1. Viral Hemorrhagic Fevers (VHF)
Amber M. Vasquez, MD
Assistant Professor, Division of Infectious Diseases
Associate Program Director, Infectious Diseases Fellowship
The Ohio State University Wexner Medical Center
Office Phone:
Welcome! My name is Dr. Amber Vasquez. Today’s lecture will introduce you to the topic of Viral Hemorrhagic Fevers. These are diseases we don’t see commonly in the United States but can cause significant disease in other countries. With the globalization of air travel, it is ever more critical that we be prepared to recognize and manage these diseases.
If you have any questions at the end of this module please don’t hesitate to contact me.

2. At the end of this module you will learn to:
Learning Objectives
At the end of this module you will learn to:
Describe the structure and microbial physiology of Hemorrhagic Fever viruses and integrate this information with the human pathophysiologic correlates
Describe physical and chemical properties of Hemorrhagic Fever viruses
Describe the replication of Hemorrhagic Fever viruses
Describe the underlying genetic mechanisms of Hemorrhagic Fever viruses
Describe the physiology of Hemorrhagic Fever viruses
Identify the normal human immune response to infection with Hemorrhagic Fever viruses
The objectives for this module will be to link the microbial physiology of the hemorrhagic fever viruses with their pathophysiologic correlates, including the epidemiology, clinical presentation, diagnosis, treatment and prevention of the hemorrhagic fever viruses. We will examine these viruses individually as well as comparing and contrasting them with one another when possible.

3. Learning Objectives
Recognize the epidemiology and ecology of infection due to Hemorrhagic Fever viruses
Describe and differentiate the principles of laboratory diagnosis for Hemorrhagic Fever viral infections
Describe the treatment, prevention and control of Hemorrhagic Fever viral infections
The objectives for this module will be to link the microbial physiology of the hemorrhagic fever viruses with their pathophysiologic correlates, including the epidemiology, clinical presentation, diagnosis, treatment and prevention of the hemorrhagic fever viruses. We will examine these viruses individually as well as comparing and contrasting them with one another when possible.

4. Hemorrhagic Fever viruses
Filoviridae
Marburg virus
Ebola virus
Flavivirdae
Yellow Fever virus
Dengue virus
Bunyaviridae
Rift Valley Fever virus
Hantavirus
Arenaviridae
Lassa Fever virus
- Japanese Encephalitis
- St. Louis Encephalitis
- West Nile Virus
…and more
We are going to focus on these 7 members of these 4 viral families but this list is not all-encompassing. There are many other specific viruses that fall under the umbrella of these viral families, some of which cause hemorrhagic fevers, some of which do not. For example, in addition to Lassa Fever virus, the Arenaviridae family also includes such viruses as Guanarito virus, which causes Venezuelan hemorrhagic fever and Machupo virus, which causes Bolivian hemorrhagic fever. Conversely, the Flaviviridae family includes Yellow Fever and Dengue but also encompasses many viruses that do not cause a hemorrhagic fever, such as Japanese, St. Louis, and West Nile Virus, all of which cause a viral encephalitis. However, for the purposes of this module we are going to focus on these seven.
- Guanarito Virus: Venezuelan hemorrhagic fever
- Machupo Virus: Bolivian hemorrhagic fever
…and more

5. Marburg and Ebola Filoviruses
Filamentous, enveloped, negative-strand RNA viruses
Severe or fatal hemorrhagic fevers
Marburg and Ebola are the two main members of the Filoviridae family of viruses. They are filamentous, enveloped, negatively stranded RNA viruses that cause a severe or fatal hemorrhagic fever and are endemic to Africa, which you’re going to find is the case for many of these hemorrhagic fever viruses. All the Filoviridae range from between about 800 and 1400 nanometers in length. On the left you see the Marburg virion, which has a similar structure to Ebola on the right but tends to be about 200 nanometers shorter and has more of a shepherd crook appearance.
Ebola virion
php.med.unsw.edu.au

6. Structure and Replication
Here is the structure of a typical filovirus on the left. The RNA genome is single stranded and encapsulated. Along the surface of the virus are glycoproteins which the virus uses to attach to the host receptors, as depicted in the upper right hand figure. Through fusion of the viral membrane with the vesicle membrane, the nucleocapsid is released into the cytoplasm of the host cell where it further replicates.
viralzone.expasy.org

7. Pathogenesis Lancet 2011;377:849-62
Ebola is the prototypical hemorrhagic fever virus and here we see part of its pathogenesis to which the Marburg virus is similar. Not shown here is the inoculation, which occurs via exposure to bats or non-human primates, then spreads to the regional lymph nodes, liver, lungs, and spleen. The virus infects monocytes, macrophages and dendritic cells where it replicates and elicits a cytokine storm of proinflammatory cytokines, such as tumor necrosis factor and interleukins. This causes tissue necrosis in those tissues that the virus had spread to - the liver, spleen, lymph nodes and lungs. All those glycoproteins on the surface of the virus that we saw in the last slide also contribute to break down of the endothelial cells in the vasculature. This cytokine storm leads to vascular injury and increased permeability, which causes the hemorrhagic symptoms typical of the disease, leading to hypovolemic shock and death from multiorgan failure.
Lancet 2011;377:849-62

