Viral Haemorrhagic Fevers (Perspectives in Medical Virology)

This process took nearly a century after the Americas were first discovered, most likely because of the length of the sea voyage. An acute infection is dependent upon mosquitoes for transmission, and those persons infected before departure succumbed well before landfall. Only by maintaining the transmission cycle several times via shipboard 1The term quarantina is derived from the Italian quaranta, meaning 40 days, a measurefirst introduced by the Venetian Republic in There is a contrary view that yellow fever moved from the Americas to Africa based on accounts of a disease resembling yellow fever before Europeans reached the New World.

This is highly unlikely, especially given the high susceptibility of the indigenous population of the time and the susceptibility of New World monkeys to yellow fever virus. This susceptibility of New World primates to yellow fever virus contrasts sharply with the situation among Old World monkeys where yellow fever virus is very much in equilibrium with its animal reservoirs. There is a myth that Africans are less susceptible to yellow fever virus, but the reality is that yellow fever is part of the normal repertoire of infectious diseases that children are exposed to and from which recovery is the normal outcome.

Thus, much of the indigenous population acquires immunity in the first years of life that extends into adulthood. Yellow fever first appeared in in Barbados, quickly spreading to other parts of the Caribbean and the Yucatan Peninsula. Over the next two centuries it spread throughout Central America, south to Brazil and as far north as New York and Boston. European ports also suffered when ships berthed on return from the Americas. The effect on troops and settlers in the Caribbean was particularly devastating.

In France sent a force of around men to take St Lucia from the British but only 89 survived 17 the ravages of yellow fever and other diseases. Nearly a century later, the English Admiral Vernon in lost nearly half of a 19, strong force intent on taking Cartegena in present-day Columbia perished as a direct result of the disease. In the closing years of the 19th century the United States government decided it was high time to root out the cause of yellow fever.

The germ theory of disease had begun to be widely accepted and new causes of infectious disease were being described with almost monotonous regularity. There he was joined among others by Carlos Finlay. Carlos Finlay, born of Scots and French parents, had experienced at first-hand the tragic effects of yellow fever when training in Philadelphia in Reed and his colleagues first explored a bacterium proposed earlier by Giuseppe Sanarelli as the likely agent: Given the ascendancy and zeal of bacteriologists at the time, Reed and his fellow investigators were under considerable pressure to confirm a bacterium as the cause.

How Reed and his team hit upon the concept of insect transmission is one of the great debates of medical endeavour. The widely quoted view is that Reed was aware of the work emanating from Ronald Ross in China whose studies on malarial transmission by mosquitoes were published a few years earlier in Two British physicians working in Havana at the same time as Reed almost certainly drew Reed's attention to the study of Ross.

Reed made the connection, perhaps, that yellow fever often occurred when malaria was also prevalent. But the input of Carlos I Finlay also needs acknowledging as he was aware of the investigations of Patrick Manson who described the role of mosquitoes in transmitting filariasis years earlier. Indeed mosquito transmission had been suggested as early as by John Crawford of Baltimore, a hypothesis reiterated by Joshua Nott in However the concept originated, Reed and his co-workers set up carefully controlled experiments, the outcome of which demonstrated unambiguously the role of mosquitoes in the transmission of yellow fever.

Importantly for the time, they also showed that the agent was a "filterable virus" and not a bacterium. Reed's work in Cuba led to intensive efforts to controlling urban yellow fever by reducing opportunities for individuals to be bitten by infected Aedes mosquitoes. Notwithstanding rural infections continued to occur and in response the Rockefeller Foundation's Commission for International Health embarked upon a programme of global eradication.

To this end, the Commission went to Guayacil in Ecuador where yellow fever was still very much in evidence. Hideyo Noguchi soon reported he had isolated the causative organism in guinea pigs, calling it Leptospira icteroides. He raised a therapeutic antiserum which he persuaded himself and those around him to have the properties of a passive prophylactic. Doubts as to the cause of yellow fever lingered, however, and in the early s the Commission decided to re-evahiate the cause of yellow fever, but this time focusing efforts on West Africa, long regarded as the source of the disease.

A permanent research station was established at Yaba, close to Lagos, and it was there that Adrian Stokes, an Irish physician appointed in to the chair in pathology at Guy's Hospital Medical School, successfully showed the viral nature of yellow fever by transmission to crown 18 monkeys Macacus simicus from India using field isolates from Latch near Accra in present-day Ghana. After an incubation period of 2 - 6 days a period of high fever followed leading to eventually to collapse and death.

Pathological changes were reproducible and re-transmission occurred after passing monkey serum from acutely ill animals through Berkefield filters. This early work in Ghana has recently been reviewed by Mortimer This important work established the foundations whereby Sawyer in New York was able to develop what we now know as the 17D vaccine strains of yellow fever virus. Tragically Adrian Stokes succumbed to yellow fever, as had Noguchi, in Accra in These strategies prompted transferral of further work to the comparatively more controlled environment of New York.

Yellow fever continued to frustrate the aspirations of Europeans in settling and exploiting the Americas and Africa until well into the 20th century. It was common for seaman to become infected visiting the shorelines of these tropical zones but the victims inevitably perished at sea.

