OVERVIEW History Organism Epidemiology Transmission Disease in Humans Prevention and Control
INTRODUCTION Two major outbreaks of disease led to the discovery of hantaviruses in the Old and New Worlds. The first outbreak occurred during the Korean War (1950 to 1953), where in more than 3,200 United Nations troops fell ill with Korean hemorrhagic fever, which is commonly referred to as hemorrhagic fever with renal syndrome (HFRS). The second outbreak of disease occurred in the Four Corners region of the United States in 1993 and was initially referred to as Four Corners disease, which is now called hantavirus pulmonary syndrome (HPS) or hantavirus cardiopulmonary syndrome (HCPS).
FOUR CORNERS OUTBREAK The converging point of Colorado, Utah, Arizona, and New Mexico‘s state borders became the location of the 1993 Four Corners Outbreak of Hantavirus Pulmonary Syndrome (HPS). The Navajo tribal belief is such that mice were responsible for bringing seeds to the earth, allowing humans to survive. Mice are thought of as the ―landlords of the world‖ and are highly respected in Navajo culture.
HISTORY In May 1993, an outbreak of an unexplained pulmonary illness occurred in the southwestern United States, in an area shared by Arizona, New Mexico, Colorado and Utah known as "The Four Corners". A young, physically fit Navajo man suffering from shortness of breath was rushed to a hospital in New Mexico and died very rapidly.
The mysterious illness A native Indian Health physician began to notice an outbreak of an unexplained illness that caused death among normal healthy young adults After calling his colleagues, he discovered that 10 people had already died of a similar respiratory disease Autopsies did not reveal any sign of viral pneumonia, influenza or any other common disease that attacked the lungs Although this disease was not specific to the Navajo people, to the media, the disease became known as the ―Navajo disease‖ When the number of cases doubled to about 20 victims, the CDC was called in
CDC In May 1993, the CDC was called in to investigate the case. 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 However, scientists were skeptical for 3 reasons The only hantaviruses known were on the Eurasian continent The diseases caused by the Eurasian hantavirus strain did not cause respiratory failure The new virus in the ―four corners‖ appeared to be 5 times as lethal as the strain in Europe!
HISTORY HPS was first recognized as a HANTAVIRUS DISEASE. 32 of the 53 people were infected & died. A warm winter allowed for an increase in the host population. Outbreak was caused by the SIN NOMBRE STRAIN(SNV, in Spanish, "Virus sin Nombre", for "nameless virus"). 30% of the mice in that area carried this strain.
FOUR CORNERS OUTBREAK Winter and spring 1993 Drought for several years followed by snow and rain Vegetation blossomed and rodent population grew tenfold Virus was isolated 1 month after the first report of cases and named as Muerto Canyon virus, then Four Corners virus, and finally Sin Nombre Virus (SNV –―virus without a name‖).
FOUR CORNERS OUTBREAK Newly emerging virus has been present since 1959. 38 year old Utah man that had died from an illness compatible with hanta in 1959. Researchers located his lung tissue and utilizing current technology, were able to isolate SNV in 1994. The earliest case of HPS to be confirmed by IHC with direct visualization of hanta viral antigens in postmortem tissue involved a patient who died in 1978.
HISTORY Hantavirus disease outbreaks have occurred as far back as American Civil War times. There are records of a hemorrhagic fever syndrome (HFRS) during World War I and II. First outbreaks of hantavirus causing HFRS were reported in Russia in 1913 and 1932. Japanese troops in Manchuria reportedly had cases in 1932 and cases referred to as Nephropathia Epidemica (NE) in Sweden appeared in 1934. Western medicine diagnosed Korean Hemorrhagic Fever (KHF) during the Korean War in the 1950‘s.
