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  1. 1. Hantavirus Loraine Wu Howard Zeng Yuka Yamaguchi
  2. 2. What is Hantavirus? <ul><li>Hantaviruses belong to the bunyaviridae family of viruses. </li></ul><ul><li>Hantavirus leads to diseases such as HPS (Hantavirus Pulmonary Syndrome) and HFRS (Hemorrhagic Fever and Renal Syndrome) </li></ul><ul><li>There are many different strains of Hantaviruses, but only a few strains of Hantavirus actually lead to disease </li></ul>
  3. 3. Old World Hantavirus vs New World Hantavirus <ul><li>Found mostly in Europe and Asia </li></ul><ul><li>Carried by rodents </li></ul><ul><li>Causes HFRS </li></ul><ul><li>Targets the kidney </li></ul><ul><li>Mortality rate is <10-15% </li></ul><ul><li>Vaccine exists for these strains </li></ul><ul><li>Discovered in the “four corners in America </li></ul><ul><li>Carried by rodents </li></ul><ul><li>Causes HPS </li></ul><ul><li>Targets the lungs </li></ul><ul><li>Mortality rate is 50-60% </li></ul><ul><li>Vaccine does not exist for these strains </li></ul>
  4. 4. The History of Hantavirus <ul><li>In 960 AD, Chinese medical records document a possible outbreak of HFRS </li></ul><ul><li>About 1000 years later (1913) Russian clinical records from eastern Siberia also document the symptoms of a mysterious disease, which is highly suspected to have been HFRS </li></ul>
  5. 5. Virus of Wars? <ul><li>An outbreak of “Field nephritis” occurred in both German and Allied troops during WWI in Flanders, Belgium </li></ul><ul><li>During WWII, Field nephritis makes another appearance. </li></ul><ul><li>During the Japanese invasion of Manchuria in 1934, Hantavirus also made an appearance </li></ul><ul><li>Throughout the war, focal outbreaks of HFRS occurred </li></ul><ul><li>From the initial encounter with HFRS, Japanese physicians immediately began a full study of the disease </li></ul>
  6. 6. Those smart Japanese doctors… <ul><li>By 1940, the Japanese had a comprehensive clinico-pathological description of the disease </li></ul><ul><ul><li>The disease had incubation period was estimated to be about 2-3 weeks </li></ul></ul><ul><ul><li>Mortality rates were found to be around 10% </li></ul></ul>
  7. 7. Is it the mouse? <ul><li>In 1944, Japanese physicians and scientists begin testing on human subjects </li></ul><ul><ul><li>Tissues from wild mice A. agrarius and from mites were injected into “volunteers” to induce the disease. </li></ul></ul><ul><ul><li>From these studies the Japanese physicians deduced that the mice and the mites were possible carriers of the disease causing virus </li></ul></ul>
  8. 8. Meanwhile….. <ul><li>Soviet scientists were also hard at work </li></ul><ul><ul><li>Annual outbreaks of HFRS (aka Tula fever) were seen in the Amur River Valley of Russia beginning in 1932 </li></ul></ul><ul><ul><li>Soviets also performed tests on human subjects to discover the natural reservoir for this disease </li></ul></ul><ul><ul><li>They deduced that 3 species of mites were incapable of transmitting the disease to humans </li></ul></ul>
  9. 9. It IS the mouse <ul><li>In 1939, an incident occurred that incriminated field mice more clearly </li></ul><ul><li>Workers in 2 different camps, 4 km apart were engaged in earth moving labor </li></ul><ul><li>31 workers in one camp and those living around that camp fell ill with HFRS </li></ul><ul><li>Only 1 other worker from the other camp fell ill </li></ul><ul><li>Rodent contact was documented to be the highest in the camp with the infected workers </li></ul>
  10. 10. Mouse genocide <ul><li>In Sept 1961, rodents ( Clethnommys glareolus and C rustlius) were sent from Kirov county in Russia to Moscow as part of a tick borne encephalitis test </li></ul><ul><li>On October 18, the first worker fell ill </li></ul><ul><li>By Nov 1, 20/23 of the workers had contracted the disease! </li></ul><ul><li>44/94 casual visitors contracted HFRS </li></ul><ul><li>All the mice were sacrificed on Nov 2, and the last case of HFRS was documented on Nov 29 </li></ul><ul><li>5 of the workers who had previously contracted HFRS did not contract the disease because of acquired immunity </li></ul><ul><li>Fortunately, there were no fatalities </li></ul>
  11. 11. Hantavirus strikes again <ul><li>America met HFRS in 1951 during the Korean War </li></ul><ul><li>By 1954, over 3,000 UN soldiers had been clinically diagnosed with this disease, which had been given the name Korean Hemorrhagic fever </li></ul><ul><li>The Japanese literature was quickly translated to gain a better understanding of this illness </li></ul><ul><li>The mortality rate for these soldiers was 7% </li></ul>
  12. 12. “Hanta” virus <ul><li>After the coded serum sample from convalescent HFRS patients in the Soviet Union were studied in 1978, scientists finally made the crucial connection between field nephritis, KHF and Tula fever. </li></ul><ul><li>Hantavirus is named for the Hantaan River which flows through the endemic region in Korea. (this is where the prototype strain was found) </li></ul><ul><li>At this point, scientist were able to isolate and propagate the virus </li></ul><ul><li>In 1983, the World Health Organization finally adopted the name HFRS to describe all the previous outbreaks of Hantavirus </li></ul>
