3. What are Viral Hemorrhagic
Fevers (VHFs)?
 A group of illnesses that are caused by several distinct
families of viruses
 A severe multisystem syndrome (multiple organ systems in
the body are affected
 Vascular system damaged : SHOCK syndromes
 Body’s ability to regulate itself (Homeostasis) is impaired
 Many cause severe and life-threatening disease.

4. Viral hemorrhagic fever (…contd)
 The prototypical viral hemorrhagic fever is Yellow Fever
 Not all viral hemorrhagic fevers are however arboviruses
 Hemorrhagic fever with Renal Syndrome (HFRS) are
also considered in relation to VHF
HFRS Caused by:
 Hantaan
 Seoul
 Dobrava
 Puumala viruses
(Ref: Mandell, Douglas and Bennett’s “Principles and Practice of Infectious Disease,
7th Ed)

5. Viral hemorrhagic fever (…contd)
 Acute infection:
fever, myalgia, malaise; progression to prostration
 Small vessel involvement:
increased permeability, cellular damage
 Multisystem compromise
 (varies with pathogen)
 Hemorrhage may be small in volume
(indicates small vessel involvement, thrombocytopenia)
 Poor prognosis associated with:
shock, encephalopathy, extensive hemorrhage

6. Viral hemorrhagic fever (…contd)
 Viruses of four distinct families
 Arenaviruses
 Filoviruses
 Bunyaviruses
 Flaviviruses
 RNA viruses
 Enveloped in lipid coating
 Survival dependent on an animal or insect host, for the
natural reservoir

7. Viral hemorrhagic fever (…contd)
Arenaviridae Bunyaviridae Filoviridae Flaviviridae
Classification
Junin Crimean- Congo Ebola Kyasanur
HF Forest Disease
Machupo Hantavirus Marburg Omsk HF
Sabia Rift Valley fever Yellow Fever
Guanarito
SFTS (China,
Dengue
2011)
Lassa
Argentine HF (In block Red:
Diseases
prevalent in
INDIA)
Venezuelan HF

8. Shapes of the above viruses

9. Dengue
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 South America.
4 serotypes: DEN 1,2,3,4
Human infections arise from a human-
mosquito-human cycle

10. Dengue (….contd)
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 (DHF)or Dengue
shock syndrome (DSS)}

11. Global Occurrence of Dengue

12. Approximate actual and potential distribution of
Aedes aegypti.
The band between the 10° C isotherms represents
potential distribution between 35 ° North – 35° South
(Ref: World Health Organization. Technical Guide for Diagnosis, Treatment,
Surveillance, Prevention, and Control of Dengue Haemorrhagic Fever, 2nd ed.
Geneva: World Health Organization; 1997.)

13. Magnitude of Problem
The reasons for this dramatic global emergence of
Dengue as a major public health problem are:
 Increased Air Travel
 Extensive vector infestations with declining
vector control: effective mosquito control is virtually
non existent in most Dengue endemic countries
 Unreliable Water supply and drainage systems
 Increasing non bio-degradable contaivers and
poor solid Waste disposal
 Major global demographic changes: Urbanization
with increasing population density in urban areas

14. Indian Scenario

15. Indian Scenario
The first recorded epidemic of clinically
Dengue like illness occurred at Madras in
1780.
 First outbreak in Indian subcontinent:
1812.
First Dengue virus isolation- Kolkata in 1943–
1944.
First outbreak in India: 1963 in Kolkata.
Ref: Jatanasen S and Thongcharoen P (1993) Dengue hemorrhagic
fever in South East-Asian countries. Monograph on dengue/dengue
haemorrhagic fever. NewDelhi: WHO 23-30.

16. Indian Scenario
Recent Dengue epidemic occurred in 1996,
2003 & 2006.
In 2008, 12,419 Dengue cases and 80
deaths were reported.
Delhi shares ~25% of dengue disease burden
of country.