8. Epidemiology Mostly Sub-Saharan Africa
Endemic in fruit bats, wild monkeys
Contact with animal reservoir
Human-to-human spread via contact with infected blood or secretions
Monkey Handlers
Healthcare exposures
Accidental Injection
Contaminated Syringes
Healthcare workers in close contact
This WHO map shows the primary distribution of reported outbreaks of Ebola virus, but this was as of The 2014 Ebola outbreak in West Africa has changed this map significantly, which we will discuss on the next slide.
Ebola is named for the river in the Democratic Republic of Congo where it was first discovered in The countries shaded light orange have reported only serologic evidence of Ebola in the population, meaning these are patients that have the antibody but had only subclinical disease rather than overt hemorrhagic fever, and it accounts for up to 18% of people in rural areas of central Africa. The dark orange countries reported actual outbreaks, which corresponds to the blue stars on the map that are marking the specific locations of these outbreaks or isolated cases. The purple dotted line is highlighting the range of the fruit bat family that carries this virus and it extends out into India and Southeast Asia off to the right. There have been some small sporadic outbreaks reported in the U.S. and parts of Europe and SE Asia that were attributable to imported monkeys from the Philippines or exported cases from Africa, but I’ve cropped them out of this map because they’re rare.
The Marburg virus was first recognized in 1967, when outbreaks occurred simultaneously in laboratories in Marburg and Frankfurt Germany. Those who were infected were laboratory workers exposed to the tissues of otherwise healthy green monkeys that had been imported from Uganda, or in the medical personnel or family members that had cared for those patients. While this WHO map doesn’t include Marburg virus, those viral outbreaks tend to occur in a similar pattern in the Sub-Saharan Africa region since they have a similar reservoir in African fruit bats and primates.
Human contact with the animal reservoir is suspected to be the means by which these outbreaks move from the natural reservoir into humans, such as being bitten by or eating the meat of an infected animal, or exposure during the slaughtering process. At that point, it is spread human-to-human via contact with infected tissues or bodily fluids.

9. 2014 Ebola Outbreak West Africa Contributing factors Sierra Leone
Guinea
Liberia
Nigeria, Senegal
United States, Mali, Spain
Contributing factors
Sheer volume of cases
Strained infrastructure
Personal Protective Equipment
Local burial customs
I have to give special attention to the ongoing Ebola outbreak in West Africa, which continues to evolve and no doubt this information will be outdated as soon as the lecture is posted, as the number of involved countries continues to rise and the case counts continue to increase exponentially. The outbreak began in March 2014 and as of late October, over 10,000 people have been infected in West Africa, with over 5,000 deaths. We can only guess what the final tally will be as the outbreak has devastated the resources in this region and strained the global community’s attempts to assist in containment, with panic begin to spread as cases spill out into other countries, including the U.S.
The outbreak is notable not only for being the largest Ebola outbreak in history (previously the largest had been 425 cases in Uganda in 2001), but also for its location. As mentioned in the previous slide, Ebola has had many sporadic outbreaks over the decades but has typically been considered a disease of sub-Saharan Africa, so it’s emergence in West Africa has been particularly devastating considering this is not a disease that region often sees.
That is a significant factor in why this particular Ebola outbreak has been so large. These are countries unaccustomed to responding to Ebola, and containment becomes harder as cases increase and stress what are already underdeveloped healthcare infrastructures. Anybody who has contact with an infected patient should be quarantined and/or monitored for symptoms in the act of contact tracing – but when hospitals and clinics don’t even have the facilities or personnel to accommodate the infected patients, it’s difficult to monitor thousands of potential contact cases as well.
Healthcare workers are at particular risk. The sicker a patient becomes, the greater the degree of their viremia, and the higher their infectivity. HCWs have very close regular contact with these patients, and as you can see in the above photo, the personal protective equipment that must be worn is not insignificant. Without proper training and vigilance, one can easily become contaminated during the process of donning and doffing the material. These precautions are stressed given the virulent nature of this special pathogen. That said, the disease is still very difficult to transmit. A patient must be showing symptoms to spread the disease and an individual must come into contact with their infected bodily fluids.
Traditional burial practices have also been one of the obstacles making this the worst Ebola outbreak in history. Families often embrace and wash the bodies of their loved ones before burial, but the presence of bodily fluids on a recently deceased Ebola patient puts that body at the height of infectivity, exposing their loved ones to the virus. Relief workers attempting to properly disinfect and dispose of an Ebola patient’s remains have met with resistance by local communities and even led to reports of families misrepresenting the cause of death of their loved ones to avoid the process.