  13. 13. Hantavirus in America <ul><li>In April, 1993 a young healthy Navajo woman in the “four corners” dies of an acute respiratory disease </li></ul><ul><li>The fiancée of the dead woman also dies on his way to her funeral </li></ul><ul><li>The “blood vessels in his lungs had spurt blood and had caused him to drown in his own blood” </li></ul>
  14. 14. The mysterious illness <ul><li>An Indian Health physician began to notice an outbreak of an unexplained illness that caused death among normal healthy young adults </li></ul><ul><li>After calling his colleagues, he discovered that 10 people had already died of a similar respiratory disease </li></ul><ul><li>Autopsies did not reveal any sign of viral pneumonia, influenza or any other common disease that attacked the lungs </li></ul><ul><li>Although this disease was not specific to the Navajo people, to the media, the disease became known as the “Navajo disease” </li></ul><ul><li>When the number of cases doubled to about 20 victims, the CDC was called in </li></ul>
  15. 15. The CDC <ul><li>In May 1993, the CDC was called in to investigate the case. </li></ul><ul><li>The CDC, using immunofluorescent techniques and their virus library, was able to positively identify this new virus as a relative of the hantavirus strains that were found on the Eurasian continent </li></ul><ul><li>However, scientists were skeptical for 3 reasons </li></ul><ul><ul><li>The only hantaviruses known were on the Eurasian continent </li></ul></ul><ul><ul><li>The diseases caused by the Eurasian hantavirus strain did not cause respiratory failure </li></ul></ul><ul><ul><li>The new virus in the “four corners” appeared to be 5 times as lethal as the strain in Europe! </li></ul></ul>
  16. 16. Those smart Navajo elders…. <ul><li>The CDC also spoke to some of the Navajo elders </li></ul><ul><li>The elders noted that because of the increase in rainfall that year, the pinon crop as well as the mice population had thrived that year </li></ul><ul><li>According to oral tradition, in 1918 and 1932, a similar outbreak had occurred, and mice were always seen as the carriers of disease </li></ul><ul><li>This information helped incriminate the mouse as a reservoir and also helped positively identify the virus as a Hantavirus </li></ul>
  17. 17. Human to human transmission? <ul><li>In 1996, there was an outbreak of HPS in Southern Argentina </li></ul><ul><li>18 incidents of HPS were discovered within a period of 3 months </li></ul><ul><li>Among the 18, 5 of the patients were physicians </li></ul><ul><li>The mouse/rat population was low in 1996, thus further suggesting that person to person transmission was very probable in this incident </li></ul><ul><li>The pattern of transmission for Andes strain does not follow that of any other hantavirus </li></ul>
  18. 18. <ul><li>Figure 1. Transmission tree for HPS cases in southern Argentina, September-December 1996, indicating dates of onset of symptoms, survivor status, and proposed lines of transmission. Lines of transmission are hypothetical since many of the patients had contact with multiple HPS patients. Bold lines denote husband and wife. The two sporadic cases, U and R, are not shown. </li></ul>                                                    
  19. 19.                                                Figure 2. Towns involved in the 1996 HPS outbreak in southern Argentina.
  20. 20. Hantavirus: HFRS <ul><li>Hantavirus named according to rodent host/First Hantavirus detection </li></ul>
  21. 21. Hantavirus: HPS
  22. 22. Hantavirus: HPS
  23. 24. Tracking HPS in the United States (1993 till 2001) <ul><li>As of April 2001, 283 hantavirus cases have been confirmed in 31 states </li></ul><ul><li>Mean age of those contracting the disease was 37, Range: 10-70 </li></ul><ul><li>Mortality rate: 50% </li></ul><ul><li>Incidents of HPS date back to 1959 and 1978 although it was unidentified at the time </li></ul><ul><li>The most popular vectors include the deer mouse and cotton rat </li></ul>
  24. 25. HPS Statistics as of March 28, 2002 Mean= 37 [10 - 75] N Male Female 335 (100%) 203 (61%) 132 (39%) White 255 (76%) American Indian 71 (21%) Black Asian 3 ( 1%) Hispanic 43 (13%) Dead 126 (38%) Age (years)
  25. 26. Topics of Molecular Biology <ul><li>Bunyaviridae family </li></ul><ul><li>Structure and properties </li></ul><ul><li>Transcription and replication </li></ul><ul><li>Pathogenesis </li></ul><ul><li>Host and epidemiology </li></ul><ul><li>Transmission </li></ul>
  26. 27. Family: Bunyaviridae
  27. 28. Bunyaviridae Family <ul><li>Similarities </li></ul><ul><ul><li>RNA viruses </li></ul></ul><ul><ul><li>Enveloped </li></ul></ul><ul><ul><li>Tri-segmented genome </li></ul></ul><ul><li>Differences </li></ul><ul><ul><li>Hantavirus transmitted through aerosolized rodent urine, feces and saliva. </li></ul></ul><ul><ul><li>Others genera transmitted through arthropod vectors. </li></ul></ul>
  28. 29. 23 Well-described Hantaviruses (Adapted from work by Simmons and Riley) <ul><li>Old World Virus </li></ul><ul><li>HFRS </li></ul><ul><li>Old World Virus </li></ul><ul><li>Non-pathogenic </li></ul><ul><li>New World Virus </li></ul><ul><li>HPS </li></ul>
  29. 30. Virus is Rodent-specific Murinae Arvicolinae Sigmodontinae Virus Strain Rodent Host Apodemus agrarius Apodemus flavicollis Rattus norvegicus Bandicota indicus Peromyscus maniculatus Sigmodon hispidus Microtus pennsylvanicus Clethrionomys glareolus Sin Nombre Black Creek Canal Bayou Prospect Hill Puumala Hantaan Dobrava Seoul Thailand Oryzomys palustris New York Peromyscus leucopus