The mosquitoes were hardier, however, and transovarial transmission ensured that infected insects could come ashore once ships had berthed in the northern hemisphere. One such episode occurred in September when the barque "Hecla" docked in Swansea when the weather was unseasonably warm Meers, The "Hecla" had loaded a cargo of copper ore in Cuba as well as infected mosquitoes.

Despite deaths amongst the crew whilst en route, the captain did not quarantine the ship before berthing. The result was a total of 27 infections and 15 deaths amongst those of the local population living or working within m of the "Hecla". Meets estimates that a maximum of 10 infected mosquitoes would have sufficed to cause this local outbreak. Although the diagnosis at this time would have been entirely on clinical observations, this outbreak almost certainly was due to yellow fever.

It illustrates vividly how readily arboviruses can be transmitted across oceans, as was seen again as recently as when West Nile virus was introduced into the USA. Epidemiology Yellow fever is confined to the tropical regions of Africa and the Americas.

Viral Haemorrhagic Fevers, Volume 11 (Perspectives in Medical Virology)

Persistence of the disease is dependent upon cyclical transmission between monkeys and humans with mosquitoes as vectors. Thus, the epidemiology of the disease is driven by a series of complex interactions between the virus, its arthropod vectors and reservoir hosts. These interactions give rise to two discrete transmission cycles, with marked variations between the cycles in Africa and South America. These interactions are summarised in Fig. In Africa, yellow fever virus infects principally Cerepithecus and Colobus species, and in West Africa is transmitted by either Aedesfurcifer-taylori or Aedes luteocephalus.

In East and Central Africa the cycle is maintained principally by Aedes simpsoni. African non-human primates are relatively resistant to the infection and most recover. The associated viraemia is relatively short lived and therefore the chances of mosquito transmission are lessened. The distinct nature of the cycle between West and East Africa reflects the evolution of yellow fever virus in association with its host over a long period of time. These profiles are in accord with the distinctive genotypes of yellow fever virus recovered from patients in these different localities on the African continent.

In South America the sylvatic cycle is quite distinct.

The virus is found in Aloutta, Ateles, Callethrix, Cebus and Saimiri monkeys and is frequently lethal. Widespread outbreaks occur centred on the river basin draining the Amazonian rain forest. Current thinking is that the virus was introduced into the rain forest ecosystem from urban outbreaks, aided by the adaptation of yellow fever virus to several species of treedwelling Haemagogus mosquitoes.

As in the African cycle, mosquitoes remain infected for life once having bitten an infected monkey, with the virus passing transovarially to larvae. Humans only become infected by the bites of such insects on clearing trees or if the insect population spills over at the margins of forested areas into rural communities. This is increasingly the case in Africa where the monkey populations have declined as human modification of their habitat has accelerated.

The margins of forest and savannah in Africa give rise to zones of emergence where during the rainy season the chance of human infection intensifies as vector numbers dramatically increase, only to decline once more during the dry season. It is at these margins, particularly after prolonged drought, that yellow fever re-emerges with serious consequences, especially among children with no immunity. The urban cycle This is maintained by peri-domestic mosquitoes such as A. Efforts to eradicate this vector in Central and South America in the second half of the 20th century were largely successful in ensuring urban areas became free of yellow fever.

Cessation of mosquito eradication programmes out of environmental concerns, however, has resulted in insect levels being restored to near pre-eradication levels in many areas. As yet, there has not been a resurgence of urban yellow fever in the Americas, mainly as a result of vigorous vaccination programmes. Humans entering forested areas infested by virus carrying infected mosquitoes become bitten by female insects.

These live from 70 to days and have a flight range of over m. Eggs are laid in still water, and can be disseminated readily in pots, crevasses and old car tires. In recent decades, yellow fever has been a far bigger public health problem in Africa, a continent where mosquito eradication has not been widely practiced and immunisation tends not to be widespread. Outbreaks in East Africa are generally few and far between, although the largest outbreak ever recorded occurred in southwestern Ethiopia in , claiming around 30, lives with more than , people infected. This outbreak had a severe impact in such a sparsely populated region of less than one million.

With the exception of the outbreaks in Nigeria when over 44, cases were recorded, outbreaks in West Africa have occurred more often but tend to be limited in scope. Yellow fever has gradually spread from countries such as Crte D'Ivoire, Burkino Faso and Cameroon to Gabon, Liberia and Kenya, countries thought to be free of infection before the middle of the 20th century. Properties of yellow fever virus Morphology Virions are spherical and approximately 50 nm in diameter with an outer lipid membrane enclosing an inner nucleocapsid Fig.

Mature virions contain two membrane proteins, E and M.

Detailed structure analysis of tick-borne encephalitis virus has revealed, for this flavivirus at least, that the E protein is arranged as dimers orientated 21 Fig. Thus, the flaviviruses do not show surface projections as is often the case for enveloped virus, such as influenza family Myxoviridae and rabies family Rhabdoviridae. Both the arrangement of these dimers and the underlying nucleocapsid conform to the principles of icosahedral symmetry.