10. Clinical Syndromes Most severe causes of VHFs
Case fatality rate
of up to 90%
Most severe causes of VHFs
Incubation period typically 5 – 10 days
(up to 21 days)
Flu-like illness (fever, malaise)
 Nausea, vomiting, diarrhea; possible cough, pharyngitis
May have photophobia, CNS symptoms (somnolence, delirium)
Day 5: Maculo-papular rash may develop on trunk
Subsequent hemorrhage from multiple sites (esp. GI tract)
Week 2: Clinical improvement vs. Death from shock with multi- organ failure
Ebola and Marburg are the most severe causes of VHF. After exposure, typical incubation period from the time of exposure to symptom onset is 5-10 days, but can be up to 21 days. Symptom onset includes a flu-like illness with fever, headaches, myalgias and malaise. This progresses to include n/v, diarrhea and possibly cough with pharyngitis. Pts may also have conjunctivitis, photophobia and other CNS symptoms such as somnolence, delirium, or coma. A maculopapular rash may develop on the trunk around Day 5 that can form into hemorrhagic bullae. Subsequent hemorrhage occurs from multiple sites, especially the GI tract as well as gum bleeding, but hemorrhage isn’t seen in every patient and Ebola is now being referred to as “Ebola Viral Disease” to reflect that fact. During the second week of illness, patients are either lucky enough to be improving or they are dying of multi-system organ failure. Given the resource poor areas in which these diseases typically occur, some outbreaks have shown a mortality rate as high as 90%.

11. Laboratory Diagnosis Biosafety Level 4 Isolation
Marburg virus – rapid tissue culture growth
Ebola virus may require animal inoculation
Eosinophilic cytoplasmic inclusion bodies
Viral antigen detection in tissue by direct immunoflourescence and in fluids by ELISA
RT-PCR amplification in secretions
IgM/IgG to filoviruses; false (+)’s  confirm testing
jamanetwork.com
These viruses are highly dangerous to work with in the laboratory setting and thus require the highest level of isolation at a Biosafety Level 4 facility, of which there are only a handful in the U.S.
Marburg virus may grow readily and rapidly in tissue culture while Ebola virus may require animal inoculation in order to recover it. Viral culture isn’t the typical means by which these diseases are diagnosed, however. More commonly, enzymed linked immunosorbent assay, or ELISA, and reverse transcriptase polymerase chain reaction, or RT-PCR, are the most likely means employed for diagnosis. These can detect specific viral antigens or antibiotics to them. The antibodies, IgM and IgG, can have some cross-reactivity across the Filoviruses, so positives tests should be confirmed with immunoflourescence or PCR to evaluate for false positives. Most testing for these diseases are not done at local hospitals, but rather through State Health Departments or the CDC.
Macrophage
Journal of Infectious Diseases 1999;179 (Suppl 1):S54-9

12. Treatment and Infection Control
No definitive management and no vaccine
Supportive care
Replacement of coagulation factors and platelets as needed
Antibody-containing serum and interferon therapy
Containment is key!
Standard precautions
Mask, gloves, gown, goggles
Appropriate cleaning of medical supplies
Proper burial techniques
There is no definitive management for Ebola or Marburg infection other than supportive care. Antibody-containing serum from those recently recovered from Ebola viral disease and interferon therapy have been used to attenuate disease progression in some patients, but this isn’t proven therapy and is not routinely recommended in treatment.
The key to the control of these infections is outbreak containment. It is essential to limit exposure to infected patients, who need to be immediately isolated and precautions should be taken to avoid exposures to infected bodily secretions. This includes gowning, gloving, and the use of masks and goggles as seen in this photo and on previous slides. There should be no area of skin exposed. In the event of patient death, proper burial techniques and limitation of exposure to the deceased is also important.
microbewiki.kenyon.edu

13. Yellow Fever and Dengue
Flaviviruses
Next we will talk about Yellow Fever and Dengue Fever, viruses that are much more common and widespread. They are Flaviviruses, which take their name specifically from the yellow fever virus, derived from the Latin word for yellow, which is “flavus” and its attack on the liver that causes jaundice. As you can see in these photos, the viruses are round and you can sort of make out the impression of glycoproteins on the viral surface.
Yellow Fever virions
Dengue Fever virions
hardinmd.lib.uiowa.edu

14. Structure and Replication
“Flavivirus” cross-section
A cross section of the flavivirus is seen on the left. Rather than a helical capsid like the Filoviruses they have an icosahedral capsid. The E glycoproteins pair up to form the outer protein layer and at the center is the nucleocapsid that holds the positively stranded RNA genome. It’s life cycle begins with viral attachment which can be enhanced by antibodies from previously being exposed to another strain of the virus. The virus is internalized into the cells via endocytosis and is uncoated by pH-dependent fusion in the endosome. The RNA is released into the cytoplasm where translation occurs and the virus is reassembled on the endoplasmic reticulum. Upon egress from the cell, the pre-membrane is cleaved and an infectious, mature virus particle is released into the extracellular space.

15. Pathogenesis Arthropod-borne viruses (arboviruses)
Aedes aegypti mosquito
Human and Nonhuman primate reservoir
Smaller mammals maintain viremia
Humans are dead-end hosts
Flaviviruses are arboviruses, meaning they utilize an arthropod vector for transmission. In this chart of the many flaviviruses, including the viral encephalitidies, Yellow Fever and Dengue have a common vector, which is the Aedes aegypti mosquito. The virus is transmitted when a mosquito feeds on a viremic host and that insect acquires the virus where it resides and replicates in the salivary glands. This is then transmitted to the next host that the mosquito feeds on. The natural hosts seen along the left side of this diagram, maintain sufficient levels of viremia long enough to act as the ongoing reservoir whereby mosquitos continue to feed and acquire the virus. Humans, on the other hand, are considered a dead-end host. While there is a window period of viremia in which a mosquito can feed on an infected human host and acquire the virus, generally humans do not maintain a persistent viremia long enough to maintain an outbreak.