HISTORY 1951-1954: Korean War 3,200 U.N. troops develop disease Hantaan River separated N. & S. Korea 1977 Hantaan agent was isolated and characterized 1990: 94% of serum samples from soldiers in 1950‘s had antibodies 1979 Seoul virus found in Japan and Europe
HISTORY Its original name was "Four Corners virus" or "Navajo Flu", but the name was changed after local residents raised objections. Its rodent host, the Deer mouse (Peromyscus maniculatus), was first identified by Terry Yates, a professor at the University of New Mexico.
HISTORY 1951-1954: HEMORAGIC FEVER WITH RENAL FAILURE FIRST RECOGNIZED AS A PATHOGEN AFTER AN OUTBREAK IN HANTAAN KOREA. 1977: DISEASE ISOLATED AND NAMED AFTER HANTAAN RIVER. 1978: IT WAS CONFIRMED THAT THE VIRUS IS CARRIED BY RODENTS. 1981: FIRST SUCCESSFUL PROPAGATION OF VIRUS IN CELL CULTURE. 1993: OUTBREAK OF HPS IN FOUR CORNER REGION OF COLORADO, NEW MEXICO, ARIZONA AND UTAH.
RECENT CASES May 2003: Montana Three cases Two deaths Contracted virus from rodents in home First cases since fall of 2001 Overall cases in Montana Virus first appeared in state in 1993 20 cases 5 deaths
BUNYAVIRIDAE Genus Human disease Bunyavirus LaCrosse encephalitis, others Phlebovirus Rift Valley fever, sandfly fever Nairovirus Crimean-Congo hemorrhagic fever Tospovirus Plant virus, no known human disease Hantavirus -Hemorrhagic fever with renal syndrome -Hantavirus pulmonary syndrome
HANTA VIRUS GENUS Hantavirus Similarities RNA viruses Lipid membrane Tri-segmented genome Hantavirus Differences Hantavirus transmitted through aerosolized rodent urine, feces and saliva. Others genera transmitted through arthropod vectors.
IMPORTANT SPECIES OF HANTA The genus Hanta contains at least four species— 1.Hantaan virus causing the severe HFRS in the Far East, North Asia and Russia, 2.Seoul virus causing a milder type of disease and probably present worldwide, 3.Puumala virus responsible for Nephropathia epidemica in Northern and Eastern Europe, and 4.Prospect Hill virus isolated from voles in the USA, which has not been associated with human illness.
INTRODUCTION Hantaviruses belong to the bunyaviridae family of viruses. Hanta viruses are rodent borne diseases transmitted from humans to humans in aerosolized urine/ saliva & occasionally by bite. International committee on taxonomy of viruses recognized 30 species in the genus Hanta virus (of which 21 are pathogenic to humans).
TAXONOMY Rodent borne Hantaviruses are divided into 3 groups based on the taxonomic assignment of their principal hosts belonging to families muridae & cricetidae. FAMILY SUBFAMILY PRINICIPAL HOSTMURIDAE Murinae old world rats & miceCricetidae Arvicolinae Voles & LemmingsCricetidae Sigmodontinae New world rats & Mice Cricetidae Neotominae
ASSOCIATION WITH DISEASE Murine rodents are the principal hosts of Hanta virus associated with severe HFRS. Voles are the principal hosts of Puumula virus, which is the cause for relative mild form of HFRS called Nephropathia epidemica. Sigmodontinae & Neotominae rodents the principal hosts of Hanta virus Known to cause HPS.
DESCRIPTION OF THE AGENT Spherical Enveloped, ss negative sense RNA virus. Virions 80-120nm in diameter with a characteristic square grid-like structure. Helical nucleocapsid. Genome consists of three RNA segments: L, M, and S. NO matrix protein. Lipid bilayer.