Immature virions differ in that prM protein replaces the M protein. Genetic organisation and gene expression The flavivirus genome is an approximately 11 kb R N A molecule of positive sense 2 with respect to protein translation. In c o m m o n with the picornaviruses, the viral genes are first expressed by synthesis of a large polyprotein Fig. This single precursor molecule then undergoes a series of cleavages thereby generating functional proteins. Cleavages are mediated either by the host signal peptidase present in the lumen of the endoplasmic reticulum or by a viral serine protease.

The complementary sequence of nucleotide bases arising from the replication process cannot be used directly for protein synthesis and is thus of negative sense with respect to ribosomal translation i. This internal ribosome entry event required for translating the viral genome is common to both flaviviruses and picornaviruses: A total of 10 proteins are expressed as a result of the processing of the polyprotein precursor.

Polyprotein processing confers the advantage that gene expression can be controlled by the rate and extent to which these cleavage events occur. In addition, the use of alternative cleavage sites results in proteins with stretches of amino acid homology but different functions. This form of viral protein synthesis is likely inefficient, however, with some gene products being produced surplus to the requirements of virus replication. Structural proteins The two viral envelope proteins, E and M, are type I integral membrane proteins with C-terminal anchor sequences.

By analogy with tick-borne encephalitis virus, the E protein consists predominantly of [3-sheets arranged in a head-to-tail configuration with the distal ends of each monomer embedded in the lipid membrane Post et al. The E protein has both receptor binding haemagglutin and acid pH-dependent cell fusion activities, and composed of three structural domains. The third domain IIl contains a fold typical of an immunoglobulin constant domain and it is this domain that is thought to represent the cell receptor.

There is considerable variation in amino acid sequence at the margins of this domain between tick- and mosquitovectored flaviviruses. Some mutations in domain III equate with changes in virulence. In the case of yellow fever virus, there is one report that suggests a region of domain II may also be involved in binding virus to receptors present in monkey brains Ni et al.

Work on the structure of dengue virus E protein has progressed substantially over the past few years see below and comparative analyses should throw light on the detail of yellow fever receptor binding and substitutions to amino acids that equate to tissue tropism and attenuation. Non-structural proteins Despite the simplicity of protein expression, almost all of the non-structural proteins serve more than one function at some stage during the replication process.

All seven are involved at various steps of RNA synthesis, although little is known as to how these interact with one another, and how each relates to those host proteins required for gene expression and RNA synthesis. The explosion of hepatitis C research, also a flavivirus, t over the past 10 years has had a beneficial effect generally on our understanding of how ftavivirus non-structural proteins function.

NS1 is an interesting protein, being glycosylated and essential for virus viability yet not found in the virus particle. NS1 does not associate with mature virions, but locates in membraneassociated RNA complexes and thus promotes negative strand synthesis. Transcomplementation studies using NS 1 deletion mutants of yellow fever virus and a replicon of Sindbis virus expressing the NS1 of dengue virus have shown that this interaction is virus specific, although variants of yellow fever virus have been detected that acquire recognition of dengue virus NS 1 as a result of a single base change in the NS4A gene Lindenbach and Rice, , NS 1 is secreted by mammalian cells as a hexarner consisting of three homodimers.

NS 1 is not, however, secreted from infected mosquito cell lines Smith and Wright, The extent of glycosylation and the processing of these sugar side chains contribute to the final structure of the resulting NS 1 dimers. Variable glycosylation may also account for the difference in secretion properties of NS 1 from mammalian and insect cells: Thus, complete processing of exposed, sugar side chains appears essential for NS 1 secretion.

Neither of these gene products contains motifs homologous to other known mammalian or viral enzymes. Two forms of NS2A are known: Mutations at the C-terminus of either form are lethal for yellow fever virus replication Kummerer and Rice, The NS2B protein has two functional activities. The second function utilises separate hydrophobic domains to facilitate the insertion of the NS2B-NS3 precursor into the endoplasmic reticulum at the time of translation.

NS4B, in contrast, is readily seen, particularly in the perinuclear region. Late in the replication cycle NS4B can be found within the nucleus. NS3 has two discrete functions. The first function is required for processing the polyprotein resulting from translation of the positive viral RNA strand. One-third of the N-terminal folds to form a serine protease.

This part of the molecule contains motifs characteristic of the trypsin superfamily of proteases but in order to fulfil this proteolytic function prior coupling to NS2B is required, as mentioned above. Furthermore, efficient polyprotein processing requires binding to cellular membranes. The protease complex removes the anchor region from the C protein and recognises several other cleavage sites along the length of the polyprotein. All share a common motif consisting of two basic amino acids followed by an amino acid with a short side chain. The heightened concern with regard to the increasing prevalence of persistent hepatitis C in the human population has stimulated work examining the NS3 protease of hepatitis C virus as a target for antivirals.

This protein, along with other hepatitis C virus proteins, has now been analysed in considerable depth. The availability of structural coordinates allows for modelling of other NS3 proteins by threading and similar algorithms. Following this approach, a model of dengue NS3 has shown there are some differences in the substrate binding domain of the dengue virus NS3 protein Brinkworth et al. A fully functioning helicase is essential for flavivirus replication.

