1. Leslie Lobel Faculty of Health Sciences Pathology Building Room 324

2. Special Pathogens and Arboviruses
Hemorrhagic Fevers

3. Arboviruses Arboviruses = viruses transmitted by insects.
Not a virus family but comprises many different viruses. There are Hemorrhagic fever viruses and encephalitis viruses that are Arboviruses.

7. Walter Reed
James Carroll

9. During the Spanish-American War of the late 19th century & the subsequent building of the Panama Canal, American deaths due to yellow fever were colossal. The disease also appeared to be spreading slowly northward into the continental United States. Through experimental transmission to mice, in Walter Reed demonstrated that yellow fever was caused by a virus, spread by mosquitoes.
Walter Reed ( )

10. The discovery that Yellow Fever was caused by a virus eventually enabled Max Theiler (1937) to propagate the virus in chick embryos & successfully produced an attenuated vaccine - the 17D strain - which is still in use today.
Max Theiler ( )

11. FAMILY/GEOGRAPHY AGENT CASE-FATALITY
Filoviridae
Sub-saharan Africa
Ebola
Marburg
50-75%
25%
Arenaviridae
West Africa (Lassa)
South America, California (Whitewater)
Old World: Lassa
New World: Junin,
Machupo, Guanarito Sabia, Whitewater arroyo
Lassa:1-2% (up to 25% in hospitalized pts)
30% for New World
Bunyaviridae
Egypt, Yemen
SW US (Hantavirus)
Phlebovirus: Rift Valley
Nairovirus: Crimean Congo
Hantavirus: Sin Nombre
Rift Valley: <1% overall
50% in hemorrhagic
Flaviviridae
Central Asia
Yellow fever
Dengue
Omsk
Kyasanur
Yellow Fever: 5-7% overall
The Viral Hemorrhagic Fevers are comprised of a number of different diseases. All are single-stranded RNA viruses and enter the bloodstream through various routes (i.e. tick, mosquito, rodent bite vectors, mucous membrane exposure. Endothelial infection occurs which leads to thrombocytopenia and endothelial dysfunction which may result in disseminated intravascular coagulation (Ebola, Marburg, Rift Valley Fever, Crimean Congo Fever) and cytokine storm (Ebola and Marburg). Vascular permeability and disregulation can occur, leading to periorbital edema, hemoconcentration and flushing, respectively. Animal models of Ebola pathogenesis suggest that the virus leads to immunosuppression and apoptosis (self-destruction) of T-lymphocytes and natural killer cells. In this table the major families of VHF are listed along with the endemic region, the specific agents and average case fatality rates for the diseases listed. For more information see the Center for Infectious Disease Reasearch and Policy and Infectious Disease Society of America (CIDRAP/IDSA) summary document on VHF which can be found on the websites: or

12. Treating Hemorrhagic Fever in the field
BSL-4
Treating Hemorrhagic Fever in the field

13. Hemorrhagic Fever Viruses: Taxonomy
Four families of lipid-enveloped viruses with single-stranded RNA genomes
Arenaviruses
Bunyaviruses
Filoviruses
Flaviviruses

14. Hemorrhagic Fever Virus Families
Arenaviridae
New World Complex
Argentine HF
Bolivian HF
Venezuelan HF
Brazilian HF
Old World Complex
Lassa Fever
Junin Virus
Machupo Virus
Guanarito Virus
Sabia Virus
Lassa Virus

15. Hemorrhagic Fever Virus Families
Bunyaviridae
Phlebovirus Genus
Rift Valley Fever
Nairovirus Genus
Crimean-Congo HF
Hantavirus Genus
HFRS
Korean or Epidemic HF
E. Europe
Nephropathia Epidemica
Rat-borne
RVF Virus
CCHF Virus
Hantaan
Dobrava-Belgrade
Puumula Virus
Seoul

16. Hemorrhagic Fever Virus Families
Filoviridae
Ebola HF
Marburg HF
Mosquito-Borne:
Yellow Fever
Dengue HF
Tick-Borne:
Kyasanur Forest Disease
Omsk HF
Ebola Virus
Marburg Virus
Flaviviridae
YF Virus
Dengue Viruses
KFD Virus
OHF Virus

17. Ebola Hemorrhagic Fever

18. Scanning electron micrograph of Ebola-infected cultured cell showing numerous virus particles (arrow) along the cell surface
(*13,500).

