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Microbiology (Dra Madrid/Reyes) Flaviviridae 13 December 07

FLAVIVIRIDAE  Flaviviridae family o Genus Flavivirus  Once classified in the Togaviridae, now constitutes one of three genera in the family Flaviviridae o Genus Pestivirus  Includes animal pathogens (bovine viral diarrhea and hog cholera viruses) that are of considerable economic importance, but contains no known human pathogens o Genus Hepacivirus o All flaviviruses that cause disease in humans are arthropod-borne viruses (arboviruses) VIRUS PRIMARY VECTOR Aedes aegypti Culex Culex Culex VERTEBRA TE RESERVOI R Humans, monkeys Birds Birds, pigs Birds GEOGRAPHIC DISTRIBUTIO N Tropics Asia Americas Africa, tropical Asia, Mediterranean Tropical Africa and the Americas Central Russia PATHOGENIC FLAVIVIRUSES  3 groups according to symptoms they cause o Meningoencephalitides  SLE, JE, MVE and TBE o Fever-arthralgia-rash syndrome  DEN, WN, KUN o Hemorrhagic fever syndrome  Omsk hemorrhagic fever, Kyasanur Forest dse virus, YF, DEN FLAVIVIRUS STRUCTURE  Virions: spherical, 40-50 nm in diameter  Nucleocapsid contains capsid protein (C)  RNA: single 405 (10.9 kilobases) positive-sense strand o Capped at the 5’ end, but, unlike alphaviruses, has no poly A segment at the 3’ end  Envelope: lipid bilayer o 1) envelope protein (E) [51,000-59,000 daltons] o 2) small nonglycosylated protein (M) [8,500 daltons] o Only E, which is glycosylated in most flaviviruses, is clearly demonstrable on the virion surface  Virions mature at intracytoplasmic membranes  Most members are transmitted by bloodsucking arthropods FLAVIVIRUS GENOME  Upon translation, several enzymes cut single polypeptide into functional protein units.

Dengue 1,2,3,4 Japanese enceph St. Louis enceph West Nile

Yellow Fever Omsk hemmorhagic fever Tick-borne encephalitis

Aedes aegypti Dermacentor

Humans, monkeys Rodents


Louping III Powassan Kyansanur Forest Disease Murray Valley encephalitis/ Kunjin Rocio

Ixodes Ixodes Haemaphysalis Culex

Rodents, birds, domesticate d animals Sheep, birds Small mammals Rodents Birds

Russia, Eastern Europe, Scandinavia British Isles Canada, US, Russia Southwest India Austrialia, New Guniea

Structural p rotein functions :  C: highly basic component of the nucleocapsid  prM: precursor of M protein  M protein: membrane-associated and serve a matrix function, linking capsid and envelope  E: major envelope protein; virion assembly, receptor binding and membrane fusion

MULTIPLICATION  Genomic RNA is capped (not polyadenylated) and serves as mRNA for all proteinds  Structural proteins are encoded at the 5’ end of the genome; nonstructural proteins (e.g., RNA-dependent RNA polymerase) are encoded in the 3’ two-thirds  Complementary (antisense) RNA, made from genomic RNA, serves as a template for progeny genomic RNA

(+) Strand parental RNA

Proteins encoded in the entire genome

Virus Dengue 1,2.3.4 Japanese enceph St. Louis enceph West Nile Yellow Fever Omsk hemorrhagic fever Tick-borne encephalitis Louping III Powassan Kyansanur Forest Disease Murray Valley encephalitis/Kunjin Rocio

Disease Fever, rash, arthralgia, myalgia Encephalitis Encephalitis Fever, rash, arthralgia, myalgia Fever, hemorrhage, jaundice Fever, hemorrhage Encephalitis Encephalitis Encephalitis Fever, hemorrhage, encephalitis Encephalitis

(-) Strand RNA (+)

Nonstructural proteins encoded in the 3’ end of genome RNA

Strand progeny RNA Structural protein encoded in the 5’