DESCRIPTION OF THE AGENT Spikes protrude from the lipid bilayer envelope. Spikes consists of glycoproteins Gn & Gc (formerly G1 &G2 respectively). Virion contains ribonucleocapsid--SS RNA complexed with nucleocapsid protein- L protein. Genomes of Hanta virus consists of trisegmented negative sense Linear ss RNA. Three segments: Large (L) codes for viral polymerase Medium (M) codes for G1 and G2 glycoproteins Small (S) codes for nucleocapsid
Membrane glycoproteins (G1 and G2) Polymerase (L)Nucleocapsidproteins (N)
Membrane Glycoproteins G1: 64-67kDa G2: 54 kDa, highly conserved Integral membrane proteins G1-G2 heterodimers form 8 nm projections on virion surface Cysteine-rich Contain asparagine-linked sugar groups Important in cell entry and pathogenesis
Nucleocapsid Protein 48 kDA Complexes with genomic vRNA in virus, as well as with cRNA after infection, but not with mRNA Necessary for virus replication and packaging
Viral Polymerase 247 kDA RNA-dependent RNA polymerase (RdRp) Complexed with ribonucleocapsids in virion Endonuclease activity to cleave host mRNA Transcriptase activity for making cRNA and mRNA from vRNA Helicase activity to unwind vRNA during transcription
Genomic Organization Tripartite negative sense genome Small (S) segment, 1.7-2.1kb, codes for N nucleocapsid protein Medium (M) segment, 3.6-3.7kb, codes for G1 and G2 glycoproteins Large (L) segment, 6.5 kb, codes for L polymerase protein
Sin Nombre Virus• Circular single stranded; RNA• Total genome 10500-22700 nucleotides long• 100 (80-120) nm in diameter• Surface projections of envelope distinct; spikes (of about 10 nm).• Reservoir for Sin Nombre virus is deer mice
Viral Replication Receptor mediated endocytosis Occurs in cytoplasm Budding at Golgi apparatus or cell membrane
Virion Assembly Membrane-bound G1 and G2 peptides are transported to Golgi apparatus and carbohydrates are attached by N-linked glycosylation vRNA complexes with N nucleocapsid protein, forms looped panhandle structure, and complexes with polymerase
Virion ReleaseTwo Mechanisms Nucleocapsid complexes G1 and G2 embed bud into the Golgi into cell membrane membrane with G1 and through Golgi G2 embedded vesicles Virion particle is formed Virions bud from cell inside Golgi apparatus membrane, not Virions are transported to through Golgi cell membrane by vesicles apparatus and released by exocytosis
Old World & New World Hantavirus Hantaviruses are commonly referred to as Old World and New World hantaviruses due to the geographic distribution of their rodent reservoirs and the type of illness (HFRS or HPS) that manifests upon transmission to humans.
Old World Hantavirus vs New World Hantavirus Found mostly in Europe and Discovered in the ―four Asia corners in America Carried by rodents Carried by rodents Causes HFRS Causes HPS Targets the kidney Targets the lungs Mortality rate is <10-15% Mortality rate is 50-60% Vaccine exists for these Vaccine does not exist for strains these strains
HANTA VIRUSES IN THE OLD WORLDSerotype Host LocationHantaan Apodemus agrarius Asia, Far (striped field mouse) East RussiaDobrava A. agrarius, A. flavicollis Europe (yellow neck mouse) BalkansSeoul Rattus norvegicus, R. rattus Worldwide (Norway brown rat, roof rat)Puumala Clethrionomys glareolus Europe (red bank vole)
HANTA VIRUSES IN THE NEW WORLDSerotype Host LocationSin Nombre Peromyscus Central & maniculatus (deer West U.S., mouse) CanadaMonongahela Peromyscus Eastern U.S., maniculatus (deer Canada mouse)New York Peromyscus leucopus Eastern U.S., (white-footed mouse) CanadaBayou Oryzomys palustris SE U.S. (rice rat)Black Creek Sigmodon hispidus SE U.S.Canal (cotton rat)