16. Immunity Humoral and cellular immunity
Viral replication  Interferon  Stimulates innate and immune responses  Rapid onset flu-like illness
IgM blocks viremic spread
Inflammation from cell-mediated response
 Weakens vasculature; causes rupture/hemorrhage
Non-neutralizing antibody can enhance viral uptake
 Worsens symptoms on repeat infection
Both humoral and cellular immunity are elicted and are important to the control of primary infection and the prevention of future infections. Replication of the virus produces a double-stranded RNA replicative intermediate that is a good inducer of interferon. When that IFN is released into the bloodstream, it limits replication, stimulates innate and immune responses and causes the rapid onset of flulike symptoms that is characteristic of mild disease. IgM antibody is produced and blocks viremic spread, potentially also providing immunity to other flaviviruses. This immunity, however, can be potentially bad. Inflammation from cell-mediated response can weaken vasculature and cause rupture. Plus, a nonneutralizing antibody that was acquired from a prior infection from a related strain of the virus can enhance uptake of flaviviruses into the cells when folks are re-infected with a different viral strain. This is particularly the case with Dengue, where there are 4 different serotypes of the virus. Initial infection tends to be fairly mild, and thus travelers to those areas have a lower case-fatality rate since they will likely only be infected the one time. People who reside in these endemic regions, on the other hand, are at risk for more serious disease since they are more likely to be infected a second time with another serotype, and those reinfections are more likely to cause hemorrhagic or shock symptoms.

17. Epidemiology – Yellow Fever
Sub-Saharan Africa
Tropical S. America
Summer months
Rainy season
Standing water, drainage ditches, open sewers
Winter – vector not present; virus dormant in arthropod larvae/eggs; migrating birds
Sub-Saharan Africa and tropical areas of South America are the highest areas of incidence, though S. America has lower levels of disease due to higher vaccination rates and lower contact between humans and the mosquito vector. As such, risk of infectivity is much higher for travelers to Africa than S. America, particularly during the summer months and rainy season. Standing water is a breeding ground for mosquitos and is a risk for exposure to infected mosquitos. During the winter, the mosquito vector isn’t present but the virus lies dormant in the larvae/eggs or maintains viremia in birds who fly away in the winter months and then migrate back to the region when it’s warm.

18. Epidemiology – Dengue www.yalescientific.org
While the distribution is somewhat similar to that of Yellow Fever, with high risk areas found where the Aedes aegypti mosquito is prevalent, as you can see from this WHO map, Dengue is a bit more widespread. It is endemic in about 100 countries in Asia, the Pacific, the Americas, Africa, and the Caribbean. Malaria is a pretty common cause of fever in a returning traveler but for those returning from the Caribbean, S. America, or Asia, the most common cause of fever is actually Dengue. This is a massively prevalent disease. Worldwide, there are approximately 100 million cases of dengue fever a year with about 300,000 cases of DHF and about 25,000 deaths. When outbreaks have been seen in the mainland U.S., they have primarily been in Texas and Florida, those border states with latin america.

19. Clinical Syndrome Yellow Fever
Most benign, self-limiting
Incubation 3-6 days
Fever, chills, myalgia, back pain, severe headache
Most resolve after this
~15% progress to severe disease
High fever, jaundice, hepatitis, hyperbilirubinemia, hemorrhage
Shock, multi-organ failure
Most infections with Yellow Fever are asymptomatic or self-limiting. You can follow along on this diagram on the right. After exposure from an insect bite, the virus incubates for a few days before producing a flu-like illness with fevers, chills, myalgias, back pain and headache. The vast majority of patients will resolve after this but about 15% will progress to a more severe form of the disease after the virus infects the liver, spleen, lymph nodes, ect. That includes symptoms of liver failure with jaundice from hepatitis and hyperbilirubinemia as well as renal failure, hemorrhage, DIC, shock and possible death from multisystem organ failure.

20. Clinical Syndrome Dengue
Most benign, self-limiting
50-80% are asymptomatic or have undifferentiated fever
4 – 7 day incubation period
Classic Dengue Fever
“Breakbone Fever”
Dengue Hemorrhagic Fever
Bruises, epistaxis, gum and GI bleed
Dengue Shock Syndrome
Hypotension
A similar process occurs with Dengue but infection with Dengue can manifest as one of a few clinical syndromes. Most patients will be asymptomatic or have an undifferentiated fever or viral syndrome. Some present with the Classic Dengue Fever, also known as breakbone fever, because of its extremely severe back and bone pain, that is accompanied by headache, high fever, and rash. The rash takes on a morbilliform maculopapular appearance sparing the palms and soles. Symptoms lasts approximately one week and then resolve on their own. The really severe presentations are Dengue Hemorrhagic Fever and Dengue Shock Syndrome. As previously mentioned, these are more likely to occur when the patient is reinfected with one of the other 4 serotypes – this increase in severity due to the nonneutralizing antibody acquired from prior infection that enhances viral uptake into the cell.