Progeny virus

Leu, virns, brim, ate candz

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Microbiology – Flaviviridae by Dra Madrid/Reyes
FLAVIVIRUS REPLICATION  Replication occurs in the cytoplasm (20-30 hrs)  Entire virus genome is translated as a single polyprotein which is then cleaved into the mature proteins o Structural proteins: encoded at the 5’ end o Nonstructural proteins (e.g., NS-1 and RNAdependent RNA polymerase) are encoded in the 3’ two-thirds  Complementary negative strand RNA is synthesized by NS proteins and then is used as a template for genomic progeny RNA synthesis  Assembly occurs characteristically into cytoplasmic vacuoles (in association with Golgi or smooth membranes)  Release occurs when cell lyses FLAVIVIRUS PATHOGENESIS AND CLINICAL MANIFESTATIONS  Flaviviruses vary widely in their pathogenic potential and mechanisms for producing human disease  It is useful to consider them in three major categories o Those associated primarily with the encephalitis Syndrome (prototype: St. Louis encephalitis) o Those associated with fever-arthralgia-rash (prototype: dengue fever), or o with hemorrhagic fever (prototype: yellow fever) o o

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Reservoir is in an animal Virus is maintained in nature in a transmission cycle involving the arthropod vector and animal. Man becomes infected incidentally. Both cycles may be seen with some arboviruses such as yellow fever

ARTHROPOD VECTORS  Mosquitoes o Japanese encephalitis, dengue, yellow fever, St. Louis encephalitis  Ticks o Various tick-borne encephalitides etc. ANIMAL RESERVOIRS  In many cases, the actual reservoir is not known. The following animals are implicated as reservoirs o Birds: Japanese encephalitis, St. Louis encephalitis o Pigs: Japanese encephalitis o Monkeys: Yellow Fever o Rodents: Russian Spring-Summer encephalitis DISEASES CAUSED  Fever and rash o This is usually a non-specific illness resembling a number of other viral illnesses such as influenza, rubella, and enterovirus infections. The patients may go on to develop encephalitis or haemorrhagic fever  Encephalitis o St. Louis encephalitis, Japanese encephalitis  Haemorrhagic fever o e.g, Yellow fever, dengue DIAGNOSIS  Serology – usually used to make a diagnosis of arbovirus infections  Culture – a number of cell lines may be used, including mosquito cell lines. However, it is rarely carried out since many of the pathogens are group 3 or 4 pathogens  Direct detection tests – e.g. detection of antigen and nucleic acids are available but again there are safety issues PREVENTION  Surveillance – of disease and vector  Control of vector – pesticides, elimination of breeding grounds  Personal protection – screening of houses, bed nets, insect repellants  Vaccination – available for a number of arboreal infections e.g. Yellow fever, Japanese encephalitis, Russian tick-borne encephalitis JAPANESE ENCEPHALITIS  First discovered and originally restricted to Japan. Now large scale epidemics occur in China, India and other parts of Asia.

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Human infection initiated by deposition of virus through the skin via the saliva of an infected arthropod  Replicates locally & in regional lymph nodes  viremia Most human infections with St. Louis enceph (SLE) and Japanese enceph (JE) viruses, there is either no apparent disease or a nonspecific febrile illness with headache Infection resolves, and lasting immunity is produced CNS lesions may develop: aseptic meningitis or encephalitis In the great majority of flavivirus infections, virus is cleared by the immune system o However, persistence in neurological tissue has been noted with tick-borne encephalitis viruses, and recent reports of recurrent encephalitic bouts in children have been associated with JE virus recovery from peripheral blood mononuclear cells

HOST DEFENSES  Lasting protection is generally restricted to the same flavivirus, and is associated with neutralizing antibodies ANTHROPOD-BORNE VIRUSES  Anthropod-borne viruses (arboviruses) WHO definition: “Viruses maintained in nature principally through biological transmission between susceptible vertebrate hosts by haematophagus arthropods.”  Arboviruses belong to three families 1. Togaviruses e.g. EEE, WEE, and VEE 2. Bunyaviruses - e.g. Sandfly Fever, Rift Valley Fever, CrimeanCongo Haemorrhagic Fever 3. Flaviviruses e.g. Yellow Fever, Dengue, Japaneses Encephalitis TRANSMISSION CYCLES  Man-arthropod-man o e.g. Dengue, Urban yellow fever o Reservoir may be in either man or arthropod vector o In the latter transovarial transmission may take place  Animal-arthropod vector-man o e.g. Japanese encephalitis, Jungle yellow fever