HANTA VIRUSES IN THE NEW WORLDSerotype Host LocationAndes Oligoryzomys Argentina/Ch longicaudatus (long- ile tailed pygmy rice rat)Oran O. longicaudatus NW ArgentinaLechiguanas O. flavescens Central ArgentinaHu39694 Unknown Central ArgentinaLaguna Negra Calomys laucha Paraguay/ BoliviaJuquitiba Unknown Brazil
Epidemiology and Rodent Hosts Each strain of hantavirus has a specific rodent host Hantavirus species appear to have co-evolved with host rodent species Rodents carrying hantavirus are asymptomatic
EPIDEMIOLOGY Hantan virus ,prototypical member of genus Hanta virus is the cause of a severe form HFRS endemic in Korea, China and Eastern Russia. Dobrava- Belgrade virus is an agent of Severe form of HFRS in the Balkans Greece and Russia Seoul Virus is endemic in Asia, Europe and America Puumala viurs is endemic in Europe and Scandinivea Saaremaa virus – in Europe . Amur virus – Eastern Russia. SinNOmbre virus is the major cause of HPS in North America . Andes virus is the major cause of HPS inSouth America
Geographical representation of approximatehanta viral disease incidence by country per year. Jonsson C B et al. Clin. Microbiol. Rev. 2010;23:412-441
TransmissionVectors Transmitted via aerosolized rodent urine, feces, and saliva Deer mouse (Peromyscus maniculatus) Cotton rat (Sigmodon hispidus) White-footed mouse (Peromyscus leucopus) Striped field mouse (Apodemus agrarius) Bank vole (Clethrionomys glareolus) Rat (Rattus)
Deer Mouse Cotton Rat House Rat (Mus musculus) Rice Rat White Footed Mouse
Hantavirus and Host Cells Virus replication typically halts host macromolecule synthesis Hantavirus replication does not affect host cell‘s natural functions Hantavirus release does not require host cell lysis Hantavirus is able to establish a persistent infection in rodent host cells
Integrins Hetero dimeric receptors composed of α and β subunits Present on endothelial cells, macrophages, and platelets – cells affected by Hantavirus infection Normally involved in regulation of endothelial cell adhesion, platelet aggregation, Ca++ channel activation, and extracellular matrix interactions, including cell migration
β3-Integrins Required for infection by pathogenic Hantaviruses β1 integrins are used by non-pathogenic strains Attachment of G1/G2 proteins of viroid to integrin initiates endocytosis, but also activates the receptor Variation in virus G1/G2 protein may account for severity of disease
Hantavirus InfectionPathogenesis Binding of Hantavirus glycoproteins to β3 integrin causes disruption of vascular integrity Capillaries become more permeable Arteriole vasoconstriction and vasodilatation are disrupted Binding to platelet receptors affects clotting and platelet function
Immune Reaction Immune system activated against Hantavirus epitopes Virus epitopes expressed on surface of host cells triggers cytotoxic T-cell attack on host tissues Symptoms are consistent with inflammatory response
Clinical Presentationof Hantavirus InfectionThree different clinical manifestations of hantavirus infection caused by different viral strainsHemorrhagic fever with renal syndrome (HFRS) Found in Europe and AsiaNephropathia Epidemica (NE) Found in EuropeHantavirus pulmonary syndrome (HPS) Found in north and south America
MAJOR DIFFERENCE B/N HFRS & HPS HFRS HPS Retroperitoneum is the Lungs & thoracic cavity are themajor site of the vascular major sites of vascular leaks in leak in HFRS. HPS.