21. Laboratory Diagnosis IgM/IgG ELISA
Primary method of diagnosis in acute illness from Dengue
IgM (+) after 5 days from symptom onset (follow seroconversion)
IgG titers for recent or past infection (4-fold increase)
False positive risk – crossreactivity with other flaviviruses or vaccinations
RT-PCR from serum, CSF, autopsy tissue in first 7 days
Serum PCR to detect viremia is test of choice for Yellow Fever
Viral culture not routinely done
Most diagnoses are made clinically in endemic regions. Short of that, IgM ELISA is the primary method of diagnosing Dengue in acute illness wherein you can follow seroconversion from negative to positive. Alternatively, you can follow the IgG titers as a 4-fold increase will be indicative of recent infection. Beware the false positive test result, though, as this can cross react from current or prior infection with other flaviviruses or from vaccination to them. Serum PCR is the test of choice for diagnosing Yellow Fever though it’s not done often for Dengue due to lack of availability. And while viral culture can be done on the same tissues as the PCR test, this is not routine.

22. Treatment and Prevention
Biosafety Level 3 or 4
Supportive care only
Yellow Fever Vaccine
Live vaccine
Confers lifelong immunity
Fever, myalgias, headache, nausea/emesis 2 – 5 days after administration
Only for those going to an endemic region
Mosquito vector control
Treatment is supportive, not only fluids and blood product replacement but treatment of secondary infections should a patient survive Yellow Fever as secondary bacterial infections are a common complication of that illness.
There is no vaccine for Dengue but there is one for yellow fever, as this child is receiving in the photo – however, this should not be undertaken cavalierly. Patients become fairly miserable after administration with a flu-like illness and there have been some deaths reported. So vaccine should only be administered to those going to a highly endemic region and never to pregnant patients or the immunosuppressed.
Mosquito vector control is of paramount importance. Disperse standing water, sleep under mosquito nets at night, wear bug spray with DEET or long sleeve shirts or long pants whenever possible.
profiles.nlm.nih.gov

23. Rift Valley Fever and Hantavirus
Bunyaviruses
“Supergroup” of 200 enveloped, segmented, negative- strand RNA viruses
The Bunyaviridae family constitutes a supergroup of over 200 enveloped, segmented, negatively stranded RNA viruses, of which we are going to focus on two of the most common hemorrhagic ones in Rift Valley Fever and Hantavirus. Like the Flaviviruses, most of the Bunyaviridae are arboviruses, including Rift Valley Fever. The exception are the hantaviruses, which are carried by rodents. This large family of viruses is further divided into multiple genuses. One of these genuses, the Phleboviruses, have Rift Valley Fever under its umbrella. The Hantaviruses are a completely separate genus. While we tend to just refer to all the Hantaviruses as just *quote* “Hantavirus” *endquote*, in actuality the Hantaviruses have a number of different species that fall under THEIR umbrella, and depending on which one the patient is infected with, they can present with a couple different clinical presentations, both of which we will discuss briefly.
Rift Valley Fever
Hantavirus particle
web.uct.ac.za/depts/mmi/stannard/emimages.html
virology-online.com/viruses/Hantaviruses

24. Structure and Replication
Nucleocapsid
L RNA – large
M RNA – medium
S RNA – small
RNA Polymerase
Replicate similar to other enveloped, negative-strand RNA viruses
The particles of the Bunyaviridae are generally spherical with a lipid envelope and spiked glycoproteins that encapsulate 3 unique negative-strand RNAs, designated L, M, and S for large medium and small. The RNA polymerase is also inside the nucleocapsid. The viruses replicate in similar fashion to other negative strand RNA viruses – the glycoproteins attached to the host cell surface, the virus is internalized by endocytosis, and after fusion of the envelope to the endosomal membranes, the nucleocapsid is released into the cytoplasm where messenger RNA and protein synthesis begin. After glycoproteins are synthesized for the surface of the virus in the golgi apparatus, the virions are assembled by budding into the golgi apparatus and released by cell lysis or exocytosis.

25. Pathogenesis Rift Valley Fever – arbovirus
Reservoir: Livestock (cattle, buffalo, sheep, goats)
Vector: mosquito (Aedes genus)
Humans infected by bite of mosquito or exposure to infected tissue of the animal (more common)
Hantavirus – NOT an arbovirus
Certain species of rodents
Deer mouse, cotton rat, rice rat, white-footed mouse
Other rodents worldwide
Hantaan, Sin Nombre, and many more!
Aerosolized urine
Deer mouse
Rift Valley Fever, like nearly all the other Bunyaviruses, is an arbovirus, transmitting from animal to animal and from animal to human via a mosquito vector. During excessive rainfall, the mosquitos breed and transfer the virus to the livestock on which they feed. This includes cattle, buffalo, sheep, etc. Once the livestock is infected, other species of mosquitoes can become infected from the animals and can spread the disease further, either biting more livestock or jumping to the human population. The vast majority of human cases, though, are due to exposure to the blood, body fluids, or tissues of infected animals. Often this kind of direct exposure occurs during slaughter or obstetric procedures. So be sure to ask patients just what sort of large animal handling they do, not simply whether they live on a farm. Your index of suspicion is going to go up if you find that a patient is sick shortly after delivering the birth of a newborne baby calf.
Hantaviruses, though, are not arboviruses. They are carried via multiple species of rodents. In the U.S., these include the adorable looking deer mouse that you see here, which is also the most common carrier, as well as the cotton rat, rice rat, and so on – all of which have a different geographic area in which they usually live but when combined spans almost the entire U.S., meaning an outbreak could occur just about anywhere in this country. Other species of rodents carry the virus worldwide, and they can carry different member viruses to cause different clinical syndromes. The virus is transmitted via exposure to the infected rodent excrement, usually by inhalation of aerosols contaminated with infected urine.