17. Indian Scenario….sharing experiences from
our centre
Primary Secondary Suspected
Total Serologically
infection infection secondary
Month Suspected Positive cases
(IgM (IgM+ IgG infection (IgG
cases (%)
Positivity) Positivity) Positivity)
August 12 3 (0.34%) 1 (0.5%) 1 (0.26%) 1 (0.32%)
September 157 68 (7.6%) 17 (8.6%) 24 (6.3%) 27 (8.6%)
October 982 583 (65.3%) 126 (63.3%) 246 (64.57%) 211 (67.4%)
November 362 230 (25.76%) 49 (24.6%) 110 (28.87%) 71 (22.68%)
December 37 9 (1%) 6 (3%) 0 (0%) 3 (1%)
Total 1550 893 (57.36%) 199 (22.28%) 381 (42.67%) 313 (35.05%)
Ref: AnitaChakravarti* and RajniKumaria: Virology Journal 2005, 2:3

18. Indian Scenario….sharing experiences from
our centre
Month Dengue-specific Antibody Positive cases
Children Adults (Positivity
Total
(Positivity %) %)
August 3 0 3 (25%)
A Clear
SeptemberCut PEAK of incidence of (41.7%)
68 18 (48.6%) 50 new
cases were seen in the (69.6%)
October 583 133 Post-monsoon
450 (57%)
November during the months of October
season 230 54 (44.3%) 176 (83.8%)
and November in cases ocurring8in or
December 9 1 (11.1%) (28.6%)
near Delhi 893
Total 206 (56.4%) 687 (58%)
Ref: AnitaChakravarti* and RajniKumaria: Virology Journal
2005, 2:3

19. Ref: J Infect Dev Countries 2011; 5(4):239-247
The National figure also corroborates with the study
from our institute, carried out in Delhi

21. Ref: J Infect Dev Countries 2011; 5(4):239-247

22. Ref: J Infect Dev Countries 2011; 5(4):239-247

23. The Vector: Aedes mosquito
 Aedes (Stegomyia)
aegypti
 Breeds in small
accumulating standing
water
 Eggs resist drying
 Domesticated mosquito
 Found within or close-by
human environments,
often biting indoors
biting is predominantly
by day

24. Dengue Transmission
1. Mosquitoes transmit
Dengue virus to human dendritic
cells. 1
2. Virus targets areas
with high WBC counts
2
(liver, spleen, lymph
nodes, bone marrow,
4
And glands)
33
3. Virus enters
WBCs & lymphatic
Tissue
4. Dengue virus enters blood
Circulation.
http://phil.cdc.gov/PHIL_Images/08051999/00004/dengue_phf/sld006.htm

25. Steps required for any Flaviviruses infection and
transmission by a mosquito

26. Dengue - Virology
Ref: Goodsell DS.
RCSB Protein Data
Bank. July, 2008.
Dengue virus is a small virus that carries a single strand of RNA as its genome. The
genome encodes only ten proteins. Three of these are structural proteins that form
the coat of the virus and deliver the RNA to target cells, and seven of them are
nonstructural proteins that orchestrate the production of new viruses once the virus
gets inside the cell. The outermost structural protein, termed the envelope protein, is
shown here from PDB entry 1k4r

27. Dengue – Virology (… Contd)
The Deadly Switch
Ref: Goodsell DS.
RCSB Protein Data
Bank. July, 2008.
When the virus is carried into the cell and into lysozomes, the acidic environment
causes the protein to snap into a different shape, assembling into trimeric spike, as
shown above from PDB entry 1ok8. Several hydrophobic amino acids at the tip of this
spike, colored bright red here, insert into the lysozomal membrane and cause the virus
membrane to fuse with lysozome. This releases the RNA into the cell and infection
starts.