19. History
In 1967, multiple cases of severe hemorrhagic fever with nosocomial transmission among European laboratory workers led to the spectacular initial identification of Marburg virus and the new virus Filoviridae virus family.
The virus came with African green monkeys imported from Uganda and the epidemic was controlled when the monkeys and contaminated materials were disposed of.
The next recognized filovirus activity was in 1976, when Ebola virus appeared simultaneously in the Democratic Republic of the Congo (DRC; then called Zaire) and Sudan. These epidemics were actually caused by two distinct forms of the virus.
There has been only sporadic filovirus activity since.

20. What is Ebola hemorrhagic fever?
Ebola hemorrhagic fever (Ebola HF) is a severe, often-fatal disease in humans
and nonhuman primates (monkeys, gorillas, and chimpanzees) that has appeared
sporadically since its initial recognition in 1976.

21. Classification
Filoviruses are taxonomically classified in the order Mononegavirales, which contains a large variety of enveloped viruses with nonsegmented negative-stranded (NNS) RNA genomes.
Morphologic, genetic, physiochemical, and virologic studies of Marburg and Ebola viruses led to the creation of a separate family, the Filoviridae. The family is divided into two genera, currently designated as “Ebola-like viruses” and “Marburg-like viruses.”
There are five species within the “Ebola-like viruses,” Zaire (type species), Sudan, Reston, Bundibugyo and Côte d'Ivoire (Ivory Coast). A single species, Marburg virus, makes up the “Marburg-like viruses.”

22. Where is Ebola virus found in nature?
The exact origin, locations, and natural habitat (known as the "natural reservoir") of Ebola virus remain unknown.
Evidence suggests that the virus is zoonotic (animal-borne) and is normally maintained in an animal host that is native to the African continent.
A similar host is probably associated with Ebola-Reston which was isolated from infected cynomolgous monkeys that were imported to the United States and Italy from the Philippines. The virus is not known to be native to other continents, such as North America.

23. Investigations of filovirus outbreaks in humans have provided circumstantial evidence that bats may be involved in the spread of filovirus disease.
Two index Marburg virus cases were known to have visited the same bat-infested cave in Kenya, near the area in Uganda that produced the infected monkeys involved in the 1967 outbreak. Recently, many cases of Marburg virus disease in the northeast region of the DRC had some connection, directly or indirectly, with a bat-infested gold mine. Also, an index case in the first Ebola Sudan outbreak worked in a bat-infested factory building.
Despite failure to detect virus in these environments, the possibility still exists that a transmission cycle among bats plays some role in the natural history of filoviruses. This is supported by laboratory transmission studies, in which persistent infection of bats for up to 21 days following inoculation, with excretion in feces, has been documented.
An extensive 3-month survey of native vertebrates following the 1995 outbreak in DRC, albeit months delayed from the purported index case, failed to identify Ebola-specific antibodies or a virus isolate in over 3,000 vertebrates sampled.

24. What are the symptoms of Ebola hemorrhagic fever?
The incubation period for Ebola HF ranges from 2 to 21 days. The onset of illness is abrupt and is characterized by fever, headache, joint and muscle aches, sore throat, and weakness, followed by diarrhea, vomiting, and stomach pain. A rash, red eyes, hiccups and internal and external bleeding may be seen in some patients.
Researchers do not understand why some people are able to recover from Ebola HF and others are not. However, it is known that patients who die usually have not developed a significant immune response to the virus at the time of death.