  30. 32. Physical Properties of Virion <ul><li>Spherical or oval-shaped. </li></ul><ul><li>Diameter: 80-120 nm. </li></ul><ul><li>Unique grid-like surface pattern -- transmembrane glycoproteins. </li></ul><ul><li>Survive 12 hours at 4  C, high salt concentration and non-physiological pH. </li></ul><ul><li>1-3 days after drying. </li></ul>(Image taken from CDC)
  31. 33. Viral Genome <ul><li>Granular, filamentous interior consists of genome and protein structure. </li></ul><ul><li>Single-stranded negative-sense RNA. </li></ul><ul><li>Approximately 13 kb. </li></ul><ul><li>Three segments: </li></ul><ul><ul><li>Small (S): 1,700 bases. </li></ul></ul><ul><ul><li>Medium (M): 3,600 bases. </li></ul></ul><ul><ul><li>Large (L): 6,500 bases. </li></ul></ul>(Original image kindly provided by Dr. Simmons of U. Missouri)
  32. 34. Panhandle Structure <ul><li>The 3’ and 5’ terminal sequences are conservative and complementary, forming panhandle structure. </li></ul><ul><li>Believed to be initiation signals for transcription and gene expression. </li></ul>(Images adapted from work by Meyer and Schmaljohn)
  33. 35. Protein Products <ul><li>Four proteins products </li></ul><ul><li>S segment  Nucleocapsid protein. </li></ul><ul><ul><li>Encapsulate vRNA and cRNA. </li></ul></ul><ul><ul><li>Regulate replication and transcription. </li></ul></ul><ul><ul><li>Nucleocapsid protein + vRNA = Ribonucleocapsids. </li></ul></ul>(Images adapted from work by Jonsson and Schmaljohn) Polymerase Protein
  34. 36. Protein Products Cont’d <ul><li>M segment  Glycoproteins, G1 and G2. </li></ul><ul><ul><li>Associated with the lipid membrane. </li></ul></ul><ul><ul><li>Interact with integrin receptors on host cell surface. </li></ul></ul><ul><ul><li>Induce neutralizing antibody response in animals. </li></ul></ul><ul><li>L segment  RNA-dependant RNA polymerase (RdRp) </li></ul><ul><ul><li>Associated with the ribonucleocapsids. </li></ul></ul><ul><ul><li>Endonuclease: Cleave host mRNA caps, which are used to prime transcription from vRNA to mRNA. </li></ul></ul><ul><ul><li>Replicase: Generate copies of the genome. </li></ul></ul><ul><ul><li>Helicase: Unwind RNA during transcription. </li></ul></ul>
  35. 37. Transcription: Viral Entry <ul><li>Virion binds to a host cell’s surface receptor. </li></ul><ul><li>Enter the cell through receptor-mediated endocytosis. </li></ul><ul><ul><li>Pathogenic hantaviruses use  3 integrin receptor. </li></ul></ul><ul><ul><li>Non-pathogenic hantaviruses use  1 integrin receptor. </li></ul></ul><ul><li>Virus uncoats in the cytoplasm, and releases the three ribonucleocapsids. </li></ul>
  36. 38. Transcription and Translation <ul><li>Primary transcription: </li></ul><ul><ul><li>For all negative-sense RNA virus, an initial burst of transcription. </li></ul></ul><ul><ul><li>Viral RNA polymerase transcribes viral RNA into mRNA. </li></ul></ul><ul><li>Viral mRNAs are translated into proteins. </li></ul><ul><ul><li>Polymerase and nucleocapsid proteins are translated on free ribosomes. </li></ul></ul><ul><ul><li>G1 and G2 proteins are translated on membrane-bound ribosomes. </li></ul></ul>
  37. 39. Replication <ul><li>An uncharacterized signal switches transcription to replication. </li></ul><ul><ul><li>Maybe cytoplasmic accumulation of nucleocapsid proteins. </li></ul></ul><ul><li>RdRp makes complementary RNA (cRNA). </li></ul><ul><li>cRNA is used as template to make viral RNA (vRNA). </li></ul><ul><ul><li>Production of vRNA results in more viral genomes for packaging into progeny virions. </li></ul></ul><ul><ul><li>Increases the number of templates for mRNA synthesis. </li></ul></ul>(Image provided by Dr. Simmons of Univ. of Missouri)
  38. 40. Prime and Realign Model <ul><li>Question 1 : How does hantavirus cap the 5’ end of its mRNA transcripts? </li></ul><ul><ul><li>Answer: “Cap snatching.” </li></ul></ul><ul><li>Question 2 : Where does hantavirus get the primers from for transcription? </li></ul><ul><ul><li>Answer: Stolen host mRNA caps are used as primers. </li></ul></ul><ul><li>Observations : </li></ul><ul><ul><li>The 5’ end of viral mRNA is heterogeneous. </li></ul></ul><ul><ul><li>5’ UAG triplets (stop codon) are deleted. </li></ul></ul><ul><ul><li>Commonly, there is a terminal G. </li></ul></ul>
  39. 41. Prime and Realign Model Cont’d <ul><li>Garcin, et al. proposes that G residue aligns with vRNA template to initiate transcription. </li></ul><ul><li>After a few RNAs are added, the mRNA slips back to realign with the template. </li></ul><ul><li>Replication has a similar prime and realign model. </li></ul><ul><li>When cRNA is made, RdRp cleaves off the terminal G base. </li></ul>
  40. 42. Virion Assembly <ul><li>Nascent vRNA associate with nucleocapsid and polymerase proteins upon transcription. </li></ul><ul><li>G1 and G2 are expressed from a single mRNA, and are cleaved cotranslationally. </li></ul><ul><li>Virus and proteins assemble in the Golgi apparatus. </li></ul>
  41. 43. Virion Release and Persistence <ul><li>Virions are transported to the cell surface within Golgi vesicles. </li></ul><ul><li>Two hantavirus strains, including Sin Nombre Virus, bud from cell membranes. </li></ul><ul><li>Infected cells are not lysed. </li></ul><ul><li>Hantavirus infection is persistent in cultured cells and in rodents. </li></ul><ul><ul><li>Maintain viral genetic information </li></ul></ul><ul><ul><li>Avoid the rodent immune system </li></ul></ul>