The consensus is that the helicase plays a role in unwinding the secondary structure at the 3 t end of the viral positive strand template prior to the commencement of RNA synthesis, and perhaps also in releasing the nascent negative RNA strand prior to commencement of positive strand synthesis. This domain overlaps the protease activity between residues and of the dengue virus NS3 molecule Li et al.

NS3 interacts also with NS5, an interaction dependent on phosphorylation. The domains responsible for this interaction in dengue virus have been mapped to amino acids on NS5, and on NS3 NS5 is the largest and most highly conserved of the non-structural proteins and constitutes the RNA-dependent RNA polymerase.

Although with an overall basic charge, NS5 has long hydrophobic stretches that are more characteristic of a membrane bound protein. A guanyltransferase would also be needed, but this activity has yet to be identified. NS5 has been detected both in the cytoplasm and in the nucleus of infected cells. The nuclear form of NS5 is extensively phosphorylated. Translocation of NS5 into the nucleus is mediated by a nuclear localisation signal sited between the methyltransferase and polymerase domains.

It is not known as what effect nuclear NS5 has on cellular functions, although differential phosphorylation of this protein during the replication cycle suggests nuclear-located NS5 has a role in the later stages of the replication cycle. Its presence in the nucleus may account for the basophilic staining bodies seen in infected cells. A further complexity is the reported interactions between NS5 and the nuclear transport receptor importin-[3. Two glycoprotein receptor molecules have been proposed for dengue 4 virus, and it may prove to be that entry requires more than one host component Salas-Benito and del Angel, As flaviviruses also infect arthropod vectors, the cellular receptor for both mammalian cells and insect cells either has to be highly conserved or involve two separate domains.

Flavivirus particles enter the host cell by a process of receptor-mediated endocytosis followed by fusion at low pH of the viral envelope with the membrane of an endosomal vesicle. The nucleocapsid is then released into the cytoplasm. As most of the replication cycle takes place at or near the perinuclear membrane, there must be some mechanism of transporting the nucelocapsid through the cytoplasm, but at present this mechanism remains obscure.

Translation usually begins as a result of internal ribosome entry at the first AUG codon of the single open reading frame, although there is evidence of occasional initiation at the next AUG some nucleotides downstream. After primary translation of the infecting viral genome, RNA synthesis begins by production of 26 negative strand copies by the NS5 protein. Negative strand synthesis continues throughout the replication cycle. These negative copies are then used as templates for the generation of further positive RNA strands.

RNA replication complexes are localised in the perinuclear endoplasmic reticulum and consist of both RNA double-stranded duplexes and replicative intermediates, the latter consisting of double-stranded regions and nascent single-stranded RNA molecules. These are either used as further plus strand templates or for translation. There are similarities here with poliovirus replication with the steady accumulation of structural proteins during the flavivirus replication cycle.

However, there seems to be a distinct compartmentalisation between polyprotein processing and RNA synthesis, and possibly moderated by such a topographical separation. Hypertrophy and proliferation of cytoplasmic membranes are characteristic of flavivirus-infected cells. Nascent virus particles first assemble on the rough endoplasmic reticulum, and then these immature virions are transported progressively through the endoplasmic reticulum compartments to the cell surface where virus particles are released by exocytosis. Immediately prior to release the prM protein located within the viral envelope is cleaved by a host furin-type protease located in the trans-Golgi network.

How this process occurs is difficult to tinalyse as visualisation of maturing yellow fever particles has proven difficult. Infected cells also release a non-infectious, subviral particle, for reasons that are unclear. These subviral particles are antigenic and represent cellular membrane fragments into which are inserted copies of the E and M proteins as well as small amounts of prM. As the E protein retains its haemagglutination properties, these particles are functionally referred to as the slowly sedimenting haemagglutinin SHA component.

Clinical disease The clinical course of yellow fever develops through three distinct stages. The acute phase is characterised by a fever Headache, back pain, nausea and vomiting constitute the major symptoms. At this stage, the patient is highly infectious with virus present in the blood from days This viraemia ensures that the likelihood of human-to-human transmission by mosquitoes is high. Remission generally follows accompanied by a lowering of the fever.

The headache disappears and the patient generally feels much recovered. During the third stage, the fever returns with many if not all of the symptoms seen on presentation, but in a more severe form.

Acknowledgments

The patient becomes increasingly anxious and agitated. Liver, heart and perhaps kidney failure follow rapidly accompanied by delirium. Jaundice is the inevitable result of the inflammation in the liver and death occurs 6 - 7 days after onset of the disease. Among those that survive, recovery can be slow. Virus cannot be recovered from the blood during this stage but anti-yellow fever virus antibodies can be detected, suggesting an immunopathological component late in the disease process. Despite suggestions to the contrary, there appears to be no link in disease severity according to ethnicity.

Other causes of viral haemorrhagic fever should always be suspected, such as CongoCrimean haemorrhagic fever, Rift Valley fever, Marburg and Ebola viruses in Africa. Meningococcal septicaemia and leptospirosis are also infections that need to be eliminated during diagnosis. Among other illnesses that can confound clinical diagnosis are the agents of viral hepatitis.