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Flavivirus, transmitted by culex mosquitoes The virus is maintained in nature in a transmission cycle involving mosquitoes, birds and pigs. Most human infections are subclinical, the unapparent to clinical cases is 300/1. In clinical cases, a life-threatening encephalitis occurs. The disease is usually diagnosed by serology. No specific therapy is available. Since culex has a flight range of 20km, all local control measures will fail. An effective vaccine is available.

Microbiology – Flaviviridae by Dra Madrid/Reyes
VIROLOGY  Japanese encephalitis (JE) serocomplex o 10 viruses o 6 human pathogens (JE, West Nile, Kunjin, Usutu, St. Louis Encephalitis, Murray Valley Ecephalitis viruses) o Most are amplified bird – mosquito – bird

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5 genotypes in Asia (most isolates in genotype 1)

HISTORY OF DISCOVERY  1871: “Summer encephalitis” epidemic in Japan  1924: Agent from human brain tissue isolated in rabbits  1934: Isolate of this virus produced experimental encephalitis in monkeys

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1938: First isolate from Culex tritaeniorhynchus 1930s: First mouse brain-derived vaccines developed 1954: “Refined” mouse brain vaccine developed


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JE is the leading cause of viral encephalitis in Asia, now that poliomyelitis has nearly been eradicated. More than 3 billion people live in areas where JE are reported to WHO each year. 10,000 to 15,000 deaths are reported each year.

CASES ARE UNDER-REPORTED  Cases are under-reported due to o Lack of good surveillance o Lack of diagnostics  Actual number of cases is probable more than 175,000 per year CLINICAL SPECTRUM OF JE DISEASE

TWO PATTERNS OF TRANSMISSION OF JE 1. Seasonal, called an epidemic pattern (e.g., southern China)

Sever Moderat Mild Asymptomati c



Year-round, called an endemic pattern (e.g., Bali, Indonesia)

DEATH AND DISABILITY FROM JE  Up to 30% of all patients with JE die  For those that survive the illness, 30% to 75% cases are left with disability  Disability is with both physical and cognitive AGE GROUPS AFFECTED BY JE  Children 1 to 15 years of age are mainly affected in endemic areas.  But people of any age can be affected. Adult infection most often occurs in areas where the disease is newly introduced.

TRANSMISSION OF JE  JE is spread by mosquitoes

Culex tritaeniorhynchus is the main vector in most of Asia, but tohe species that breed in rice paddies, ditches, and ground pools are also important.

Microbiology – Flaviviridae by Dra Madrid/Reyes

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Dengue haemorrhagic fever and shock syndrome appear most often in patients previously infected by a different serotype of dengue, thus suggesting an immunopathological mechanism. Diagnosis is made by serology. No specific antiviral therapy is available. Prevention of dengue in endemic areas depends on mosquito eradication. The population should remove all containers from their premises which may serve as vessels for egg deposition. A live attenuated vaccine is being tried in Thailand with encouraging results.

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PEOPLE AT RISK  People living at rural areas have the highest risk of disease because the mosquitoes that spread JE breed in rice paddies and pool water.  Cases in urban areas also occur. YELLOW FEVER  Flavivirus, mainly found in West Africa and S America

DENGUE PREVENTION  Currently, the only effective way to avoid dengue virus infection in areas where the disease is endemic or epidemic is to avoid being bitten by infected mosquitoes throught he use of personal insect repellant and other insect barriers. REPLICATION AND TRANSMISSION OF DENGUE VIRUS 1. Virus transmitted to human in mosquito saliva 2. Virus replicates in target organs 3. Virus infects white blood cells and lymphatic tissues 4. Virus released and circulates in blood 5. Second mosquito ingests virus with blood 6. Virus replicates in mosquito midgut and other organs, infects salivary glands 7. Virus replicates in salivary glands