  42. 44. The Big Picture (Image kindly provided by Dr. Simmons of Univ. of Missouri)
  43. 45. Pathogenesis <ul><li>In both cell cultures and rodents, hantavirus does not cause clinical symptoms, despite the presence of large amounts of antibodies. </li></ul><ul><li>In human, the virus causes HFRS and HPS. </li></ul><ul><li>The pathogenesis of HFRS and HPS are incompletely understood. </li></ul><ul><ul><li>Lack of animal models for human symptoms. </li></ul></ul><ul><ul><li>The requirement for Biosafety Level 3 or 4 containment limits the number of laboratories that can work on it. </li></ul></ul>
  44. 46.  3 Integrins and Pathogenesis <ul><li>Observation : All pathogenic hantaviruses use  3 integrin receptors to infect host cells. </li></ul><ul><ul><li>Experiment: When  3 receptors are blocked by integrin-specific antibodies, cells are not infected by Sin Nombre Virus (SNV), New York Virus (NY-1), Hantaan Virus (HTN) or Seoul Virus (SEO). </li></ul></ul><ul><li>Hantavirus pathogenesis is related to the use of  3 integrin receptor. </li></ul>
  45. 47.  3 Integrins <ul><li> 3 integrins are critical surface receptors on endothelial cells and macrophages in human. </li></ul><ul><ul><li>platelet activation and adhesion </li></ul></ul><ul><ul><li>endothelial cell adherence </li></ul></ul><ul><ul><li>capillary integrity and vascular permeability </li></ul></ul><ul><li>Hantavirus dysregulates normal  3 functions, which are fundamental to pathogenesis. </li></ul><ul><li>HFRS- and HPS-causing hantaviruses have different G1 and G2 composition </li></ul><ul><ul><li>Interactions between viral G proteins and  3 receptors contribute to pathogenic differences. </li></ul></ul>
  46. 48. Immune Response & Pathogenesis <ul><li>Observation : The onset of hantavirus symptoms coincide with immune response. </li></ul><ul><li>Autopsy results showed higher than normal numbers of interleukin (IL), tumor necrosis factor (TNF), interferons (INF), and activated T cells. </li></ul><ul><li>Hantavirus pathogenesis is a result of the host inflammatory response to the virus. </li></ul><ul><li>Hantavirus pathogenesis awaits for a suitable animal model. </li></ul><ul><ul><li>Hooper, et al. reported Syrian hamsters inoculated with Andes Virus strain develop compatible HPS symptoms. </li></ul></ul>
  47. 49. Rodent Hosts and Epidemiology <ul><li>Carried by peri-domestic and wild rodents. </li></ul><ul><li>Each strain of hantavirus is generally associated with a single rodent species. </li></ul><ul><ul><li>100 rodent species have been screened, and 23 hantaviruses found  1/4 of the rodents are carriers. </li></ul></ul><ul><ul><li>There are about 2000 species of rodents. 2000 x 1/4 = 500 different strains of hantaviruses. </li></ul></ul><ul><ul><li>Not all hantaviruses cause disease, e.g., Prospect Hill virus found in the US. </li></ul></ul><ul><li>HPS-causing rodents in the US belong to the Muridae family, Sigmodontinae subfamily. </li></ul>
  48. 50. Rodent Hosts in the United States <ul><li>Deer mouse ( Peromyscus maniculatus ). </li></ul><ul><li>Carrier of Sin Nombre strain, primary agent of HPS in the US. 250~300 cases since discovery. </li></ul><ul><li>> 50% mortality rate. </li></ul>
  49. 51. White-footed Mouse ( Peromyscus leucopus ) <ul><li>Carrier of New York strain. </li></ul>
  50. 52. Cotton Rat ( Sigmodon hispidus ) <ul><li>Carrier of Black Creek Canal strain. </li></ul>
  51. 53. Rice Rat ( Oryzomys palustris ) <ul><li>Carrier of Bayou strain. </li></ul><ul><li>> 40% mortality rate. </li></ul>
  52. 54. Rodent Transmission Infected rodent Virions in excretion (urine, feces, saliva). Biting, fighting, sexual behavior. Naive rodent
  53. 55. From Rodent to Human <ul><li>Breathing contaminated air, touching contaminated surface, and bite from infected rodent. </li></ul>
  54. 56. Person-to-Person Transmission <ul><li>In general, hantavirus is not transmitted among human. </li></ul><ul><li>The Andes strain found in the 1995 outbreak in Argentina may be an exception. </li></ul>
  55. 57. Summary <ul><li>Negative-sense RNA virus. </li></ul><ul><li>Family: Bunyaviridae. 23 strains so far. </li></ul><ul><li>Simple structure: tri-segmented genome; 4 proteins. </li></ul><ul><li>Simple replication and transcription. </li></ul><ul><ul><li>Prime and realign model. </li></ul></ul><ul><li>Pathogenesis is unclear. </li></ul><ul><ul><li>Dysregulate  3 integrin receptor. </li></ul></ul><ul><ul><li>Immunoresponse. </li></ul></ul><ul><li>Carried by rodents. </li></ul><ul><li>Transmitted through inhalation or contact with rodent excretion. </li></ul>
  56. 58. Topics <ul><li>Clinical Manifestations </li></ul><ul><ul><li>HFRS </li></ul></ul><ul><ul><li>HPS </li></ul></ul><ul><ul><li>Comparison </li></ul></ul><ul><li>Diagnostics </li></ul><ul><li>Treatments </li></ul><ul><li>Vaccines </li></ul>
  57. 59. Clinical Manifestations: <ul><li>Hantavirus is associated with two frequently fatal human diseases: </li></ul><ul><li>a. Hemorrhagic Fever with Renal Syndrome (HFRS): -15% fatality </li></ul><ul><li>b. Nephropathia Epidemica (NE): a mild form of HFRS </li></ul><ul><li>Hantavirus Pulmonary Syndrome (HPS) </li></ul><ul><li>-50% fatality </li></ul>
  58. 60. Stages of Hemorrhagic Fever with Renal Syndrome (HFRS) <ul><li>After an incubation period of 1 or 2 weeks (4-40 days)… </li></ul><ul><li>1)Febrile Phase </li></ul><ul><li>2)Hypotensive Phase </li></ul><ul><li>3)Oliguric Phase </li></ul><ul><li>4)Diuretic Phase </li></ul><ul><li>5)Convalescent Phase </li></ul>