Stages of Hemorrhagic Fever with Renal Syndrome (HFRS)1)Incubation (4-40 days)2)Febrile Phase (3-5 days):Characterized by fever, chills, headache, severe myalgia (muscle pain), nausea3)Hypotensive Phase (hours to days): Decrease in blood pressure, hypovolemia (decreased blood volume), shock4)Oliguric Phase (3-7 days):Marked by decreased urine production due to renal (kidney) dysfunction
Stages of Hemorrhagic Fever with RenalSyndrome (HFRS)Recovery:5)Diuretic Phase (2-21 days):Beginning of recovery, 3-6 liters of urine/ day; return to normal renal activity6)Convalescent Phase (2-3 months):Progressive improvement in glomerular filtration, renal blood flow, and urine concentrating ability
Clinical Testing forHFRS Thrombocytopenia (low platelet count) is a signifier Urine tests for albuminuria (abnormally high amounts of the plasma protein albumin in the urine) Urine tests for microhematuria (microscopic amounts of blood in the urine)
Problems Diagnosing HFRS Early symptoms resemble influenza More serious symptoms of hypotensive phase have acute onset
PATHOLOGY In HFRS, pathology findings are: 1.Effusions in the body cavities, 2.Retroperitoneal edema, 3.Enlarged, congested & hemorrhagic kidneys.
Nephropathia Epidemica (NE) Puumala hantavirus strain Common mild form of HFRS in Europe Similar sequence of symptoms as HFRS, but much milder Only 6% of serologically confirmed cases require hospitalization
HPS 1993 four corners outbreak Cases found in almost all parts of the Americas ~50% fatality
Stages of Hantavirus Pulmonary Syndrome (HPS)1)Incubation (4-30 days)2)Febrile phase (3-5 days): Characterized by fever, myalgia, malaise, headache, dizziness, anorexia, nausea, vomiting, and diarrhea.3)Cardiopulmonary phase ( 4-24 hours): Presentation and rapid progression of shock and pulmonary edema (4-24h non- productive cough and tachypnea (shortness of breath)4)Diuretic phase5)Convalescent phase: Results in chronic decreased small- airway volume and diminished alveolar diffusing capacity
HPS Radiographic Findings Bilateral interstitial infiltrates Moderate to rapid progression Bilateral alveolar infiltrates Pleural effusion Normal heart size
Clinical Testing for HPS Many lab tests and radiographs appear normal Serological tests more effective ELISA IgM capture assay, using either SNV, Laguna Negra, or Andes antigens are used in all countries that have previously detected cases Immunofluorescent test for the presence of antibodies Blood analysis also may find thrombocytopenia with platelet count less than 150,000 mm in 98% of cases
PATHOLOGY In HPS, pathology findings are: 1.Copious amounts of frothy fluid in bronchi & other airways, 2.Edematous lungs & 3.Pleural effusions.
COLLECTION,TRANSPORT & STORAGE OFSPECIMENS SPECIMENS: Blood, Serum, Urine, CSF, Respiratory secretions. Specimens are collected during acute phase of illness. Precautions must be taken while handling the specimens of Hanta virus. 1)Sera from HFRS/ HPS patients should be handled at BSL-2. 2) Potentially infectious tissue specimens should be handled at BSL-2 using BSL-3 practices. Blood, Serum/plasma samples for serology should be strored at 4º C & sent to lab on ice packs. Samples for RNA isolation & subsequent testing by RT PCR are stored continuously at -70º C.
LABORATORY DIAGNOSIS 1.Direct detection—a. Microscopy. b. Antigen detection. c. Nucleic acid detection. 2.Virus isolation. 3.Serologic tests. 4.Identification– a. Serologic methods. b. Genetic methods. 5.Typing Systems.—Sequence analysis.
DIRECT DETECTION Microscopy : It has limited diagnostic value. Electron microscopic examination of autopsy tissues from HFRS & HPS patients showed more number of HANTA viral inclusion bodies containing mature intact virions. Antigen detection : Immunohistochemistry: can test formalin fixed tissues with specific monoclonal (murine) and polyclonal antibodies retrospective diagnosis. Nucleic acid detection : By RT PCR, Nested PCR, real time RT PCR. Use of genus specific Oligonucleotide primers that anneal to regions of ‗S‘& ‗M‘ genomic segments confirms genotype of the infecting virus. Exponential production of product that may be sequenced for viral characterization .