28. What laboratory tests are used to diagnose Ebola hemorrhagic fever?
Antigen-capture ELISA testing
IgM ELISA
Polymerase chain reaction (PCR)
Virus isolation
Above can be used to diagnose a case of Ebola HF within a few days of the onset of symptoms.
Can also be diagnosed retrospectively in deceased patients by immunohistochemistry, virus isolation or PCR.

29. How is Ebola hemorrhagic fever treated?
There is no standard treatment for Ebola HF.
Patients receive supportive therapy. This consists of balancing the patient’s fluids and electrolytes, maintaining their oxygen status and blood pressure, and treating them for any complicating infections.

30. During the 1995 Kikwit outbreak, seven of eight patients receiving whole blood transfusions from surviving patients lived.
There were no controls and the transfusions were usually given to younger patients later in the epidemic and sufficiently late in their clinical course that some patients had already died—all factors that favor survival.
The apparent success should prompt research into alternative approaches that might involve soluble substances or cells in the transfused blood.

31. Marburg and Ebola virus infectivity is quite stable at room temperature (20°C) and can resist desiccation, but it is largely inactivated in 30 min at 60°C.
Infectivity is also greatly reduced or destroyed by high doses of ultraviolet light and gamma irradiation, lipid solvents, -propiolactone, and commercial hypochlorite and phenolic disinfectants.

32. The primary gene product of the Ebola GP gene is not the GP but rather a smaller, nonstructural, secreted glycoprotein (SGP) that is released from
infected cells in large quantities.
The transcriptional editing event that leads to GP production occurs at a series of seven Us on the genomic RNA template and results in the insertion of an additional single A, which connects the GP open coding frames by insertion of a single nucleotide at the editing site, which appears to occur with a high degree of fidelity.
Transcriptional editing is not unprecedented in the order Mononegavirales, as a similar type of editing has been demonstrated for the P gene of certain paramyxoviruses. However, the editing of Ebola virus GP gene transcripts is the only example of a virus glycoprotein that is expressed through this type of mechanism.
Sequence analysis of the GP genes of Marburg virus isolates indicates that a nucleotide sequence that corresponds to the editing region of Ebola virus GP genes is absent, and efforts to detect a similar secreted form of the Marburg virus GP have been unsuccessful.

33. Schematic representation of the structure of a filovirus virion and the genome organization of Ebola virus (Zaire species) and Marburg virus (Musoke strain). The virion is depicted as a full-length particle with the internal nucleocapsid exposed, and also as a cross-section.

34. The extragenic sequences at the extreme 3´ (leader) and 5´ (trailer) ends of the genomes of filoviruses are conserved, show a high degree of complementarity, and can form stem-loop structures.
For all NNS RNA viruses, these sequences are important in the
initiation of transcription and the replication of full-length genome RNA, and they serve as promoters for the virus polymerase.
Analyses of defective interfering particles have shown the Ebola virus promoters to be contained within 156 and 177 nucleotide regions of the genomic and antigenomic RNA 3´ termini, respectively.

36. Schematic representation of a proposed structure for the Ebola virus (Zaire species)
surface peplomer (spike) predicted from genetic, biochemical, and x-ray diffraction studies. The left side of the figure shows the spike structure in which three molecules of GP1 are covalently linked by a single disulfide bond to GP2 molecules.

37. Arenaviruses and Lassa Fever

40. All arenaviruses are enveloped and have a bi-segmented RNA with a unique
ambisense genomic organization
The genome of arenaviruses consists of two single-stranded RNA segments designated S (small) and L (large). In virions, the molar ratio of S to L RNAs is roughly 2:1.
The S RNA segment contains two genes that encode three final gene products—the nucleoprotein (NP or N) and the envelope glycoproteins GP1 and GP2 (also termed GP-1 and GP-2, or G1 and G2). GP1 and GP2 are first expressed as a precursor protein, GPC (or GP-C), which is cleaved posttranslationally.
The L RNA segment contains two genes that encode two gene products, the viral polymerase (L protein) and the Z protein, a small protein of undetermined function. On both segments, the genes are arranged in an ambisense orientation.