26. Epidemiology Rift Valley Fever Sub-Saharan and North Africa
Kenya
Somalia
Tanzania
Saudi Arabia and Yemen
Yet another infectious disease related reason to be glad you don’t live in Africa, because that is where Rift Valley Fever is endemic. Some of the largest outbreaks have occurred in Kenya, Somalia, and Tanzania – some of them very deadly a few decades ago. Recently in 2000, cases of Rift Valley Fever were reported in Saudi Arabia and Yemen, two countries in the Middle East off the Northeast border of Africa and that are also highlighted blue on the map. This raised concerns that the virus could potentially spread to Asia and Europe from there, but thankfully we haven’t seen that happen.
cdc.gov

27. Epidemiology Hantavirus Worldwide “Old World” “New World”
Hantaan, Dobrava
Europe, Asia, Africa
Hemorrhagic fever with renal syndrome
“New World”
Sin Nombre
N. and S. America
Hantavirus pulmonary syndrome
Hantaviruses, while having some characteristics based on geography, are found worldwide. With somewhat archaic terminology these have been separated into two groups, The Old World species of hantaviruses and The New World. The Old World species, such as Hantaan or Dobrava virus, are primarily seen in Europe, Asia, and Africa. These species cause hemorrhagic fever with renal syndrome. The New World species, such as Sin Nombre virus (which caused a deadly outbreak in the U.S. in 1993 at the Four Corners region of the Southwest), affect North and South America and cause Hantavirus pulmonary syndrome. As I previously mentioned, because the various species of mice or rats that carry hantavirus in the U.S. have a wide distribution, there have been cases reported in 34 different states. Thankfully, Ohio is yet to become one of them and in general you can see by the map that the western states are the place to be if you want to see this disease some day.
Indeed, I’ve starred the area of Yosemite National Park in California. You may have heard about this Hantavirus outbreak late last year and so I thought I would touch upon it as an illustration of how these outbreaks occur in the U.S. I actually went to the Epidemiologic Intelligence Service conference this year at the CDC and they reported their findings on this outbreak since the EIS officers were naturally dispatched to try and contain it. A handful of people who had recently stayed at Yosemite National Park were presenting to area hospitals with symptoms of pneumonia. Moreover, they had all stayed in the area of Curry Village. Now, Curry Village was an encampment of about 400 new, canvas-sided signature tent cabins that were a pretty big deal for the park because they were an upgrade from the old soft vinyl walled tents elsewhere in the park. These tents were double wall plywood which allowed for the use of heaters, thus warmer and quieter, and allowed campers to store food inside, neither of which was the case with the old tents because they were just flimsy soft walls and there were strict food storage regulations about not keeping food in your tent unless you wanted to be attacked by a bear. As it turns out, these thick walls were also more susceptible to becoming the new home of a bunch of deer mice carrying hantavirus, as they burrowed in to be close to warmth and food. Ultimately at least 10 people became ill, 3 of whom died, and after attempts to control the rodent population in that area failed, the Curry Village signature tent cabins were demolished.
Curry Village tent cabins

28. Mortality as high as 50% with hemorrhagic disease
Clinical Syndromes
Mortality as high as 50% with hemorrhagic disease
Rift Valley Fever
Incubation period of 48 hours
Flu-like illness from viremia; fever lasts about 3 days
Can be mild or progress to severe illness with hemorrhage
Petechial hemorrhages, ecchymosis, epistaxis, GI and gum bleeding
Hantavirus
Hemorrhagic Fever with Renal Syndrome
Similar to Rift Valley Fever but with acute renal failure
Hantavirus Pulmonary Syndrome
Flu-like illness (fever, headache, myalgias, nausea/vomiting, diarrhea)
Rapid progression to cough, short of breath, pulmonary edema, respiratory failure, and death within days
As far as clinical syndromes, Rift valley fever causes a flu-like illness a few days after infection with the virus. The fever lasts about 3 days and most patients will have a mild illness and just get better. Some, however, can progress to hemorrhagic disease characterized by what you would expect: petechiae, ecchymoses, bleeding from the nose, gums, and GI tract with bloody vomitus and stool.
Hantavirus species that cause hemorrhagic fever is similar to rift valley fever but includes renal failure in addition to the previously mentioned symptoms. Hantavirus pulmonary syndrome, which is what you are most likely to see since the species that cause this are what we have in the U.S., starts out as a flu-like illness as well, with fever, myalgias and GI symptoms. It can then rapidly progress to pulmonary symptoms with subsequent pulmonary edema, respiratory failure, and eventual death.

