Emerging Killer Viruses

ARBOVIRUS DISEASES / AETIOLOGY / EPIDEMIOLOGY / TRANSMISSION / IMMUNITY / ACTIVE IMMUNITY / PASSIVE IMMUNITY / GENERAL CLINICAL FEATURES / ECOLOGICAL FACTORS / HANTAAN VIRUS / SEOUL VIRUS / "FOUR CORNERS VIRUS" / EBOLA VIRUS / EBOLA SEROLOGY / TREATMENT OF EBOLA / PREVENTION AND CONTROL OF EBOLA /

By Byron T. Weeks, M.D.


RECENT EXAMPLES OF EMERGING AND POTENTIALLY EMERGENT VIRUSES

Most emergent viruses are zoonotic, with natural animal reservoirs a more frequent source of new viruses than is the spontaneous evolution of a new entity. The most frequent factor in emergence is human behaviour that increases the probability of transfer of viruses from their endogenous animal hosts to man. Rodents and arthropods are most commonly involved in direct transfer, and changes in agricultural practices or urban conditions that promote rodent or vector multiplication favour increased incidence of human disease. Other animals, especially primates, are important reservoirs for transfer by arthropods. Because arthropod transmission plays a very large part in infectious animal disease, specifically potential emergent virus epidemics, I will dedicate the next part of this essay to a discussion of them.

ARBOVIRUS DISEASES

Approximately 100 of the more than 520 known arthropodborne viruses (arboviruses) cause human disease. At least 20 of these might fulfill the criteria for emerging viruses, appearing in epidemic form at generally unpredictable intervals (Morse and Schluederberg 1990). These viruses are usually spread by the bites of arthropods, but some can also be transmitted by other means, for example through milk, excreta or aerosols. The arbovirus infections are maintained in nature principally, or to an important extent, through biological transmission between susceptible vertebrate hosts by blood-sucking insects; they multiply to produce viremia in the vertebrates, multiply in the tissues of the insects and are passed on to new vertebrates by the bites of insects after a period of extrinsic incubation. The names by which these viruses are known are often place names such as West Nile or Rift Valley, or are based on clinical characteristics like yellow fever.

AETIOLOGY

Most arboviruses are spherical, measuring 17-150 nm or more, a few are rod-shaped, measuring 70 x 200 nm. All are RNA viruses. Many circulate in a natural environment and do not infect man. Some infect man only occasionally or cause only a mild illness; others are of great clinical importance causing large epidemics and many deaths. Specifically, these belong to the Togaviridae, the alphaviruses, flaviviruses, the Bunyaviridae, nairoviruses, phleboviruses and other subgroups. A range of arboviruses are listed in Table 2, while some patterns of transmission are shown in Figure 1.

EPIDEMIOLOGY

Vertebrate hosts

Maintenance, incidental, link and amplifier hosts are categorized according to Stickland Hunter's Tropical Medicine (1991) as the following:

Maintenance hosts are essential for the continued existence of the virus, usually living in symbiosis with the viruses, without actual disease, but they do develop antibodies. These include birds such as the prairie chicken, pigeon and wood thrush which transmit Eastern and Western equine encephalitis; heron and egrets transmitting Japanese encephalitis and migrating birds which travel over long distances carrying these and other similar viruses; rodents and insectivores such as rats, hedgehogs, lemmings and chipmunks are known to carry louping ill and Colorado tick fever; primates such as monkeys which carry Dengue fever; Leporidae (rabbits and hares) which carry Californian encephalitis; Ungulates (cattle and deer) which are implicated in the transmission of European tick-borne encephalitis; bats which carry Rio Brava virus; and marsupials, reptiles and amphibia such as kangaroos and snakes which also harbour encephalitis-causing viruses.

Incidental hosts become infected, but transmission from them does not occur with sufficient regularity for stable maintenance. Man is usually an incidental host, often, but not always, being a dead end in the chain. These hosts may or may not show symptoms. Link hosts bridge a gap between maintenance hosts and man, for example, between small mammals and man by goats (via milk) in tick-borne encephalitis. Amplifier hosts increase the weight of infection, as is the case with pigs which act between wild birds and man in Japanese encephalitis.