  59. 61. HFRS: Febrile Phase <ul><li>Persists 3-5 days </li></ul><ul><li>Sudden onset of fevers and chills </li></ul><ul><li>Accompanied by headache, severe myalgia, nausea </li></ul><ul><li>Blurred vision, photophobia, pain on ocular movement </li></ul><ul><li>Flushing of face, V-area of the neck and back </li></ul><ul><li>petechiae </li></ul><ul><li>Abdominal pain and back pain. </li></ul><ul><li>Vascular leak syndrome : thirst, edema, hemoconcentration, postural hypotension </li></ul>www.emedicine.com/emerg/topic887.htm#section~pictures
  60. 62. HFRS: Hypotensive phase <ul><li>- Lasts for hours or days </li></ul><ul><li>- Blood pressure decrease, hypovolemia, shock </li></ul><ul><li>-worsening of bleeding manifestations: petechiae, epistaxis, gastrointestinal and intracranial bleeding </li></ul><ul><li>-levels of urea and creatinine in blood rise, proteinuria </li></ul><ul><li>- leukocytosis, thrombocytopenia </li></ul>33% of all HFRS deaths are linked to multi-organ hypoperfusion at this stage www.emedicine.com/emerg/topic887.htm#section~pictures
  61. 63. HFRS: Oliguric Phase <ul><ul><li>Lasts 3-7 days </li></ul></ul><ul><ul><li>Elevation of blood pressure </li></ul></ul><ul><ul><li>Hypervolemia leading to hypertension </li></ul></ul><ul><ul><li>Urine output decreases (renal dysfunction) </li></ul></ul><ul><ul><li>Blood electrolyte imbalance </li></ul></ul><ul><ul><li>Continuation of hemorrhagic symptoms </li></ul></ul><ul><ul><li>Severe complications: cardiac failure pulmonary edema, and cerebral bleeding </li></ul></ul><ul><ul><li>50% of fatalities during this phase </li></ul></ul>
  62. 64. HFRS: Recovery <ul><li>Diuretic Phase: </li></ul><ul><li>Lasts a few days to a few weeks </li></ul><ul><li>Clinical recovery begins </li></ul><ul><li>3-6 liters of urine/ day </li></ul><ul><li>Anorexia, lassitude due to dehydration </li></ul><ul><li>Convalescent Phase: </li></ul><ul><li>Lasts 2-3 months </li></ul><ul><li>Progressive improvement in glomerular filtration, renal blood flow, and urine concentrating ability </li></ul>
  63. 65. Nephropathia Epidemica (NE) <ul><li>Puumala strain </li></ul><ul><li>Most common form of HFRS in Europe </li></ul><ul><li>Generally a milder form of HFRS </li></ul><ul><li>Similar sequence of symptoms, but attenuated </li></ul><ul><li>Only 6% of serologically confirmed cases require hospitalization </li></ul>
  64. 66. Stages of Hantavirus Pulmonary Syndrome (HPS) <ul><li>After asymptomatic incubation of 4-30 days… </li></ul><ul><li>1) Febrile Phase </li></ul><ul><li>2) Cardiopulmonary Phase </li></ul><ul><li>3) Diuretic Phase </li></ul><ul><li>4) Convalescent Phase </li></ul>
  65. 67. HPS: Febrile phase <ul><li>Lasts 3-5 days (1-12 days) </li></ul><ul><li>Fever, myalgias, malaise </li></ul><ul><li>Other symptoms: headache, dizziness, anorexia, nausea, vomiting, and diarrhea. </li></ul><ul><li>Difficult to diagnose as HPS at this stage, since the symptoms are similar to many other viral prodromes </li></ul>Image from CDC
  66. 68. HPS: Cardiopulmonary Phase <ul><li>Non-productive cough and tachypnea appear </li></ul><ul><li>Presentation and rapid progression of shock and pulmonary edema (4-24h). </li></ul><ul><ul><li>Hypovolemia due to progressive leakage of high protein fluid from blood to lung interstitium and alveoli </li></ul></ul><ul><ul><li>Myocardial failure </li></ul></ul><ul><li>Hypotension and oliguria </li></ul><ul><li>Death within 24-48 hours due to hypoxia and/or circulatory compromise . </li></ul>
  67. 69. HPS <ul><li>Diuretic Phase </li></ul><ul><li>Rapid clearance of pulmonary edema </li></ul><ul><li>Resolution of fever and shock </li></ul><ul><li>Early sign: spontaneous diuresis </li></ul><ul><li>Convalescent Phase (up to 2 months) </li></ul><ul><li>Slow but full recovery </li></ul><ul><li>Short term finding: pulmonary dysfunction . </li></ul><ul><ul><li>Decreased small-airways flow and diminished diffusing capacity </li></ul></ul>
  68. 70. HPS <ul><li>Clinical Laboratory Findings </li></ul><ul><ul><li>Thrombocytopenia (decrease in number of platelets): this is consistent in almost all cases in Americas </li></ul></ul><ul><ul><li>Normal or elevated white cell count on presentation of symptoms which increases to high values as disease progresses (leukocytosis). </li></ul></ul><ul><ul><li>A left shift and presence of immunoblasts from late in febrile phase. </li></ul></ul><ul><ul><li>Hemoconcentration </li></ul></ul>
  69. 71. HPS <ul><li>Radiologic Findings </li></ul><ul><ul><li>Radiography of chest show progression from slight interstitial edema to bilateral alveolar edema </li></ul></ul>Images from CDC
  70. 72. HPS: Differential Diagnosis <ul><li>During prodromal phase difficult to differentiate HPS from other acute febrile conditions . </li></ul><ul><ul><ul><li>Blood picture: circulating immunoblasts and thrombocytopenia </li></ul></ul></ul><ul><li>Cardiopulmonary phase: </li></ul><ul><ul><li>Common cause of diffuse pulmonary edema is silent myocardial infarction </li></ul></ul><ul><ul><ul><li>Obtain ECG and echocardiogram </li></ul></ul></ul><ul><ul><li>Bilateral pneumonia with sepsis, adult respiratory distress syndrome complicating systemic infections, sepsis syndrome complicated by disseminated intravascular coagulation or alcohol toxicity </li></ul></ul>