28. Dengue – Virology (… Contd)
Ref: Goodsell DS.
RCSB Protein Data
Bank. July, 2008.
Each of these enzymes performs a different part of the life cycle. The polymerase
builds new RNA strands based on the viral RNA, the helicase helps to separate these
strands, and the methyltransferase adds methyl groups to the end of them, protecting
the RNA strands and coaxing the cell's ribosomes to create viral proteins based on
them. The viral proteins are created in one long polyprotein chain, which is finally
clipped into the functional units by the protease. The little chain coloured blue is a
portion of another viral protein, NS2B, that assists with the protease activity.

29. Dengue – Virology (… Contd)
Ref: Goodsell DS.
RCSB Protein Data
Bank. July, 2008.
The one shown here, from PDB entry 2r6p6, shows the envelope
protein on the surface of the virus (in white) with many antibody Fab
fragments (in blue) bound to the viral proteins. By looking carefully at
this structure, researchers have discovered that the antibodies distort
the arrangement of the envelope proteins, blocking their normal
action in infection.

30. Clinical Presentation Of Dengue
Dengue Virus Infection
Asymptomatic Symptomatic
Dengue hemorrhagic
Undifferentiated Dengue fever fever
fever syndrome (plasma leakage)
(viral syndrome)
Without With unusual No shock Dengue shock
hemorrhage hemorrhage syndrome
Dengue fever Dengue
hemorrhagic Fever
WHO 95629

31. Clinical spectrum, pathophysiology, and
classification of dengue hemorrhagic fever.
At the top are key clinical findings; in the center,
pathophysiologic mechanisms; and on the side, the World
Health Organization classification of cases:
Grade 1: Fever accompanied by nonspecific constitutional
symptoms; the only hemorrhagic manifestations are a
positive tourniquet test result, easy bruising, or both.
Grade 2: Spontaneous bleeding in addition to the
manifestations of grade 1, usually in the form of skin
hemorrhages or other hemorrhages.
Grade 3: Circulatory failure manifested by a rapid, weak
pulse and narrowing of pulse pressure or hypotension, with
the presence of cold, clammy skin and restlessness.
Grade 4: Profound shock with undetectable blood
pressure or pulse.
(Ref: WHO. Technical Guide for Diagnosis, Treatment, Surveillance, Prevention, and
Control of Dengue Haemorrhagic Fever, 2nd ed. Geneva: 1997.)

32. Dengue (cont…)
 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.

33. Clinical Case Definition for
Dengue Hemorrhagic Fever
4 Necessary Criteria:
1. Fever, or recent history of acute fever
2. Hemorrhagic manifestations
3. Low platelet count (100,000/mm3 or less)
4. Objective evidence of “leaky capillaries” :
 elevated hematocrit (20% or more over
baseline)
 low albumin
 pleural or other serosal cavity effusions

34. Dengue Clinical Syndromes
Undifferentiated fever
Classic dengue fever
Dengue hemorrhagic fever
Dengue shock syndrome

35. Hemorrhagic Manifestations
of Dengue
Skin : petechiae, purpura, ecchymoses
Gingival bleeding
Nasal bleeding
Gastro-intestinal bleeding:
hematemesis, melena
Hematuria
Increased menstrual flow

36. Four Grades of DHF
 Grade 1
 Fever and nonspecific constitutional symptoms
 Positive tourniquet test is only hemorrhagic
manifestation
 Grade 2
 Grade 1 manifestations + spontaneous bleeding
 Grade 3
 Signs of circulatory failure (rapid/weak pulse, narrow
pulse pressure, hypotension, cold/clammy skin)
 Grade 4
 Profound shock (undetectable pulse and BP)

38. Laboratory Tests in Dengue Fever
 Clinical laboratory tests
 CBC--WBC, platelets, hematocrit
 Albumin
 Liver function tests
 Urine--check for microscopic hematuria
 Dengue-specific tests
 Virus isolation
 Serology

39. Laboratory Diagnosis of Dengue
Fever: Virus detection
 Detection of virus by culture is obviously the
definitive diagnostic test.
 By the time a person infected with Dengue develops
fever, the infection is widely disseminated.
 The virus is found in serum or plasma, in circulating
blood cells and in selected tissues, especially those
of the immune system, for approx. 2-7 days, roughly
corresponding to the period of fever.
 Detection of dengue RNA using specific
oligonucleotide primers, reverse transcriptase and
thermostable polymerase are Faster and are applied
in many Laboratories.