29. Laboratory Diagnosis RT-PCR to detect viral RNA
Most common diagnostic tool
IgM antibodies by ELISA in acute illness
IgG with four-fold increase in titers – recent infection
ELISA may be able to detect antigen in very viremic patients, such as those infected early with Rift Valley Fever
Diagnosis is primarily made by RT-PCR to detect viral RNA. Seroconversion of a negative to positive IgM or fourfold increase in IgG titers can likewise make the diagnosis. ELISA can detect viral antigen in the blood in very viremic patients, and the virus can even be isolated in them such as those with Rift Valley, but this is not really done.

30. Treatment, Prevention, and Control
Biosafety Level 3 or 4
Supportive management
Rift Valley vaccine not licensed or commercially available
Has been used for veterinary and laboratory personnel at high risk of exposure
Vector control!!
Remember these are dangerous viruses, so if you have to isolate than specimens should be contained to BSL 3 or 4 facility
As far as treatment, there isn’t really any. Some reports of Ribavirin being used are still lacking in solid data, and Ribavirin is attempted in nearly every viral illness these days. Ultimately, supportive management is the key. There is a Rift Valley vaccine that has only really been used for veterinary and laboratory personnel who are at high risk of exposure because the vaccine is not licensed or commercially available. The best means of prevention is going to be vector control in both cases, either with the typical water dispersion and bug spray techniques with rift valley and mosquitos or by rodent control with Hantavirus.

31. Lassa Fever Arenavirus Pleomorphic, enveloped viruses
Greek word “arenosa” = “sandy”
Lassa Fever is a member of the arenaviridae family of viruses, which also includes a number of less common hemorrhagic fevers as well as lymphocytic choriomeningitis virus which is a central nervous system infection. These viruses cause persistent infections in specific rodents and can be transmitted to humans as zoonoses. Arenaviruses are pleomorphic, enveloped viruses that have a sandy appearance with what you can see on this electronmicrograph are dense interior granules visible in the lassa fever virion. If you’re fluent in Greek this is easy to remember because the word arenavirus is derived from the greek word “arenosa”, meaning “sandy”.
Lassa fever virion

32. Structure and Replication
Two single-stranded RNA
- L segment: encodes polymerase
- S segment: nucleoprotein and glycoproteins
Inside the nucelocapsid are two single-stranded RNA circles, designated S and L. The L strand is negative-sense RNA and encodes the polymerase. The S strand encodes the nucleoprotein and the glycoproteins. Arenaviruses replicate in the cytoplasm and acquire their envelope by budding from the host cell plasma membrane.
Vhfc.org/lassa fever/virology

33. Epidemiology Endemic to West Africa African rodent population
Mastomys natalensis
Rodent urine, droppings
Colonize human homes
Inhalation of aerosols
Contaminated food
Contact with open cuts, sores
Person-to-person spread
Infected secretions, bodily fluids
Lassa Fever is endemic to West Africa. The virus is carried by the Mastomys rodent which breeds in West, Central and East Africa, putting a larger geographic area at risk though most of the actual reported disease is focused to West Africa. These rodents tend to colonize around human homes and shed the virus in their urine and droppings, similar to Hantavirus. Humans are exposed via aerosols, food that becomes contaminated with these excretions due to these rodents scavenging around human food sources, or contact via open cuts and sores. The virus is then propagated via person to person transmission from contact with infected secretions or bodily fluids of those who have taken ill.

34. Clinical Syndrome Incubation: 1 – 3 weeks
Fever, sore throat, retrosternal pain, abdominal pain, vomiting, diarrhea
Facial swelling, proteinuria, conjunctivitis
Coagulopathy, petechiae, occasional visceral hemorrhage
Hemorrhage and Shock
Highest rates of death in 3rd trimester pregnancy
Varying degrees of deafness in approximately 1/3
After contact with the virus, symptoms generally begin after an incubation period of 1-3 weeks. Symptoms are initially fairly nonspecific: fever, sore throat, back pain, abd pain, vomiting, diarrhea, etc. There can be facial swelling as seen in the photo on the left and conjunctivitis seen on the right. It can then progress to coagulopathy and hemorrhage with petechiae. Studies have shown that the combination of fever, pharyngitis, retrosternal pain, and proteinuria can correctly predict 70% of laboratory confirmed Lassa Fever. Mortality is not as high as in some of the other VHFs but the greatest risk is for pregnant women in their 3rd trimester of pregnancy. If the patient does survive, beware the longterm sequela of varying degrees of hearing loss, an effect seen in approximately 1/3 of patients.

35. Laboratory Diagnosis Biosafety Level 3 or 4 Precautions
Throat and urine specimens for isolation
Takes 7-10 days to grow
ELISA: IgM/IgG or Lassa antigen
RT-PCR
Given the dangers of isolating this organism, it needs at least a Biosafety level 3 or 4 facility. Throat and urine specimens are the preferred sources for isolating the organism, which can take over a week to grow. ELISA test for IgM or the Lassa antigen is rapid, sensitive, and the most commonly used diagnostic tool for Lassa fever given the high degree of viremia these patients maintain compared to other Arenaviruses. There is a PCR that can be used but this is primarily a research tool at this point and not readily available.