  71. 73. Similarities between HFRS and HPS <ul><li>Febrile illnesses with acute onset </li></ul><ul><li>Generalized vascular involvement (capillary leak, vasodilation) </li></ul><ul><li>Common laboratory features: thrombocytopenia, proteinuria, and leukocytosis with occurrence of activated lymphocytes in peripheral blood. </li></ul><ul><li>Theory : same pathophysiological events, different location . </li></ul><ul><ul><li>The main difference between the two conditions seems to be the difference in location of the particular vascular beds afflicted . </li></ul></ul>
  72. 74. Similarities: Post-mortem Analysis <ul><li>The lack of histological lesions in both HFRS and HPS cases to explain disordered organ function </li></ul><ul><li>Hantaviruses induce altered endothelial function without overt cell death </li></ul><ul><li>Functional derangement of vascular endothelium </li></ul><ul><li>Increased permeability of microvascular beds </li></ul>Image from CDC
  73. 75. Similarities: Spectrum of Illnesses <ul><li>There is no clear delineation; the symptoms of some HFRS and HPS causing strains overlap </li></ul><ul><ul><li>HFRS cases without apparent renal involvement </li></ul></ul><ul><ul><li>Renal involvement in HPS cases: </li></ul></ul><ul><ul><ul><li>HPS associated with Bayou and Black Creek Canal viruses display moderate to prominent renal involvement. </li></ul></ul></ul><ul><ul><li>HPS associated with SNV without pulmonary symptoms </li></ul></ul><ul><ul><li>Pulmonary manifestations in HFRS </li></ul></ul><ul><ul><ul><li>Pulmonary complications recorded in 6% of 828 patients in 1954 outbreak of HFRS; fatal complications in 2% </li></ul></ul></ul>
  74. 76. Etiological Diagnosis <ul><li>Serologic </li></ul><ul><li>Detection of circulating immunoglobulins. </li></ul><ul><ul><ul><li>Usually there is a robust immune response by the time symptoms are present (24hours –within one week of infection) </li></ul></ul></ul><ul><ul><ul><li>IgM present 3-6 months after infection </li></ul></ul></ul><ul><ul><ul><li>IgG can be detected for years post infection </li></ul></ul></ul><ul><ul><li>*ELISA IgM capture assay, using either SNV, Laguna Negra, or Andes antigens depending on location </li></ul></ul><ul><ul><ul><li>Broad cross-reactivities </li></ul></ul></ul><ul><ul><li>*Western blot assay using recombinant antigens and isotype specific conjugates for IgM/IgG differentiation </li></ul></ul><ul><ul><li>Indirect Immunofluorence </li></ul></ul><ul><ul><li>Rapid immunoblot strip assay (RIBA) an investigational prototype assay to identify serum antibody to recombinant proteins and peptides specific for different strains of hantavirus </li></ul></ul>
  75. 77. Etiological Diagnosis <ul><li>Immunohistochemistry: can test formalin fixed tissues with specific monoclonal and polyclonal antibodies </li></ul><ul><li>-retrospective </li></ul><ul><li>RT-PCR: Demonstration of hantavirus antigen in tissues by immunochemistry </li></ul><ul><ul><ul><li>Use of genus specific primers </li></ul></ul></ul><ul><ul><ul><li>Confirms genotype of the infecting virus </li></ul></ul></ul><ul><ul><ul><li>Exponential production of product that may be sequenced for further study </li></ul></ul></ul>
  76. 78. Treatment: Bad News… <ul><li>There is NO CURE for HFRS or HPS!! </li></ul>
  77. 79. Treatment: <ul><li>Aggressive supportive care </li></ul><ul><ul><li>Fluid management </li></ul></ul><ul><ul><li>Hemodynamic monitoring </li></ul></ul><ul><ul><li>Ventilatory support </li></ul></ul><ul><ul><li>Peritoneal dialysis </li></ul></ul><ul><ul><li>Pressor agents (blood pressure support) </li></ul></ul><ul><ul><li>Inotropic agents (cardiac support) </li></ul></ul><ul><ul><ul><li>Increases cardiac muscle contractility </li></ul></ul></ul><ul><li>Broad spectrum antibiotic therapy until HPS is proven (to cover for differential diagnoses) </li></ul><ul><ul><li>Intravenous ceftriaxone or aminoglycoside </li></ul></ul><ul><ul><li>Doxycycline </li></ul></ul>
  78. 80. Experimental Treatment: Extracorporeal Membrane Oxygenation (ECMO) <ul><li>CASE REPORT : University of New Mexico Hospital (UNMH) </li></ul><ul><li> ECMO as rescue therapy: </li></ul><ul><li>- Physicians reviewed hemodynamic and oxygenation data from the 14 HPS patients treated at UNMH during the 1993 Four Corner’s outbreak </li></ul><ul><li>- Identified the findings that correlate with 100% mortality from HPS related cardiopulmonary failure </li></ul><ul><li>- Identified three patients with confirmed HPS and severe cardiopulmonary failure, with predicted 100% mortality to be put on ECMO </li></ul><ul><li>- 2 of 3 survived with good outcome </li></ul>
  79. 81. ECMO: how does it work? <ul><ul><li>Three components: </li></ul></ul><ul><ul><li>1)membrane artificial lung that adds oxygen and removes carbon dioxide </li></ul></ul><ul><ul><li>2) roller pump that moves the patient’s deoxygenated blood to the membrane and back into the body </li></ul></ul><ul><ul><li>3) heat exchanger that warms the blood back to body temperature </li></ul></ul><ul><ul><li>Takes over the function of heart and lungs while the patient recovers from initial cause of pulmonary/cardiac failure </li></ul></ul><ul><li>Venoarterial ECMO: A catheter takes blood from a major vein, runs it through the ECMO machine, and then replaces the blood, under pressure, to arterial circulation. </li></ul>http://biomed.brown.edu/Courses/BI108/BI108_2001_Groups/ECMO