40. Laboratory Diagnosis of Dengue
Fever: Virus detection
Drawbacks and limitations of Viral isolation
 The period of illness when the dengue virus can be
successfully detected is brief
 Within a day or 2 after subsidence of fever, the
rising level of antibody interfere with virus culture
 Dengue virus is heat-labile and special precautions
must be taken against the thermal inactivation of
specimens.
 Laboratories equipped and staffed to culture viruses
are expensive to develop and maintain.

41. Laboratory Diagnosis of Dengue
Fever: Virus detection

42. Laboratory Diagnosis of Dengue Fever:
Virus detection
Inoculation into mosquitos
 Most sensitive dengue viral culture technique
 Serum, Plasma, CSF, Pleural fluid, Peripheral blood
leucocytes & tissue homogenates can be used
 Toxorhynchites mosquitos generally used
 They are not hematophagus and their large size facilitates
inoculation
 Infection is detected by Immunofluorescence of a tissue
smear prepared from the crushed head of the mosquito
(Head Squash)
 High sensitive culture requires 5-20 mosquitos per specimen
 adult male Aedes aegypti & Ae. Albopictus can also be used.

43. Laboratory Diagnosis of Dengue Fever:
Virus detection
Inoculation into mosquitos
Toxorhynchites Ae. aegypti & Ae.
Albopictus
 Large, easy to inoculate Small, difficult to
inoculate
 Raising is labour Easier to maintain
intensive, as the larvae are
carnivorous & needs a
second mosquito species
larvae as food source
 Non Hematophagus, Female spp can’t be used
hence safe to handle due to ability to act as
vector

44. Laboratory Diagnosis of Dengue Fever:
Virus detection
Inoculation into mosquito cell lines
 C6/36 and AP-61 cell lines can be used
 Less sensitive than direct inoculation into live
mosquitoes
 Cell cultures to be screened for specific evidence
of infection by an immunoassay as the cytopathic
effects might be absent in many dengue virus
isolates
 As mosquito cell lines are propagable in ambient
tropical temperatures (25-34° C), it is easier to
maintain and practice

45. Laboratory Diagnosis of Dengue Fever:
Virus detection
Inoculation into vertebrate cell lines
 VERO and LLC-MK2 cell lines can be used
 Least sensitive than other direct inoculation methods
All cultures are examined using serotype-specific anti-
Dengue monoclonal Abs tagged to a second labelled Ab.
Positive control: Dengue-complex-reactibe MAb
 Intracerebral inoculation into newborn mice is also
tried in certain laboratories : but have proven to be
very less sensitive

46. Laboratory Diagnosis of Dengue Fever:
Antigen detection in fixed tissue
Sample:
 Peripheral Blood Leukocyte
 Autopsy Lung, Liver specimen
 Less commonly: Autopsy Thymus, Spleen, Lymph
node, Bone marrow
Mainly for epidemiological purpose and
confirmation of epidemic / outbreak.
Immunohistochemistry examined using serotype-
specific anti-Dengue monoclonal Abs tagged to a
second labelled Ab.

