to cross-reaction with that related virus, which had generated a massive outbreak just several years prior to the serosurvey. This same situation was found during a serosurvey during 1966–68 in neighboring Kenya where both ONNV and CHIKV antibody levels were found to be high in regions where ONNV had reached during the 1959–62 outbreak (Geser et al., 1970). CHIKV-specific antibodies were also found in coastal regions of Kenya where ONNV was never reported indicating CHIKV was indeed in this region. The spread of CHIKV was not limited to East Africa as evidenced by the finding of the virus in mosquitoes as well as antibodies to the virus in man and monkeys in the Lagos area, Nigeria (Boorman and Draper, 1968), in 1962. Using sera from these areas as far back as 1955 also showed evidence of CHIKV-specific antibody suggesting the virus was already broadly distributed through Africa by the time of its original discovery in 1952. Evidence of the virus in Southern and Western Africa was also found by discovering antibodies in primates (McIntosh et al., 1964) and in other vertebrates in Nigeria and Senegal (Moore et al., 1974).
As scientists were working to characterize the epidemiology of the newly discovered CHIKV in Africa, there were also surveys being conducted in Southeast Asia to determine if the dengue-like illnesses in this area were also perhaps due to CHIKV. Interestingly, the discovery of CHIKV in Southeast Asia came during outbreaks of hemorrhagic fever in pediatric cases hospitalized in the Thailand in 1958 (Hammon et al., 1960; Hammon and Sather, 1964). This clinical presentation of hemorrhage was not reported in East Africa, but in CHIKV-infected children in Thailand, hemorrhage was found as frequently as in dengue cases. Later outbreaks of CHIKV in Thailand (Halstead et al., 1969a; Nimmannitya et al., 1969) also showed hemorrhagic manifestations in children albeit milder than those found in dengue patients. Surveys of individuals throughout the country performed in 1962 revealed CHIKV seroprevalence rates as high as 84% (Halstead et al., 1969b) and provided strong evidence of high levels of broad endemicity. Nearly simultaneously, the virus was identified in cases of dengue-like illness in Cambodia; however, CHIKV infections with hemorrhagic presentation were not reported there (Chastel, 1963). Hemorrhage was also not a common feature found in soldiers serving in Vietnam who were found to be infected with CHIKV in 1966 (Deller Jr. and Russell, 1968).
In addition to widespread endemic transmission in Thailand, India also exhibited significant CHIKV activity in the form of multiple epidemics around the country. Particularly large outbreaks were recorded in the early 1960s in Calcutta (Shah et al., 1964; Sarkar et al., 1965a, b; Chatterjee et al., 1967; Sarkar, 1967) and in Vellore and Madras (Dandawate et al., 1965; Jadhav et al., 1965; Myers et al., 1965; Rao et al., 1965; Carey et al., 1969) in the South of the country. Interestingly, the outbreaks were clinically more similar to the presentation reported in Africa and were rarely associated with hemorrhagic manifestations. Follow-up testing of serum collected from the 1950s in these outbreak-affected areas showed that CHIKV had been in India as least this long (Pavri, 1964; Banerjee, 1965; Rao, 1966). These repeated findings that the virus was very widely distributed even before the earliest detection in Tanzania are in agreement with phylogenetic estimates suggesting that CHIKV may have entered Asia in the later part of the 19th century (Powers et al., 2000; Volk et al., 2010) and had spread throughout sub-Saharan Africa long before this. As was common of arboviruses discovered during the Rockefeller period of exploration, outbreaks were one of the primary ways these long present viruses were discovered.
In 1947, intensive efforts were underway to understand the zoonotic transmission dynamics of yellow fever virus. The Entebbe peninsula near Lake Victoria in Uganda was an area where there was a high incidence of immunity to yellow fever in the local monkeys. Therefore, a small forest area called Zika in this region was established as a study site for these efforts (Dick et al., 1952). To obtain isolates of yellow fever virus (YFV), monkeys were placed on platforms in the canopy of trees in the Zika Forest. When one of these monkeys developed a fever, blood was collected from the animal to use in virus isolation efforts using laboratory mice and another monkey. The monkey and all mice inoculated intraperitoneally remained healthy while the mice inoculated by the intracranial route became ill 10 days postinoculation. Neutralization testing was performed to determine if this virus was indeed YFV. However, there was no cross-neutralization providing strong evidence that this agent, named Zika virus after the area where it was obtained, was distinct from YFV and other related viruses (Dick et al., 1952). Interestingly, a virus isolated from Ae. africanus mosquitoes in the same forest several months later was not YFV but found to also be ZIKAV suggesting this virus was continuously maintained in the small Zika Forest.
Upon determining that ZIKAV was a unique pathogen, the researchers wanted to determine if it caused human disease. Human serum from four regions of Uganda (primarily collected for YFV studies) was tested to identify if ZIKAV-specific antibodies were present. Overall, 6% of the sera evaluated were positive for ZIKAV antibody. Curiously, the positive sera were not from the Zika region but were from Bwamba and West Nile regions, far to the west of the Zika Forest. In spite of finding antibody against ZIKAV in humans, there was no evidence of clinical illness associated with infection. However, prophetically, the scientists performing this work warned against drawing conclusions as to the lack of detection of human disease stating that this did “not necessarily mean that the disease is either rare or unimportant” (Dick, 1952). They noted that YFV was not typically detected during acute infection even though the virus was known to be endemic among the population of Bwamba. Additional studies in Uganda, Tanganyika, and Nigeria (Smithburn, 1952; Dick, 1953) also showed evidence of ZIKAV antibody prevalence but no disease.