  80. 82. ECMO: Why isn’t it used? <ul><li>Complications of the therapy itself </li></ul><ul><li>Cost: tens of thousands to a million dollars per patient depending on length of time necessary </li></ul><ul><li>Only specialized tertiary care institutions have these units. </li></ul>
  81. 83. Experimental Treatment: antiviral agents (Ribavirin) <ul><ul><li>RNA virus mutagen: antiviral guanosine analog </li></ul></ul><ul><ul><li>Inhibits hantavirus growth in vitro </li></ul></ul><ul><ul><li>Mechanism is unknown </li></ul></ul><ul><ul><ul><li>Severson et al (2003) hypothesize that ribavirin challenges the fidelity of the hantavirus polymerase, causing error catastrophe </li></ul></ul></ul><ul><li>Effective in treating HFRS if administered 5 days after onset of disease. </li></ul><ul><ul><ul><ul><li>-Lessens renal failure </li></ul></ul></ul></ul><ul><ul><ul><ul><li>-Decreases bleeding manifestations </li></ul></ul></ul></ul><ul><ul><ul><ul><li>-Decreases overall mortality </li></ul></ul></ul></ul><ul><li>Not proven effective in HPS </li></ul>www. sch-plough.com/prod/prod03_anti.htm
  82. 84. Ribavirin:HPS <ul><li>Open label protocol treating 30 patients with HPS with IV ribavirin (June 4, 1993 to September 1, 1994), comparing outcome to 34 untreated HPS patients : </li></ul><ul><ul><ul><ul><li>Treated: mortality rate of 47% (14/30) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Untreated: mortality rate of 50% (17/34) </li></ul></ul></ul></ul><ul><li>INCONCLUSIVE : </li></ul><ul><ul><li>Most enrolled were ill in the early phase of the epidemic or presented in nonepidemic areas where diagnosis may have been delayed. </li></ul></ul><ul><ul><li>Treatment with ribavirin may have come too late. </li></ul></ul><ul><li>Currently in progress : an NIH sponsored double-blinded placebo controlled trial of intravenous ribavirin for presumed HPS: </li></ul><ul><ul><li>Designed to treat patients in the earliest stage of illness before the onset of shock. </li></ul></ul><ul><li>And so we await the results… </li></ul>
  83. 85. Treatment <ul><li>Future strategies for treatment </li></ul><ul><li>Target the hantavirus/ β -3 integrin interaction </li></ul><ul><li>Immunologic approach </li></ul><ul><ul><li>Target host inflammatory responses </li></ul></ul><ul><ul><li>In progress: work to find inhibitors of TNF-  </li></ul></ul>
  84. 86. Vaccines <ul><li>No hantavirus vaccines are currently approved for common use in the U.S. </li></ul><ul><li>Inactivated virus vaccines in Asia </li></ul><ul><ul><li>Cell culture derived vaccines in China </li></ul></ul><ul><ul><ul><li>Vaccination trial with >100,000 participants showed that four years after primary vaccination, average prevention rates were >90% </li></ul></ul></ul><ul><ul><li>Formalin inactivated rodent brain derived vaccines for HFRS (from SEOV and HTNV infections). </li></ul></ul><ul><ul><ul><li>Example: Hantavax, commercially produced in South Korea </li></ul></ul></ul><ul><ul><ul><li>Seroconversion of 97% one month after second vaccination </li></ul></ul></ul><ul><ul><ul><li>Safe, only minor side effects </li></ul></ul></ul>
  85. 87. Vaccines <ul><ul><li>U.S. is now focusing on recombinant DNA approaches. </li></ul></ul><ul><ul><li>Investigational vaccinia HTNV vaccine is currently offered to laboratory workers at USAMRIID. </li></ul></ul>
  86. 88. Summary <ul><li>HFRS: febrile illness with acute renal dysfuntion </li></ul><ul><li>HPS: febrile illness with acute pulmonary dysfunction. </li></ul><ul><li>Both characterized by vascular leakage. </li></ul><ul><li>Treatment is mainly supportive </li></ul><ul><li>Inactivated virus vaccines are available for HFRS in Asia </li></ul><ul><li>There are no vaccines for HPS </li></ul>
  87. 89. <ul><li>“ Based on current human population growth and </li></ul><ul><li>development trends, hantavirus diseases will become </li></ul><ul><li>more common in the near future unless public health </li></ul><ul><li>measures are taken to curtail or eliminate rodents from </li></ul><ul><li>human communities.” </li></ul><ul><li>-JA Lednicky Department of Pathology, Loyola University Medical Center, </li></ul>
  88. 90. Hantavirus as a bioweapon? <ul><li>In more recent years, it has been more common for outbreak investigators to consider the possibility of a terrorist event when they investigate the cause of an outbreak </li></ul><ul><li>The 1993 outbreak of Hantavirus in the Four Corners represented an incident in which a bio-terrorist attack was suspected </li></ul><ul><li>Throughout the investigation there were rumors that a biological agent had been released as an act of genocide against the Navajo people </li></ul>
  89. 91. Speculation of Hantavirus use? <ul><li>The outbreak in Korea in 1950 is suspected to have been caused by bioterrorist attack </li></ul><ul><li>In 1995, an outbreak in Bosnia infected over 250 people (outbreak) </li></ul><ul><li>Because this virus has made an appearance at almost every major war of the 20 th century, it has definitely been suspected to have been used as a biological weapon </li></ul><ul><ul><li>However, it is unknown whether this occurs mainly because of increased exposure during wartime or a disruption in the ecosystem resulting in an increase in the mouse population </li></ul></ul>
  90. 92. HFRS as a bio-weapon <ul><li>HFRS is categorized as a Biological Agents Category A: High priority </li></ul><ul><ul><li>Easily disseminated or transmitted person to person </li></ul></ul><ul><ul><li>High mortality - major P.H. impact </li></ul></ul><ul><ul><li>Cause public panic, social disruption </li></ul></ul><ul><ul><li>Special action for P.H. preparedness  </li></ul></ul>