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Yellow fever occurs in 2 major forms: urban and jungle (sylvatic) yellow fever. Jungle YF is the natural reservoir of the disease in a cycle involving nonhuman primates and forest mosquitoes. Man may become incidentally infected on venturing into jungle areas. The urban form is transmitted between humans by the Aedes aegypti mosquito Classically Yellow Fever presents with chills, fever, and headache. Generalized myalgias and GI complaints (N+V). Some patients may experience an asymptomatic infection or a mild undifferentiated febrile illness. After a period of 3 to 4 days, the more severely ill patients with a classical YF course will develop bradycardia (Faget's sign), jaundice, and haemorrhagic manifestations. 50% of patients with frank YF will develop fatal disease characterized by severe haemorrhagic manifestations, oliguria and hypotension. Diagnosis is usually made by serology There is no specific antiviral treatment An effective live attenuated vaccine is available against yellow fever and is used for persons living in or traveling to endemic areas.


Aedes aegypti Mosquito

Dengue is the biggest arbovirus problem in the world today with over 2 million cases per year. Dengue is found in SE Asia, Africa and the Caribbean and S America. Flavivirus, 4 serotypes, transmitted by Aedes mosquitoes which reside in water-filled containers. Human infections arise from a human-mosquitohuman cycle Classically, dengue presents with a high fever, lymphadenopathy, myalgia, bone and joint pains, headache, and a maculopapular rash. Severe cases may present with haemorrhagic fever and shock with a mortality of 5-10%. (Dengue haemorrhagic fever or Dengue shock syndrome.)     Dengue transmitted by infected female mosquito Primarily a daytime feeder Lives around human habitation Lays eggs and produces larvae preferentially in artificial containers

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Microbiology – Flaviviridae by Dra Madrid/Reyes
    Undifferentiated fever Classic dengue fever Dengue hemorrhagic fever Dengue shock syndrome

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 UNDIFFERENTIATED FEVER  May be the most common manifestation of dengue  Prospective study found that 87% of students infected were either asymptomatic or only mildly symptomatic  Other prospective studies including all age-groups also demonstrate silent transmission CLINICAL CHARACTERISTICS OF DENGUE FEVER  Fever  Headache  Muscle and joint pain  Nausea/vomiting  Rash  Hemorrhagic manifestations SIGNS AND SYMPTOMS OF ENCEPHALITIS/ENCEPHALOPATHY ASSOCIATED WITH ACUTE DENGUE INFECTION  Decreased level of consciousness: lethargy, confusion, coma  Seizures  Nuchal rigidity  Paresis HEMORRHAGIC MANIFESTATIONS OF DENGUE  Skin hemorrhages: petechiae, purpura, ecchymoses  Gingival bleeding  Nasal bleeding  Gastrointestinal bleeding: hematemesis, melena, hematochezia  Hematuria  Increased menstrual flow CLINICAL CASE DEFINITION FOR DENGUE HEMORRHAGIC FEVER

o Grade 1 manifestations + spontaneous bleeding Grade 3 o Signs of circulatory failure (rapid/weak pulse, narrow pulse pressure, hypotension, cold/clammy skin) Grade 4 o Profound shock (undetectable pulse and BP)

DANGER SIGNS IN DENGUE HEMORRHAGIC FEVER  Abdominal pain - intense and sustained  Persistent vomiting  Abrupt change from fever to hypothermia, with sweating and prostration  Restlessness or somnolence WARNING SIGNS FOR DENGUE SHOCK

HYPOTHESIS ON PATHOGENESIS OF DHF (PART 1)  Persons who have experienced a dengue infection develop serum antibodies that can neutralize the dengue virus of that same (homologous) serotype Homologous Antibodies Form Non-Infectious Complexes