The first evidence of human ZIKAV disease occurred in Nigeria during an outbreak of jaundice in 1954 (Macnamara, 1954). Three individuals visiting an outpatient clinic reported fever, joint pain, and headache. Virus was isolated from one patient (with no signs of jaundice) and a rise in ZIKAV-specific antibody titers was found in the other two patients. A human volunteer was later experimentally infected with the virus isolated in Nigeria and developed mild fever, headache, and malaise before fully recovering on day 7 postinfection (Bearcroft, 1956). Given the mild symptoms associated with this infection, the investigators noted that it would not be unexpected that few human cases were found. A laboratory worker in Uganda became infected with ZIKAV and showed similarly mild illness exhibiting low-grade fever, rash, and slight malaise; the patient completely recovered within 5 days (Simpson, 1964). Over the next several years, researchers in both Africa and Asia continued to look for evidence of human disease due to ZIKAV. ZIKAV-specific antibodies were found as early as 1953 in a range of locations including Malaya, Borneo, India, Vietnam, Thailand, and Angola (Smithburn, 1954; Smithburn et al., 1954; Pond, 1963; Kokernot et al., 1965), but human disease was not associated with ZIKAV. Significant human disease caused by ZIKAV would only be reported 7 decades after the initial discovery of the virus as will be described in Chapters 5 and 7.
While the descriptions provided here are the first published reports describing the discovery of both CHIK and ZIKA viruses, the viruses certainly existed before these discovery events and maintained at least zoonotic cycles if not epidemic periods. Because these agents had not been named, described, or characterized earlier, any recordings of illnesses with similar clinical patterns may have (either correctly or incorrectly) been attributed to other agents such as dengue viruses. Outbreaks may also have been caused by related viruses that exhibit similar symptoms further contributing to the confusion of whether these viruses “emerged” long before their initial scientific discovery. For example, one extensive review of written descriptions of outbreaks of “breakbone fever” postulates that many of these episodes may actually have been due to CHIKV rather than the dengue viruses (Carey, 1971) due to the descriptions of severe or incapacitating joint pain. Interestingly, some of these outbreaks occurred in the Western Hemisphere suggesting that CHIKV may have been in the Americas centuries before its official documentation of local transmission in the Caribbean in 2013. These speculations were convincingly revived as CHIKV was reported in the Americas and again led to questions about the initial entry of this virus into the West (Halstead, 2015; Kuno, 2015). (See Chapter 3). Of note, Mayaro virus (a close relative of CHIKV), which also causes painful arthralgia, has been known to exist in the Americas and could possibly have been the cause of these pre-“discovery” outbreaks of dengue-like illness. With ZIKAV, it would be even more challenging to attribute any early disease to this virus due to the high rate of asymptomatic cases combined with the mild nature of the illness in most individuals and the lack of documented disease in humans for years after the discovery. Thus, while it is intriguing to speculate on whether or not these viruses may have moved about the globe and caused large outbreaks hundreds of years before their official discoveries, it is important to note that lack of scientific and laboratory data means that these suggestions will remain only speculation.
With CHIKV, while epidemics haven’t been occurring continuously since the original description of the virus, there have been reports of sporadic human cases and small clusters of illness even during interepidemic periods. For example, after the large outbreaks of CHIKV in India in the 1960s, no major epidemics were reported for ~ 3 decades. However, serosurveys conducted in numerous countries of SE Asia and the Pacific islands indicated not only an expanded geographic range for CHIKV but evidence of recent activity in some areas (Tesh et al., 1975). Another study in sites across Indonesia demonstrated 24 distinct “outbreaks” of CHIKV over a period of 5 years; some of these were based only on clinical descriptions and some had fewer than 50 cases (Laras et al., 2005). This same report also chronicled periodic CHIKV activity during the 1970–80.
In spite of this semicontinuous CHIKV activity, true “re-emergence” would next occur when the virus caused outbreaks in two communities in Kenya in 2004 before moving to novel locations in the islands of the Indian Ocean. After this, the virus continued to move around Asia for the next decade. In 2013, the virus emerged in the Western hemisphere on the island of Saint Martin (Cassadou et al., 2014) and spread throughout the Caribbean and the Americas (Fischer et al., 2014).
ZIKAV had a true emergence event in 2007 when the first ever human epidemic due to this virus was reported on the island of Yap in the Federated States of Micronesia (Duffy et al., 2009). Over the course of the outbreak, it was estimated that 73% of the island inhabitants had been infected with ZIKAV, yet all cases were either mild or asymptomatic. After this outbreak, the virus again disappeared until 2013 when outbreaks were reported in numerous islands of the South Pacific followed by detection of the virus in Brazil in 2015 (Campos et al., 2015; Musso et al., 2015; Zanluca et al., 2015). Here, the virus not only caused epidemics of unprecedented size but caused never previously documented forms of the disease including Guillain-Barré Syndrome and microcephaly in infants infected in utero (Musso et al., 2014; Cardoso et al., 2015; Schuler-Faccini et al., 2016). Chapter 7 will describe the emergence of these viruses in the Americas in detail. The paths that chikungunya and Zika viruses followed through the Americas as they emerged are strikingly similar. Understanding the factors in common with both of these events may help us better prepare for the next arboviral priority pathogen.