  91. 93. However… <ul><li>However, there are currently quick and efficient diagnostic tests </li></ul><ul><li>Treatments for HFRS available </li></ul><ul><li>Vaccines are against HFRS are also available </li></ul>
  92. 94. HPS as a bio-weapon <ul><li>Hantavirus causing HPS are classified under Biological Agents Category C: third priority </li></ul><ul><ul><li>Emerging Pathogens   </li></ul></ul><ul><ul><li>Availability </li></ul></ul><ul><ul><li>Ease of production </li></ul></ul><ul><ul><li>Potential for high morbidity, mortality and major public health impact  </li></ul></ul>
  93. 95. However…  <ul><li>SNV is highly lethal in its aerosolized form (four corners incident) </li></ul><ul><li>Certain forms (the Andes virus) are suspected of being able to transmit through human contact </li></ul><ul><li>There are no vaccines </li></ul><ul><li>Natural immunity to HPS is low </li></ul><ul><li>Mice populate the entire United States </li></ul><ul><li>Can pose a worldwide threat because it is carried by all types of rodent </li></ul>
  94. 96. If hantavirus were to be used… <ul><li>The hantavirus can only exist 1-3 days outside of the host because of its weak lipid envelope </li></ul><ul><li>However, the symptoms will take anywhere from 4-40 days to show, thus delaying the impact of the weapon release </li></ul><ul><li>This may cause secondary and tertiary waves of illness, especially if a strain that has properties that allow for human-human transmission </li></ul>
  95. 97. The good news <ul><li>The Hantavirus can be destroyed with a simple detergent </li></ul><ul><li>The disease itself is considered rare, as the virus is not very infectious except under certain circumstances (like an attack!) </li></ul><ul><li>The detection methods are improving, thus allowing physicians and other health personnel to catch the disease at an earlier time, thus increasing the chances of full recovery </li></ul>
  96. 98. Staying Safe <ul><li>As usual, PREVENTION is the BEST method </li></ul><ul><li>Even if Hantavirus is not used as a bioterrorist method, because of the nature of the rodent reservoir, everyone is potentially at risk </li></ul><ul><li>Furthermore, education would help prevent or contain an outbreak or an attack </li></ul>
  97. 99. Keep away from… <ul><li>occupying rodent-infested vacant cabins or other dwellings; </li></ul><ul><li>cleaning barns or other outbuildings; </li></ul><ul><li>disturbing rodent infested areas while hiking or camping; </li></ul><ul><li>planting or harvesting fields, </li></ul><ul><li>living in or visiting areas where there has been an increase in rodents. </li></ul>
  98. 100. Lowering the risk of contracting HPS
  99. 101. Preventing the spread of a possible bio-terrorist attack or outbreak <ul><li>Maintaining surveillance systems for emerging diseases allows possible infectious diseases to be detected quickly and efficiently </li></ul><ul><li>If the weapon is transmissible from person to person, there may only be a short window of opportunity to identify the organism and prevent further spread before a second wave of illness strikes </li></ul><ul><li>Training emergency personnel and more experienced in addressing the cases of unexplained illness (ie. It’s NOT the flu) </li></ul><ul><li>Improving diagnostic techniques </li></ul><ul><li>Making sure that resources for outbreak investigations are readily available </li></ul>
  100. 102. References <ul><li>Simmons, J, Riley, L (2002). Hantaviruses: an overview, Comparative Medicine 52 , 97-110. </li></ul><ul><li>Garcin, D, Lezzi, M, Dobbs, M, Elliott RM, Schmaljohn C, Kang CY, Kolakofsky D (1995). The 5’ end of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis. J. Virio 69 , 5754-5762. </li></ul><ul><li>Centers for Disease Control and Prevention (2002). Hantavirus Pulmonary Syndrome - United States: Updated Recommendation for Risk Prevention. MMWR 51 . </li></ul><ul><li>Schmaljohn, C. Hantaviruses (Bunyaviridae), Academic Press, 1999. </li></ul><ul><li>Meyer B, Schmaljohn C (2000). Persistent hantavirus infections: characteristics and mechanisms. Trends in Microbio 8 , 61-67. </li></ul><ul><li>National Center for Infectious Diseases. Preventing hantavirus disease education video, September 1994. </li></ul><ul><li>Zhao X, Hay, J (1997). The epidemiology of hantavirus infections. Clinical Microbiology Newletter 19, No. 7. </li></ul><ul><li>Crowley, MR., et al (1998). Successful treatment of adults with severe Hantavirus pulmonary syndrome with extracorporeal membrane oxygenation. Critical Care Medicine 26 , 409-414. </li></ul><ul><li>Khaiboullina, Svetlana F. and Stephen C. St. Jeor (2002). Hantavirus Immunology, Viral Immunology 15 , 609-625. </li></ul><ul><li>Peters, C.J., Gary L. Simpson and H Levy. (1999). Spectrum of Hantavirus Infection: Hemorrhagic Fever with Renal Syndrome and Hantavirus Pulmonary Syndrome, Annu. Rev. Med. 50 , 531-45. </li></ul><ul><li>Severson, William E., Connie S. Schmaljohn, Ali Javadian, and Colleen B. Jonsson (2003). Ribavirin causes error catastrophe during hantaan virus relication, Journal of Virology 77 , 481-488. </li></ul><ul><li>Peters, Clarence J. Infections Caused by Arthropod –and Rodent Borne Viruses (2001-2003). Harrison’s Principles of Internal Medicine. McGraw Hill Companies. </li></ul>
  101. 103. The End Thank you!