42. In convalescence, deafness is common; this is an important feature of Lassa fever, as it impacts on the community, and it provides an important diagnostic clue. Late in the course of disease or early in convalescence, unilateral or bilateral hearing loss was noted in 29% of prospectively studied patients. No treatment is available and the effects may be transitory or often permanent.

43. Dengue Fever
*The first cases of Dengue Fever (DF) were recorded in 1779 in Indonesia, and Cairo. In 1780, there was an epidemic reported in Philadelphia, PA.
*For the past 200 years, pandemics have been recorded in tropical and subtropical climates at 10 to 30 year intervals.
*In 1944, Albert Sabin successfully isolated the virus that causes DF and found that it belongs to the Flavivirdae virus family. There are more than 70 known members of the Flavividae family. Some examples include Yellow Fever and Japanese Encephalitis Virus.

44. Pathogenesis: Dengue Hemorrhagic Fever
Four different serotypes of Dengue virus
Initial infection: Neutralizing Ab vs. intial strain
Re-infection due to different serotype: Non-neutralizing Ab
Immune complexes with live virus
Enhanced uptake by monocytes
Infection/lysis of monocytes-release of cytokines, anticoagulants, procoagulants
Implications re vaccine development

45. VHF Vaccines YELLOW FEVER ARGENTINE HEMORRHAGIC FEVER live, attenuated
licensed 17D vaccine safe and efficacious
cannot be used in persons with egg allergy
ARGENTINE HEMORRHAGIC FEVER
live, attenuated
safe and efficacious; used in 150,000
protects monkeys against Bolivian HF

46. VHF Vaccines formalin-inactivated live, attenuated MP-12
RIFT VALLEY FEVER
formalin-inactivated
safe but requires 3 shots, intermittent booster
limited supply
live, attenuated MP-12
Phase II testing
HFRS (HANTAAN)
vaccinia vectored recombinant vaccine

47. Encephalitis viruses

48. Meningitis and Encephalitis
Enteroviruses are the main recognized cause of aseptic meningitis in both children and adults in developed countries, and were identified in 85% to 95% of cases in which a specific pathogen was cultured.
In one study, 62% of infants less than 3 months old with aseptic meningitis had coxsackievirus group B as the etiologic agent.

49. Enterovirus 71 is a major cause of poliomyelitis-like disease in the post polio period.

50. Arboviruses
Classified as those viruses that alternately infect insect and vertebrate hosts.
Viruses maintained in extrahuman cycles and humans are the dead-end hosts.

53. Flaviviruses – Enveloped + Stranded RNA viruses
a) St. Louis Encephalitis Virus
b) Japanese Encephalitis Virus
c) West Nile Fever Virus
d) Murray Valley Encephalitis Virus
e) Central European and Russian Spring-Summer (Tick- borne Encephalitis) Viruses
Alphaviruses – Enveloped + Strand RNA Viruses
a) Eastern Equine Encephalitis
b) Western Equine Encephalitis
c) Venezuelan Equine Encephalitis

55. Bunyaviruses
Three single-stranded RNA genome segments designated large (L), medium (M), and small (S).
All three gene segments of a virus have the same complementary nucleotides at their 3´ and 5´ termini.
Viral RNAs use negative sense and ambisense coding strategy.
a) Bunyavirus genus - California encephalitis, La Crosse virus
b) Phlebovirus genus – Rift Valley Fever

59. Some Closing Thoughts
Coxsackievirus is the most common cause of aseptic meningitis.
Arboviruses are a serious health problem and can be agents of bioterror/biowarfare. Rift Valley Fever is high on the list for biowarfare agents.
Botulinum toxin produces nervous system symptoms and is high on the list for biowarfare agents.