Hepatitis A is common in endemic areas, indeed in Columbia and neighbouring countries hepatitis A is more common than other forms of viral hepatitis. Hepatitis A is rarely accompanied by a high fever, however, and serological tests for anti-hepatitis A IgM antibodies are readily available to differentiate this agent from yellow fever virus. The Rockefeller Foundation conducted surveillance measures in Brazil for many years based on the taking of liver tissue from fatal cases using a viscerotome. Diagnosis As with many virus infections, there is an emphasis on the detection of IgM antibodies during the early acute phase.

Immunofluorescence using infected cells fixed previously in acetone is an easy method to adopt for field use and can be modified to detect either IgM or IgG antibodies, although the assay for IgG antibodies is sensitive to the presence of rheumatoid factor. A more definitive diagnosis is the presence of virus in the early viraemic period.

Intracerebral or intraperitoneal inoculation of suckling mice is a sensitive method for virus detection but results may take up to 3 weeks. Intrathoracic inoculation of mosquitoes is also possible, but tissue culture isolation is perhaps preferable. Cell lines derived from either Aedes albopictus C cells or Aedes pseudoscutellaris MOS61 cells can readily support growth of yellow fever virus.

Detection of virus replication by application of monoclonal antibodies can produce results in a few days. There is a risk that virus in samples is already complexed with antibodies thus dissociation of antigenantibody complexes by treatment with the reducing agent dithiothreitol is recommended prior to inoculation of cell cultures.

Sequencing of yellow fever isolates has revealed that there are at least two, possibly three genotypes of yellow fever currently in circulation. These distinctions are based on phylogenetic analyses of the E protein. The relatively 28 close association of strains from West Africa and the Americas is consistent with what we know of the historical origins of the virus in the New World, but it should be stressed that there are many phenotypic differences between isolates grouped in genotypes IIa and IIb. More needs to be done in defining to what extent such variation can result in virus adaptation to new hosts and mosquito vectors, and thus fundamentally alter the nature of the transmission cycle.

Despite these differences, the presently available yellow fever vaccines protect against all three genotypes. Pathology The taking of a liver biopsy is not advisable given the high risk of haemorrhage. Howe Ier, liver tissue taken at autopsy is useful for epidemiological purposes, as menfi,, ned above.

The hepatitis associated with yellow fever virus is manifested by the presei: The portal tracts become extensively infiltrated with inonocytes. In it was found that monkeys were susceptible to yellow fever virus, an observation that led directly to the isolation of Asibi strain of yellow fever virus from present-day Ghana together with the Dakar strain obtained from a Senegalese patient. Shortly after, Theiler discovered that Swiss white e: Two live attenuated vaccines were developed concurrently using these isolates.

French workers passaged the Dakar isolate times in mice brains to derive what became known as the French neurotropic vaccine. The second vaccine lineage was derived from the Asibi strain. Present day vaccines all have their origin in this virus originally recovered in West Africa. It is important to note that both the French neurotropic vaccine and the 17D strains have lost both the capacity to produce viscerotropic disease and an ability to replicate in mosquitoes. The 17D vaccine strains were originally developed by Theiler and colleagues by passage first in mouse brain and then in chick embryo tissue.

Two substrains of the 17D attenuated virus form the basis of present vaccines. The first is based upon virus derived at the th passage 17D and is used predominantly in Europe and Africa at the passage level. The second originates from the th passage of the lineage, being subsequently passaged independently in embryonated eggs and used at the 2 8 6 passage level 17DD: Although differing in lineage, both are equally effective, despite evidence of phenotypic differences between yellow fever virus circulating in the Americas and Africa.

Protection against yellow fever indeed against any human flavivirus infection--is mediated by the presence of neutralising antibodies. Immunity is probably lifelong although revaccination after 10 years is required under the International Health Regulations. The vaccine is delivered subcutaneously and is well tolerated: It can be given simultaneously with other live attenuated vaccines such as measles and polio as well as together with DPT, oral cholera, typhoid, hepatitis A and hepatitis B vaccines.

The vaccine is contraindicated for pregnant women unless demanded by local circumstances. Inadvertent vaccination in early pregnancy can lead to anxiety, but a study by Robert et al. There is no evidence of the vaccine crossing the placenta. A study by Nasidi et al. The issue of immunosuppression in patients with HIV and responsiveness to yellow fever vaccine has not been sufficiently investigated.

As the vaccine is produced in eggs, residual egg protein may prove difficult in those with allergies to eggs but again this has not been borne out in practice, with allergic reactions being as infrequent as one per million of individuals vaccinated. The viraemia following vaccination is low, normally below 2 logm pfu, and is of short duration. Thus, the risk of vaccine virus transmission by insect bite is low, but in any event the 17D virus is substantially attenuated to the extent that it is unable to replicate in mosquito vectors.

Presently vaccine continues to be manufactured in embryonated chicken eggs. Attempts to replace the egg-derived products with virus grown in cell monolayers such as chick fibroblasts have been frustrated by the low yields that result from infection of cell cultures with the 17D virus. There is also the question of cost. Egg-grown virus is cheap to produce and the technology readily transposed into endemic areas where investment in expensive cell culture facilities is limited and skilled personnel rarely available.