The populations and characters of the vertebrate hosts and their threshold levels of viraemia are important. Small rodents multiply rapidly and have short lives, thus providing a constant supply of susceptible individuals. In contrast, monkeys and pigs multiply slowly, and once they have recovered from an infection, remain immune for life. African monkeys are relatively resistant to Yellow Fever, but Asian and American monkeys are susceptible, probably because, unlike the African monkeys, they have not been exposed continuously for centuries to the infection. Also, possibly related arboviruses may offer partial immunization.

Invertebrate hosts

Mosquitoes, sandflies and ticks may imbibe virus from a vertebrate in a state of viraemia, after which the virus undergoes an incubation period within the arthropod, known as the extrinsic incubation period. In mosquitoes this period is short: 10 days at 30o C ambient temperature and longer at lower temperatures. Mosquitoes remain infective for life without any apparent ill-effects. In fact, their infectivity appears to increase with time after infection and their effectiveness as transmitters depends upon the frequency with which they bite. It is also possible that arthropods, whose mouth parts are contaminated by virus in the act of feeding, could transmit the virus mechanically if they feed soon afterwards on another animal. For instance, chikungunya virus can be transmitted mechanically by A. aegypti for 8 hours after infection. In general, mosquito-borne viruses may not use ticks as vectors nor can tick-borne viruses reside in mosquitoes.

TRANSMISSION

Arthropod transmission involves several stages:

1) ingestion by the arthropods of virus in the blood (usually) or tissue fluids of the vertebrate hosts;

2) penetration of the viruses into the tissue of the arthropods, in the gut wall, or elsewhere after passing through the gut barrier;

3) multiplication of the viruses in the arthropod cells, including those of the salivary glands.

Stage 2 and part of stage 3 represent the extrinsic incubation period of the disease (Hunter 1991).

The quantity of blood, and therefore the amount of virus ingested, seems to be important as each arthropod species must ingest a minimum quantity of a given virus before multiplication can take place. The same mosquito species can have two different thresholds for two different viruses and if one species has a low threshold, other species may have high thresholds or may be completely resistant. This threshold phenomenon is extremely important in determining the efficiency of a vector and may also vitally affect the course of an epidemic. Viruses reportedly persist in overwintering mosquitoes, while transovarial passage of virus has been seen in some tick species. For mosquitoes the availability of suitable breeding places (and therefore rainfall) is a major factor. An efficient vector may have a wide range of animals on which to feed, but if the arthropod species is abundant, and even if it bites man only infrequently in the presence of other (and preferred) animals, the large numbers enable it to maintain transmission to man. For example, Culex tritaeniorhynchus, which mostly bites birds, Bovidae, dogs and especially porcines, and only to a limited extent man, can maintain transmission of Japanese encephalitis from pigs to man by sheer numbers.

Although transmission of arboviruses usually takes place through the bites of arthropods, Lassa virus, for example, may be transmitted through contact with excreta of infective rodents, and others via urine or faeces infecting the nasopharynx, some through aerosol from a patient or others by one bird pecking another.

IMMUNITY

After a vertebrate has been infected, the arbovirus probably multiplies first in the regional lymph glands where the earliest formation of antibodies also probably takes place. Some do not produce high titres of antibodies in man and some antibodies are short-lived or appear late. In diagnosis, haemagglutination-inhibiting and complement-fixing antibodies are important, but the only protective antibody is of the neutralizing type, which is also the most specific.

Arboviruses are grouped according to antigenic characters, but after inoculation of one virus into a fresh animal, not only the homologous antibodies, but also heterologous antibodies reacting with other viruses of the same group tend to appear. Recovery from an infection by a member of one group of arboviruses may provide some degree of resistance to a susbsequent infection by another member of the same group. For example, infection with West Nile virus may have modified the Ethiopian epidemic of Yellow Fever in 1962. Again, the effect of prior infection with Zika, Uganda S and other related viruses in the forest belt of Nigeria, leading to a high incidence of related antibodies, is suggested as the explanation of the absence of epidemic Yellow Fever in man in that area. These related infections probably modify the disease rather than prevent infection.