36. Treatment, Prevention, and Control
Supportive care
Fluids, electrolytes, oxygen and blood pressure support
Limited Ribavirin activity
Has successfully decreased mortality in prior studies
Most effective if given in the first 6 days of treatment
Prevention
Rodent control; trapping
Proper food storage
Contact precautions and equipment sterilization
As usual, supportive care is the mainstay of treatment. However, patients in Sierra Leone have been treated with intravenous Ribavirin with improved mortality from 55% to 5% when the drug was given within the first 6 days of disease onset. Now, IV Ribavirin is not currently licensed in the U.S. and treatment of Lassa fever is not one of the FDA indications for the inhaled or oral forms which are licensed and we have on formulary here at OSU. Otherwise, this would be an appropriate drug to consider for any serious arenavirus infection. Prevention, however, is the key. The rodent population should be controlled and food properly stored to avoid scavenging. Like some of the other VHFs, there has been nosocomial spread of the disease during outbreaks to HCWs caring for the sick and not using proper infection control precautions or sterilization procedures.
N Engl J Med Jan 2:314:20-6

37. Summary Marburg and Ebola – Filoviruses
Sub-Saharan Africa; fruit bats, wild monkeys
Human-to-human spread via infected blood/tissue
Severe hemorrhagic fever syndrome
Yellow Fever and Dengue Fever – Flaviviruses
YF: Sub-Saharan Africa; Tropical S. America (less)
DF: Similar to YF, plus Asia, Caribbean, the Pacific
Mosquito vector transmission (arboviruses) – Aedes aegypti
YF: Flu-like; progress to jaundice, hemorrhage
DF: Breakbone fever, Hemorrhagic fever, Shock Syndrome
To summarize some key points:
marburg and ebola viruses are filoviruses, endemic to subsaharan africa in fruit bats and wild monkeys; human to human spread occurs via exposure to infected blood/tissue of ill patients; primarily causes the most severe forms of hemorrhagic fever syndrome
Yellow fever and dengue are flaviviruses; yellow fever primarily seen in subsaharan africa and less so in south america; presents with a flulike illness progressing to liver failure with jaundice and hemorrhage. Dengue found in similar distribution to yellow fever but also seen in asia, caribbean and the pacific where it is the most common cause of fever in a returning traveler. Symptoms can range from a nonspecific febrile illness to breakbone fever, hemorrhagic fever, or dengue shock syndrome

38. Summary Rift Valley Fever and Hantavirus – Bunyaviruses
Rift Valley: mosquito vector; livestock reservoir; sub-Saharan and North Africa
Hanta: infected urine from rodents; U.S. (pulmonary syndrome); worldwide (hemorrhagic fever + renal failure)
Lassa Fever – Arenavirus
West Africa
Rodents; aerosolized urine, contaminated food
Person to person spread – infected blood/tissue
Fever, sore throat, retrosternal pain, proteinuria
Hemorrhage and Shock
Rift valley fever and hantavirus are bunyaviruses. Rift valley is also an arbovirus with a mosquito vector; livestock are the reservoir and it is endemic to subsaharan and north africa. Hantavirus is transmitted via the urine of infected rodents, usually from aerosolized droplets contaminated with infected urine. In the U.S. the strains we see cause a pulmonary syndrome. Worldwide, those strains are more likely to cause a hemorrhagic fever plus renal failure.
Lassa fever is an arenavirus endemic to west africa. Also spread by infected excrement from rodents then further spread person to person by infected blood or tissues. Presents with a potentially broad range of nonspecific symptoms but the combination of fever, sore throat, retrosternal pain, and proteinuria can predict about 70% of those infected. Symptoms can progress to hemorrhage and shock.

39. Summary All require Biosafety Level 3 or 4
Most common means of diagnosis:
ELISA antibody testing (IgM/IgG)
RT-PCR
All are primarily managed by supportive care
Prevention: vector control (insect or rodent); isolation precautions; sterile medical equipment
Vaccines available for:
Yellow Fever (commonly used)
Rift Valley Fever (not commonly used)
All these viruses require either Biosafety level 3 or preferrably 4 given their special pathogen status.
While viruses can be isolated in culture, most commons means of diagnosis is usually either ELISA antibody testing or RT-PCR
All are managed by supportive care
And prevention is key with vector control and prevention of nosocomial transmission during outbreaks
Vaccines are available for both yellow fever and rift valley fever, but the latter is not really used.

40. Viral Hemorrhagic Fevers Quiz

41. Thank you for completing this module
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42. References
Medical Microbiology, 7th Ed. Murray, Rosenthal & Pfaller; Chapter 58, pages 537 – 538; Chapter 60, pages 549 – 556; Chapter 61, pages 561 – 566.
Principles and Practice of Infectious Diseases, 7th Ed. Mandell, Douglas, and Bennett; Chapters 153, 164, 166, 167.

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