Some embryonated hens' eggs are contaminated with avian leucosis virus; although there is no evidence to suggest the presence of this contaminant has any effect on efficacy or the health of vaccines, it is thought desirable to eliminate this agent from yellow fever vaccine products. The long history in the use of the 17D substrains presents a unique opportunity to define the molecular basis of attenuation and obtain indicators as to how vaccines could be developed against dengue and other flaviviruses, particularly vaccines against those infections that as yet have proved too difficult to develop for various reasons.

The genome of yellow fever virus was first sequenced using 17D virus held by the American Type Culture Collection Rice et al. This landmark study has since been followed by completing the sequence of both the wild type Asibi virus as originally isolated by Stoker and the complementary 17DD virus subjected to an independent lineage beyond passage in chick embryos. These studies together with related work have revealed a number of differences at the molecular level: Parallel studies of the Dakar isolate and its derived French Neurotropic vaccine strain have similarly demonstrated a proportionally high mutation rate in the E and NS2A proteins Wang et al.

Only two substitutions are common: The fact that monoclonal antibody studies show cross-reactivity for viruses with dissimilar linear amino acid sequences indicates the importance of conformational epitopes in eliciting neutralising antibodies. Interestingly, the 17DD strain possesses an additional glycosylation site on the E protein at position not present on 17D virus, and the new master seed lot approved by WHO possesses 32 a new glycosylation site close by at position The significance of such differences is unclear but could affect either the conformation of a critical determinant or its immunogenicity.

An important issue is the potential of vaccine strains to revert to neurovirulence. There have been a handful of cases reported in vaccines, mainly in children less than 1 year of age. Only one fatal case has been reported: This case is notable as virus was reisolated from the child and subjected to antigenic analysis, showing that the virus had regained reactivity for a monoclonal antibody normally reactive only with wild type virus.

The isolate was also neurovirulent for a single cynomolgus monkey. Sequencing of this isolate has shown a number of mutations Barrett, Interestingly one of the changes was at position on the E protein, close to the glycosylation site discussed above. Notwithstanding this and rare instances of encephalitis in vaccinees, it needs to be stressed that yellow fever vaccination remains one of the greatest medical achievements in the control of infectious diseases. The experience of over 60 years of routine use has provided substantial evidence that the 17D virus is both safe and highly efficacious.

For this reason, the 17D vaccine strain makes an ideal candidate for use as a live vector for genes of other, heterologous antigens, most notably for genes coding for the structural proteins of other flaviviruses, such as hepatitis C, dengue and West Nile viruses. Studies using chimeric yellow fever--dengue constructs are described later in this chapter. This platform technology owes its origins to the work of Pletnev et al.

This work established the important principle that the chimera construct had lost peripheral invasiveness as a result of the vector construction. Dengue Colonisation of the New World brought with it infections previously unknown to the indigenous populations of the Americas. These included smallpox, measles, scarlet fever, malaria and yellow fever. It is probable that cases of the latter could have well been mistaken for dengue fever as examination of historical records makes it impossible to distinguish the two infections apart until at least the end of the 18th century.

Between and detailed descriptions were made of outbreaks resembling present-day dengue fever from the nascent United States, Africa and the Dutch East Indies. The most celebrated of these descriptions is due to Benjamin Rush, a physician of Philadelphia and a close friend and colleague of the American Founding Fathers Fig. Rush is generally credited as being the first to provide a detailed description of what we now know as dengue fever. This was largely based on an outbreak occurred in Philadelphia in the summer of which Rush described as a bilious remittent fever. The description has remained little improved over the centuries, so fastidious was Rush in ensuring he committed to record an accurate and objective observation as to the course of the disease.

His descriptions were all the more remarkable given that a number of illnesses were circulating in Philadelphia at that time. Although he did not to make 33 the connection with a possible vector, he noted that " That he failed to make the connection is all the more remarkable given that Rush recorded the epidemic as subsiding as soon as the temperature dropped sharply in October. The locals referred to the malady as "breakbone fever". Coincidentally Rush was also renowned for his interest in mental disorders, and records the case of two afflicted sisters, both of whom suffered from dejection and depression, pressed Rush to change the name to "breakheart fever".

Concurrently David Bylon, a physician in the Dutch East Indies noted a similar disease which he referred to as knokkel-koorts "Knuckle fever". Humphreys has pointed out that the prominence of knuckle pain means this could have been an outbreak of Chikungunya virus, not breakbone fever as was supposed by his contemporaries. The name dengue came into use after reports of an outbreak in the Spanish West Indies between and when the Swahili term Ki denga pepo was first used to describe the disease.

Dengue fever epidemics in the 18th and 19th centuries occurred in the Americas and elsewhere in regular cycles, reflecting the fact that transport by sea took many weeks, if not months, between Europe and the newly emerging empires of the Netherlands and Britain. The rapid changes of the 20th century changed dramatically the epidemiology of dengue virus, especially the upheavals accompanying the Second World War and its aftermath.