4 Necessary Criteria: o Fever, or recent history of acute fever o Hemorrhagic manifestations o Low platelet count (100,000/mm3 or less) o Objective evidence of “leaky capillaries:”  elevated hematocrit (20% or more over baseline)  low albumin  pleural or other effusions This slide presents a graphic depiction of the development of homologous antibodies. When an individual is infected with any dengue serotype—here called dengue type A, to indicate that the infection may be with DEN-1, 2, 3 or 4—the immune system responds by producing anti-A antibodies. These anti-A antibodies form homologous complexes with the type A virus, which results in the neutralization of the virus. HYPOTHESIS ON PATHOGENESIS OF DHF (PART 2)  In a subsequent infection, the pre-existing heterologous antibodies form complexes with the new infecting virus serotype, but do not neutralize the new virus Heterologous Antibodies Form Infectious Complexes


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4 criteria for DHF Evidence of circulatory failure manifested indirectly by all of the following: o Rapid and weak pulse o Narrow pulse pressure (< 20 mm Hg) OR hypotension for age o Cold, clammy skin and altered mental status Frank shock is direct evidence of circulatory failure

FOUR GRADES OF DHF  Grade 1 o Fever and nonspecific constitutional symptoms o Positive tourniquet test is only hemorrhagic manifestation  Grade 2

Microbiology – Flaviviridae by Dra Madrid/Reyes

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In this depiction of the second step in antibody-dependent enhancement, dengue type B represents the new serotype. The anti-A antibodies produced in response to the original infection can form complexes with the new serotype, as we see here. However, the new virus serotype is not neutralized by these heterologous antibodies. The virus retains its capacity to become active and replicate. HYPOTHESIS ON PATHOGENESIS OF DHF (PART 3)  Antibody-dependent enhancement is the process in which certain strains of dengue virus, complexed with nonneutralizing antibodies, can enter a greater proportion of cells of the mononuclear lineage, thus increasing virus production Heterologous Complexes Enter More Monocytes, Where Virus Replicates

LABORATORY TESTS IN DENGUE FEVER  Clinical laboratory tests  CBC—WBC, platelets, hematocrit  Albumin  Liver function tests  Urine—check for microscopic hematuria * CBC. In patients with dengue, the leukocyte counts are often low, and the patient may even be neutropenic. Platelet levels should also be checked, and you should do serial hematocrits for hemoconcentration.  Dengue-specific tests  Virus isolation  Serology LABORATORY METHODS FOR DENGUE DIAGNOSIS, CDC DENGUE BRANCH  Virus isolation to determine serotype of the infecting virus  This can be performed either by using mosquito cell cultures or by mosquito inoculation. o IgM ELISA test for serologic diagnosis DENGUE: MANAGEMENT  No hemorrhagic manifestations and patient is wellhydrated: home treatment  Hemorrhagic manifestations or hydration borderline: outpatient observation center or hospitalization  Warning signs (even without profound shock) or DSS: hospitalize  Fluids  Patients should be encouraged to take small, frequent sips of fluids. If the patient cannot be rehydrated by mouth, fluids should be administered intravenously. At times large amounts of intravenous fluids are needed.  Rest  Antipyretics (avoid aspirin and non-steroidal antiinflammatory drugs so that platelet function will not be impaired)  Monitor blood pressure, hematocrit, platelet count, level of consciousness DENGUE VACCINE?  No licensed vaccine at present  Effective vaccine must be tetravalent  Field testing of an attenuated tetravalent vaccine currently underway  Effective, safe and affordable vaccine will not be available in the immediate future

This slide shows the same information graphically. We can see that the heterologous antibody-virus complexes can enter a greater proportion of monocytes than the virus alone. The greater proportion of infected cells results in greater virus production. HYPOTHESIS ON PATHOGENESIS OF DHF (PART 4)  Infected monocytes release vasoactive mediators, resulting in increased vascular permeability and hemorrhagic manifestations that characterize DHF and DSS PETECHIAE

TOURNIQUET TEST  Inflate blood pressure cuff to a point midway between systolic and diastolic pressure for 5 minutes  Positive test: 20 or more petechiae per 1 inch² (6.25 cm²) Positive Tourniquet Test

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