ACTIVE IMMUNITY

With Yellow Fever, neutralizing antibodies can be found as early as a few days after the beginning of the disease and are found constantly for many years in the sera. The persistence of immunity does not depend on exogenous reinfection. It is probable that a mosquito infected with Yellow Fever is not harmed by it, but continues to excrete the virus throughout life. This means a continuous supply and release of virus, probably from the epithelial cells of the salivary glands. The virus enters man (or other animals) and gains the liver and other epithelia, provoking the early antibodies in the blood, which neutralize circulating viruses. But, as suggested by Hunter (1991), antibodies which can be detected for so many years in man must stem from a continuing stimulus, and the sensitive cells and their progeny probably have a prophase equivalent of the virus incorporated into their genome, with occasional reversion to productive development which provides the stimulus for further antibody formation. A degree of immunity of this kind may possibly be provided when a related virus invades epithelial cells.

PASSIVE IMMUNITY

Infant rhesus monkeys and human infants born of mothers immune to Yellow Fever have transient protective antibodies in their sera at birth which persist for several months. They are probably placentally transferred, rather than coming from the mother's milk, because antibodies may disappear from infant sera while they are still suckling. Passive immunity induced by injection of homologous immune serum, has been used for protection against tick-borne encephalitis in cases of special risk and similar sera could be used against other infections, particularly after laboratory or hospital accidents.

GENERAL CLINICAL FEATURES

Most arbovirus infections are inapparent, that is they produce no symptoms or often only mild ones (fever and occasional rash). For example, in an epidemic of Japanese encephalitis it was estimated that for each case of apparent disease there were 500-1000 inapparent infections. If clinical manifestations arise after infection they do so after an intrinsic incubation period lasting from a few days to a week or more. Some arboviruses damage the endothelial lining of the capillaries increasing permeability which allows the virus to pass the blood brain barrier causing meningoencephalitis. Others damage the parenchymatous organs by direct damage to the cells in which they are situated, while with others damage is caused by the immune system of the host from the formation of antigen- antibody complexes and disordered complement formation which damage the renal tubules and alter the coagulation and fibrinolytic systems of the body causing haemorrhage (viral hemorrhagic fevers). There is a general pattern of biphasic illness, the first phase associated with viremia ending when antibodies appear in the blood and the second phase when the virus is located in organs, such as the liver or brain.

The onset of clinical manifestations is usually abrupt, generally occurring after the onset of viraemia. Fever is usual and is sometimes the only sign. In many cases the clinical manifestations last only while the virus is disseminated, but in other cases there is remission, short or long. If long, the disease is biphasic. After this, fever returns with signs indicating localization of the virus in certain organs. If the period of viraemia has been symptomless and the virus becomes localized in the central nervous system, encephalitis appears. In hemorrhagic cases there is a special risk of shock which can rapidly become irreversible unless promptly treated (Hunter 1991).

ECOLOGICAL FACTORS

Microorganisms and viruses are adapted to extremely diverse econiches. One of the most complex sets of adaptive characteristics concern arthropod transmission of viruses. The arthropod-borne viruses are spectacular examples of emergence and re-emergence resulting from innocent environmental manipulation or natural environmental change. Deforestation, amateur irrigation and the introduction of new species (usually livestock) gives rise to many virus disease threats of humans and animals. Important aspects of ecological change and their relation to arbovirus life cycles are:

1) Population movements and the intrusion of humans and domestic animals into new arthropod habitats, particularly tropical forests;

2) Deforestation, with development of new forest-farmland margins and exposure of farmers and domestic animals to new arthropods;

3) Irrigation, especially primitive irrigation systems, which are oblivious to arthropod control;

4) Uncontrolled urbanization, with vector populations breeding in accumulations of water (tin cans, old tires etc.) and sewage;

5) Increased long distance air travel, with potential for transport of arthropod vectors;

6) Increased long-distance livestock transportations, with potential for carriage of viruses and arthropods (especially ticks); and

7) New routing of long-distance bird migration brought about by new man-made water impoundments (Murphy 1994).