It was at this time that the first extensively documented outbreak of dengue haemorrhagic fever DHF occurred in the Philippines in This was followed 5 years later by a much larger outbreak in Thailand. Although outbreaks are known to occur in Africa, less is known about these, most likely because if and when they do occur the infrastructure for reporting such outbreaks is invariably poor. Although dengue fever as an acute illness has a low mortality rate, its impact, like that of yellow fever, extends beyond its clinical importance.

In centuries past, the introduction of infection into a community could mean economic disaster for fledgling colonies. In modem times, the impact of an infectious disease such as dengue can still result in serious economic loss in a country heavily dependent upon tourism.

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The four serotypes of dengue virus extend over regions inhabited by over 2. This makes dengue arguably the most important of virus diseases spread by arthropods. Epidemiology Dengue viruses are transmitted to humans by the bite of an infected female Aedes mosquito. The principal vector is A. The female needs regular blood meals in order to provide nutrients for its eggs, which it lays in containers of still water commonly found 34 close to domestic dwellings, for example open water barrels, flower pots and old tins and containers.

Old car tyres provide ideal vessels for still water and at least one outbreak in Taiwan has been attributed to Aedes larvae imported in a cargo of recycled tyres.

Viral Haemorrhagic Fevers, Volume 11 (Perspectives in Medical Virology) - PDF Free Download

The adults are difficult to detect, feeding off humans in daylight, with bites often going unnoticed. Civic programmes aimed at eliminating the vector by removing open containers of still water have done much to reduce, if not entirely eliminate, the risk of transmission in urban areas. These measures are often backed by the force of law as in the case of Singapore, for example. Epidemics first occurred in the s in South'East Asia and over the ensuring 30 years spread first through the Philippines to the South Pacific islands, and from there spread to Central and South America, parts of Africa, India and south to Queensland in Australia.

Dengue viruses have a considerable propensity to spread fin'ther, particularly as A. The disease threatens the southern United States, the populous areas of Brazil and could spread to much of Africa Fig. Once a female mosquito imbibes blood from a viraemic human, the virus replicates in the gut of the insect, moving to the salivary glands by 8 - 1 1 days after ingestion. This species has recently invaded the southern USA, setting the stage for possible future outbreaks.

The denser the shade, the greater the incidence of disease courtesy of the World Health Organisation. This cycle is likely complex as A. In Africa, Erythrocebus monkeys are the principal hosts in the rain forests, with other Aedine mosquitoes also contributing to maintenance of the transmission cycle, for example A. There is evidence of all four serotypes of dengue virus 3 originating in monkeys, with adaptation to humans having occurred both relatively recently and independently for all four serotypes.

Although the sylvatic cycle has been demonstrated for all four serotypes in Asia, only a dengue 2 sylvatic cycle has been demonstrated in Africa Rodhain, Understanding more regarding the sylvatic cycle would boost our understanding of genetic variation between and within the four virus serotypes: Clinical features Seroprevalence studies have shown that the majority of dengue virus infections are asymptomatic.

Clinical disease is of two distinct types.

1. Viral Haemorrhagic Fevers, Perspectives in Medical Virology, Volume 11.
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The first is dengue fever, the "classical" disease seen in the vast majority of cases among adults who have not been previously exposed to the virus. Other neurological signs include severe depression, apathy and complaints of disturbing dreams. However, the most distinctive feature is the severe muscle and bone pain, particularly in the lower back. Joint pains, a lymphadenopathy and vomiting all develop over the next 3 - 7 days. A diffuse and discrete macropapaular rash develops immediately prior to recovery. Although incapacitating, the acute illness is relatively short lived and patients eventually make a full recovery although many remain incapacitated for many weeks during convalescence.

The more severe disease of DHF is seen in children below the age of 15 and in those infected previously with dengue virus. Clinically, DHF resembles yellow fever in that the initial stages of the disease--similar to uncomplicated dengue fever is followed by a brief respite when body temperature returns to near normal, only to be followed by a sharp onset of fever once more and a rapid deterioration in the patient's condition.

The patient displays profound prostration and shows progressively all the manifestations of haemorrhage and shock that result from circulatory collapse and hypotension. The liver may become enlarged with signs of jaundice. Petechiae appear in the skin and patients give a positive tourniquet test. Ecchymoses, gastrointestinal bleeding and haemorrhagic pneumonia become evident. This case definition has been the subject of much debate, with many experts emphasising that the disease profile differs according to the age of the patient and geographical location. Although severe muscle and bone pain together with a sudden onset of fever are highly suggestive of dengue fever, the disease on presentation may resemble many other febrile illnesses.

The more severe form, DHF, may be confused clinically with other causes of haemorrhagic disease, although haemoconcentration and indications of a coagulopathy may be useful in pointing towards dengue virus as a cause. Although the risk of DHF and DSS increases significantly with secondary infections, the risk does not increase further on a Subsequent third exposure to another serotype.

If anything, the risk appears to decline, perhaps as a result of previous infection stimulating a sufficiently broad immune response that replication by a third serotype is contained. Diagnosis In contrast to most flaviviruses that cause human disease, virus isolation can prove difficult from cases of dengue fever. Direct intracerebral injection into suckling mice often requires several blind passages for an isolate to become evident.