VIRUS ISOLATION Virus isolation is not commonly used for diagnosis of Hanta viral infections in humans. Vero E6 cell line (ATCC CRL-1586) is used to isolate hanta virus from blood, urine, tissue samples of HFRS patients and from serum &urine of patients with Andes virus HPS. A monolayer cell cultures of Vero E6 cells are inoculated with a crude / clarified tissue homogenate & maintained under a fluid overlay for 10-14 days.
VIRUS ISOLATION Hanta viruses are neither cytopathic in cultured cells nor pathogenic in laboratory rodents. Detection of infection in cultured cells & in tissues of laboratory rodents is by an indirect method i.e 1.Fluorescent antibody testing for viral antigen. 2.RT PCR assay for Hantavirus specific RNA.
SEROLOGIC TESTS Detection of circulating immunoglobulins. Usually there is a robust immune response by the time symptoms are present (24hours –within one week of infection) IgM present 3-6 months after infection. IgG can be detected for years post infection. IgG & IgM responses are directed first against nucleocapsid protein, then against the glycoproteins. Neutralizing antibodies appear during acute phase of HFRS & HPS—Reactive against glycoproteins Gn & Gc. IgG against Gn is more specific than anti nucleocapsid protein IgG.
SEROLOGIC TESTS Methods to detect antibodies against hanta viruses in serum/ plasma are; 1.High particle density agglutination. 2.Indirect Immunofluorescence assay. 3. Immunoprecipitation. 4.RadioImmunoassay. 5.Haemagglutination inhibition. 6.Plaque & Focus reduction Neutralization -Foci of infected cells(viral antigen in focus) is revealed by IHC staining/ Chemiluminescence. 7.Western Immunoblotting-*using recombinant antigens and isotype specific conjugates for IgM/IgG differentiation. 8.µ capture (IgM) ELISA. 9.IgG ELISA.
IgM Capture ELISA Highly sensitive in detecting antiHantavirus IgM, but not specific for virus. It uses a Hanta virus infected cell lysate as test antigen. Uninfected cell lysate as control. Appropriate positive & negative control.
Treatment ofHantavirus Infection General care, alleviation of symptoms Ribavirin (HFRS) ECMO (HPS). Hyper immune (Neutralizing ) serum.
GENERAL CARE HFRS HPS General treatment for General treatment for renal failure pulmonary pathology Hydration Administration of Dialysis oxygen
THERAPYAggressive supportive care Fluid management Hemodynamic monitoring Ventilatory support Peritoneal dialysis Pressor agents (blood pressure support) Inotropic agents (cardiac support) Increases cardiac muscle contractility Broad spectrum antibiotic therapy until HPS is proven (to cover for differential diagnoses) Intravenous ceftriaxone or aminoglycoside Doxycycline
Ribavirin Administered intravenously Shown to be effective against Hemorrhagic Fever with Renal Syndrome Not shown to be effective against Hantavirus Pulmonary Syndrome causing strains
ECMO-Takes over the function of heart and lungs while the patientrecovers from initial cause of pulmonary/cardiac failure Three components: 1)membrane artificial lung that adds oxygen and removes carbon dioxide 2) roller pump that moves the patient‘s deoxygenated blood to the membrane and back into the body 3) heat exchanger that warms the blood back to body temperature
VACCINES No WHO approved vaccines are available. Inactivated vaccines have been developed in Asia & used locally in Korea for protection of humans against HFRS. Inactivated vaccines are prepared from brains of suckling rats/ mice or from cell cultures infected with Hantaan virus/ seoul virus.----Hantavax, commercially produced in S.korea. STRATAGIES for development of new Hanta virus vaccines include: 1.Recombinant nonpathogenic viruses, 2. Rodent/ cell culture derived inactivated virus. 3. Naked DNA 4.E. Coli expressed truncated nucleocapsid as an immunogen
PREVENTION & CONTROL Rodent Control - control measures should be aimed at reducing contact between humans and rodents. Prevent aerosolization of virus from rodent excrement Dampen surfaces with detergent before cleaning General hygiene