To illustrate the effect ecological change can have on the emergence of a new disease and the course of it afterwards one can look to dengue, one of the most rapidly expanding diseases in tropical parts of the world, with millions of cases occurring each year. For example, Puerto Rico had five dengue epidemics in the first 75 years of this century, but has had six epidemics in the past 11 years, at an estimated cost of over $150 million. Simultaneously, Brazil, Nicaragua and Cuba have had their first major dengue epidemic in over 50 years, involving multiple virus types. At the lethal end of the dengue spectrum is dengue haemorrhagic fever, first occurring in the Americas in 1981. Since then, 11 countries have reported cases, and since 1990 over 3000 cases have been reported annually. Figure 3 illustrates the extent of dengue occurrence globally.

The primary reason that dengue is emerging and re-emerging is vector control. National priority lists are political in nature and tend to emphasize daily problems, not episodic ones. Expensive mosquito control tends to fall off the bottom of the list. Meanwhile, as older cheaper chemicals lose effectiveness or are banned, new and expensive chemicals replace them. Before 1970, A. aegypti, the vector of dengue and Yellow Fever, was targeted for regional or even global eradication through the use of DDT (dichlorodiphenyltrichloroethane) (Murphy 1993). Obviously, this solution is no longer applicable, but nothing has effectively supplanted it.

CASES IN POINT

HANTAAN VIRUS

The hantavirus (mentioned above) is also the focus of much international attention. During the Korean War of 1950-1952, thousands of United Nations troops developed a mysterious disease marked by fever, headache, hemorrhage and acute renal failure; the mortality rate was 5-10%. Despite much research, the agent of this disease remained unknown for 28 years, when a new virus, named Hantaan virus, was isolated in Korea from field mice. Recently, related viruses have been found in many parts of the world in association with different rodents and as the cause of human diseases with a variety of little-known local names. Epidemic haemorrhagic fever, one of the most important diseases in China, causes more than 100000 cases per year. Transmission to humans is primarily by inhalation of aerosolized excreta. In May 1993 a cluster of deaths in the southwestern United States set in motion a multiagency local, state and federal investigation that led to the discovery of a highly pathogenic hantavirus and to the definition of a new clinical syndrome (Peters 1994).

SEOUL VIRUS

Another virus of current interest in the USA, Seoul virus, was identified about 10 years ago in Korea as a Hantaan-like virus whose natural host is the urban rat. Serologic surveys detect it worldwide, including seroprevalence rates of 12% in urban rats in Philadelphia and about 64% in Baltimore rats (Le Duc 1986). Although acute hemorrhagic fever was not identified in inner-city Baltimore, 1.3% of 1148 local residents were antibody-positive and the possibility of viral association with chronic renal disease is under study.

"FOUR CORNERS VIRUS"

The disease hantavirus pulmonary syndrome (HPS), is characterized by an initial fever followed by the abrupt onset of acute pulmonary edema and shock. After recognition of the initial cases by observant clinicians in the Southwest, investigations were swiftly mounted by local university and public health workers but, in spite of efficient and competent studies, failed to find the cause. By the time the CDC became involved, a number of possible causative agents had been ruled out, leading most of the investigators to believe they were dealing with a new entity. This observation led to a broadly based approach to the field epidemiology and the laboratory study of the disease. Samples from the field investigations were distributed among many different laboratories of the National Center for Infectious Disease (NCID) for analysis by the most sensitive classic and modern molecular biological tests for a wide range spectrum of infectious agents.

Somewhat surprisingly, successful results were obtained after only a few days of straightforward serologic tests for hantaviruses. The hemoconcentration, thrombocytopenia and shock observed in some of the patients had raised speculation about the involvement of these viral agents; however they had been previously known as associated with renal syndromes only. The serologic results came from established techniques such as indirect fluorescent-antibody assays and enzyme-linked immunosorbent assays. The next steps utilized reverse transcription and PCR amplification of RNA in postmortem tissue samples (60% of confirmed cases to date have been fatal), using consensus primers based on known hantavirus RNA sequences. These yielded products with sequences typical of hantavirus but clearly different from any known member of the genus. This provided additional evidence for the hantavirus etiology and linked the new hantavirus closely to the human disease by its presence in the tissues of people dying of the infection. Using the genomic sequences from human tissues, investigators were subsequently able to implicate the deer mouse as the principle reservoir of the virus.