An alternative approach is the intracerebral inoculation of Toxorhynchites ticks, with successful isolation possible in less than 3 days. Intrathoracic inoculation of mosquitoes is another possibility, with isolation taking somewhat longer at up to a week. Virus isolation in cell culture is to be preferred, however. Insect cell lines are very sensitive to dengue virus: Regular inspection of replicate cultures by addition of fluorescent- or peroxidase-labelled monoclonal antibody specific for dengue virus usually produces a positive response within 3 days of inoculation.

Specific serology is vital to making an accurate diagnosis. ELISA assays designed to detect IgM are increasingly replacing haemagglutinin inhibition for the detection of 37 anti-dengue virus antibodies. IgM antibodies are present from as early as the third day of infection and may persist for as long as 3 months. Rapid tests for IgG antibodies are common, but care needs to be taken to ensure that tests are adequately controlled for unwanted cross-reactions with other flaviviruses.

Immunofluorescence is useful as each visual field contains a number of uninfected cells that can serve as negative controls. The complement-fixation test has now largely fallen out of use, mainly because of its complexity, its relative insensitivity compared to other methods, and the difficulties in standardising reagents. Tests for measuring neutralising antibodies are particularly useful in confirming both the diagnosis and for determining the serotype of an isolate.

Properties of dengue virus The dengue virus genome is a single-stranded RNA molecule of positive sense with respect to gene expression. Approximately 11 kb in length, the genome organisation is similar to that of yellow fever virus see Fig.

Ebola : replication - virus mutation of the virus

Electron microscopy of extracellular virus shows a particle of approximately 50 nm in diameter with clearly visible surface projections. The core protein exists in an ordered structure less well defined compared to that of the alphaviruses, where the core assumes an icosahedral symmetry dictated by the envelope glycoproteins as the virus buds from an infected cell, a process mediated by interactions with the alphavirus E2 envelope glycoprotein.

Such interactions appear to be absent in dengue ar d other flaviviruses.

Viral Haemorrhagic Fevers, Perspectives in Medical Virology, Volume 11

Knowledge of the three-dimensional structure is important, for identifying regions that can be targeted to block virus entry and for designing new vaccines, as well as elucidating just how heterologous neutralising antibodies can cause antibody-mediated enhancement. The structure of the tick-borne encephalitis virus E protein was determined in Rey et al.

The class II fusion E protein of dengue virus has a distinctly different structure to the class I fusion protein of the haemagglutinin of influenza virus. In common with the E protein of tick-borne encephalitis virus, the dengue E protein is ordered as 90 dimers fat-packed on the surface of an icosahedral-shaped virus particle. The dimers are so closely packed on the surface of dengue virus that the viral membrane is inaccessible at physiological pH and thus fusion cannot occur. The net effect is an increase in particle diameter and the exposure of the underlying viral membrane.

The chapters cover epidemiology, clinical disease, immunology and pathology, molecular virology, diagnosis, and all other aspects of the science and threat to public health of a disparate group of viral infections. A compendium of information of this kind is a useful concept for those who need an overview of and an introduction to the field of viral hemorrhagic fevers. The author has also added general chapters about safety and perspectives on bioterrorism, diagnostic techniques, vaccine development, and other topics. The early history of each viral disease discussed is well covered.

The author's considerable background in molecular virology allows some useful, in-depth discussion of viral structure, replication, and functions of the various molecular components of viral hemorrhagic fevers. These discussions are mostly in text, and it would have greatly increased understanding particularly for the general reader to have had many more illustrations. The author is clearly less comfortable with epidemiology and ecology of the viruses; both topics are rather underserved by this book.

For example, aerosol transmission of Lassa fever is discussed, even though this means of transmission has been exclused in most published articles. The importance of Marburg virus is dismissed, and the author fails to mention the major outbreak that occurred in the Democratic Republic of Congo in the late s. This omission is important in light of the major outbreak in Angola in — Also of much concern is the misnaming of countries.

Lack of attention to detail in this respect seems unnecessary and is unsettling. In general, many of the concepts presented are dated and fail to address much of the most recent literature, which has changed and advanced the concepts presented in this book. In addition, there are many errors and unsubstantiated and unreferenced statements that are misleading, at best.

For instance, ticks are described as breeding in stagnant pools, and nonhuman primate species are misnamed. Descriptions of human infection with Whitewater Arroyo virus are treated as fact but are unreferenced—for the excellent reason that definitive articles were never published, possibly because of inability to confirm the authenticity of isolation.

Other articles that present irreproducible data, such as the report of reverse transcriptase activity associated with arenaviruses, are quoted uncritically. Disappointingly, even the section on diagnostic techniques is out of date. Today, RT-PCR is widely available, even in developing countries, so discussions about immunofluorescent antibody assays—the staple of diagnostic techniques during the s and s—being the primary approach are overemphasized to the detriment of newer approaches. Even some sections that discuss molecular virology are seriously out of date, particularly those concerning filoviruses.

Many major articles about filovirus that have been published during the past few years are not referenced; complete bodies of work from some of the current leading experts have been omitted. The problem of not including findings from the most recent literature are compounded when discussing pathophysiology particularly, immunopathology. Much of what is discussed is limited to old articles about histopathology.