Hantaviruses have traditionally been difficult to propagate, and this one was no exception. Thus a full-length cDNA clone of the small RNA segment of the virus was synthesized. This technique provided a diagnostic reagent of increased sensitivity that could be made widely available. Eventually, full length RNA sequences were developed for the medium segment and a partial sequence was determined for the large segment, permitting the definitive determination that the new virus, isolated weeks later and registered as Muerto Canyon virus, was not a reassortant of any known hantavirus.

Immunohistochemical identification of hantavirus antigens and in situ hybridization with genomic sequences also confirmed the hantavirus etiology of the syndrome. The extensive presence of antigen in pulmonary capillaries provided an explanation for the pathophysiology and target organ specificity differing from that of other known disease-causing hantaviruses. This method, when applied to paraffin- imbedded tissues, has also served as a retrospective diagnostic tool, firmly identifying fatal cases from 10 to 15 years ago.

The rapid recognition of the hantavirus etiology of this disease was important in that it alleviated heightened fear among the general American population, and saved lives by focusing public health recommendations on the avoidance of contact with potentially infected rodents. Different hantaviruses have been isolated in Louisiana, Florida and also Brazil, indicating the uncommon, yet widespread nature of this disease. Recently (Diglisic 1994), isolation of a hantavirus from Mus musculus captured in Yugoslavia was reported.

As stated by C.J. Peters, chief of the Special Pathogens Branch of the Division of Viral and Rickettsial Diseases at NCID, the crucial role of modern techniques in virology was possible only in a context of past hantavirus research, and as part of efforts of a multidisciplinary team of clinicians, epidemiologists, field ecologists and classic microbiologists. The need for basic research is highlighted by the applied practical success which resulted from it, as was the case in identifying a new strain of hantavirus. Future research will need to investigate the molecular mechanisms for induction of pulmonary edema and an appropriate blocking therapy. The evolutionary relationships of the hantaviruses and their rodent host specificity must be understood to predict the future course of transmission, and finally the basis for the different tropisms of the viruses must be examined at a molecular level.

EBOLA VIRUS

Ebola virus, a member of the Filoviridae, burst from obscurity with spectacular outbreaks of severe, haemorrhagic fever. It was first associated with an outbreak of 318 cases and a case-fatality rate of 90% in Zaire and caused 150 deaths among 250 cases in Sudan. Smaller outbreaks continue to appear periodically, particularly in East, Central and southern Africa. In 1989, a haemorrhagic disease was recognized among cynomolgus macaques imported into the United States from the Philippines. Strains of Ebola virus were isolated from these monkeys. Serologic studies in the Philippines and elsewhere in Southeast Asia indicated that Ebola virus is a prevalent cause of infection among macaques (Manson 1989).

These threadlike polymorphic viruses are highly variable in length apparently owing to concatemerization. However, the average length of an infectious virion appears to be 920 nm. The virions are 80 nm in diameter with a helical nucleocapsid, a membrane made of 10 nm projections, and host cell membrane. They contain a unique single-stranded molecule of noninfectious (negative sense ) RNA. The virus is composed of 7 polypeptides, a nucleoprotein, a glycoprotein, a polymerase and 4 other undesignated proteins. Proteins are produced from polyadenylated monocistronic mRNA species transcribed from virus RNA. The replication in and destruction of the host cell is rapid and produces a large number of viruses budding from the cell membrane.

Epidemics have resulted from person to person transmission, nosocomial spread or laboratory infections. The mode of primary infection and the natural ecology of these viruses are unknown. Association with bats has been implicated directly in at least 2 episodes when individuals entered the same bat-filled cave in Eastern Kenya. Ebola infections in Sudan in 1976 and 1979 occurred in workers of a cotton factory containing thousands of bats in the roof. However, in all instances, study of antibody in bats failed to detect evidence of infection, and no virus was isolated form bat tissue.

The index case in 1976 was never identified, but this large outbreak resulted in 280 deaths of 318 infections. The outbreak was primarily the result of person to person spread and transmission by contaminated needles in outpatient and inpatient departments of a hospital and subsequent person to person spread in surrounding villages. In serosurveys in Zaire, antibody prevalence to Ebola virus has been 3 to 7%. The incubation period for needle- transmitted Ebola virus is 5 to 7 days and that for person to person transmitted disease is 6 to 12 days.

The virus spreads through the blood and is replicated in many organs. The histopathologic change is focal necrosis in these organs, including the liver, lymphatic organs, kidneys, ovaries and testes. The central lesions appear to be those affecting the vascular endothelium and the platelets. The resulting manifestations are bleeding, especially in the mucosa, abdomen, pericardium and vagina. Capillary leakage appears to lead to loss of intravascular volume, bleeding, shock and the acute respiratory disorder seen in fatal cases. Patients die of intractable shock. Those with severe illness often have sustained high fevers and are delirious, combative and difficult to control.

EBOLA SEROLOGY

The serologic method used in the discovery of Ebola was the direct immunofluorescent assay. The test is performed on a monolayer of infected and uninfected cells fixed on a microscopic slide. IgG- or IgM-specific immunoglobulin assays are performed. These tests may then be confirmed by using western blot or radioimmunoprecipitation. Virus isolation is also a highly useful diagnostic method, and is performed on suitably preserved serum, blood or tissue specimens stored at -70oC or freshly collected.

TREATMENT OF EBOLA

No specific antiviral therapy presently exists against Ebola virus, nor does interferon have any effect. Past recommendations for isolation of the patient in a plastic isolator have given way to the more moderate recommendation of strict barrier isolation with body fluid precautions. This presents no excess risk to the hospital personnel and allows substantially better patient care, as shown in Table 2. The major factor in nosocomial transmission is the combination of the unawareness of the possibility of the disease by a worker who is also inattentive to the requirements of effective barrier nursing. After diagnosis, the risk of nosocomial transmission is small.

PREVENTION AND CONTROL OF EBOLA

The basic method of prevention and control is the interruption of airborn, and contact by person to person spread of the virus. However, in rural areas, this may be difficult because families are often reluctant to admit members to the hospital because of limited resources and the culturally unacceptable separation of sick or dying patients from the care of their family. Experience with human disease and primate infection suggests that a vaccine inducing a strong cell- mediated response will be necessary for virus clearance and adequate protection. Neutralizing antibodies are not observed in convalescent patients nor do they occur in primates inoculated with killed vaccine. A vaccine expressing the glycoprotein in vaccinia is being prepared for laboratory evaluation.

SUMMARY of Symptoms: Fever is prominent. Intially the patient coplains of severe aching, scratchy throat, headache, fatigue and fever. Physicians generally diagnose "flu." However, the fever and aching worsen. Headache rapidly becomes severe, and small hemorrhages called petichiae appear on the skin, usually where clothing is tight, such as the beltline and under the bra. As the virus attacks the lining of small blood vessels, internal bleeding supervenes. Then severe vomiting with black and red blood, and bloody diarrhea. Due to marked general capillary hemorrhage and seepage of serum into the body spaces, there will be progressive fall in blood pressure and shock. The organs are attacked and dissolve rapidly. the kidneys first usually, with failure of function, then the liver, heart, and brain with rapid onset of coma, convulsions, shock and death. The body becomes a blackened sack of skin containing bloody fluid, dissolved muscles and organs, and bones. TREATMENT: CONSISTS OF ICE BAGS, MORPHINE FOR SEVERE TOTAL BODY PAIN, INTRAVENOUS FLUIDS, AND BLOOD TRANSFUSIONS. There is no specific therapy. Antibiotics are ineffective. Death rate in the recent outbreak in Zaire was 90%.

Byron T. Weeks, MD