47. Laboratory Diagnosis of Dengue Fever:
Reverse transcriptase-PCR amplification of
Dengue RNA
 High potential to detect dengue virus during
convalescence, when circulating antibodies otherwise
preclude its detection
 2 step nested RT-PCR and 1 tube multiplex RT-PCR
are among the most widely used methods
Experience at our centre have shown that the 1 tube
multiplex RT-PCR is more sensitive and specific than
the other available methods.
(Ref: Kumaria R, Chakravarti A. Diagn Microbiol Infect Dis 2005)

48. Laboratory Diagnosis of Dengue Fever:
Serology: IgM capture ELISA
 The IgM Capture or the MAC-ELISA is the most
widely used serological test
 Serum, Saliva, dried blood sample collected in Filter
paper and CSF can be used as sample
 Can even detect a rise in dengue-specific IgM in
acute phase at 1-day to 2-day interval
 Specimens collected at an interval of 2-3 days
spanning the day of defervescence are usually
diagnostic

49. Laboratory Diagnosis of Dengue Fever:
Serology: IgM capture ELISA
Experience at our centre
 MAC-ELISA is regularly practiced at our centre
 IgM and IgG detection from non-invasive Saliva samples
were carried out at our centre which yielded wonderful
reproducible results:
 Salivary IgM antibodies were detected in 100% of the serum
IgM-positive samples and in 30% of the serum samples that
were negative for IgM antibodies. Salivary IgG antibodies
were detected in 93.3% of the serum samples that were
positive for anti-dengue IgG antibodies and in none of the
serum IgG-negative cases (Ref: Chakravarti A, Matlani M,
Jain M. 2007, Curr Microbiol)
 IgM/IgG detection from reconstitution of dried blood
samples from clinically suspected cases collected in filter
paper are being carried out at present.

50. Laboratory Diagnosis of Dengue Fever:
Serology: IgM capture ELISA
The Interpretation of MAC-ELISA results
Ref: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.
2nd edition. Geneva : World Health Organization

51. Laboratory Diagnosis of Dengue Fever:
Rapid NS1 Antigen detection
 Extensive study taking place to establish rapid
diagnosis and shorten the window period of misdiagnosis
by detecting the NS1 antigen of dengue virus
 Most of the studies have shown that a combination
rapid test comprising immunochromatographic assay for
detection of both the NS1 Antigen and the anti-dengue
Igm together yields satisfactory clinical results,
instead of sole NS1 antigen detection.
(Ref: 1. Tontulawat P et al, Southeast Asian J Trop Med Public Health. May,
2011.
2. Fry SR et al, PLoS Negl Trop Dis. June, 2011.)

52. Laboratory Diagnosis of Dengue Fever:
Serology: Haemagglutination-Inhibition
test (HAI)
 Simple, sensitive and reproducible
 Reagents may be prepared locally
 Disadvantages:
 Pretreatment of serum samples reqd with acetone/
kaolin and then adsorbed with type O human RBCs to
remove non-specific inhibitors of agglutinin and non-
specific agglutinins.
 Paired sera are required with a gap of at least 7 days.
 Can’t reliably distinguish between closely related
Flaviviruses: Between Dengue and Jap Encephalitis or
West Nile viruses

53. Laboratory Diagnosis of Dengue Fever:
Serology: Haemagglutination-Inhibition
test (HAI)
The Interpretation of HAI results
Ref: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.
2nd edition. Geneva : World Health Organization

54. Progress toward a Dengue Vaccine
 Control of dengue by widespread vaccination has
been a priority of WHO for three decades (Ref:Brandt
WE. J Infect Dis 1990)
Background:
 Robust neutralising antibody responses develop after
dengue infection and are believed to provide lifelong
protection against reinfection with the same dengue
serotype and short-lived protection, of several
months, against a heterologous dengue serotype.
 This naturally acquired immunity provides optimism
for the feasibility of a dengue vaccine.

55. Progress toward a Dengue Vaccine
Vaccine development and the issue of Immunopathogenesis
The fear:
The pathogenesis of severe dengue results from a complex
interaction between the virus, the host, and, at least in part,
immune-mediated mechanisms. Vaccine development has
been slowed by fears that immunisation might predispose
individuals to the severe form of dengue infection.
The assurance:
Whatever the role of antibody-dependent enhancement, it
seems that a vaccine inducing a long-lived neutralising
antibody response against all four serotypes simultaneously
should not induce any risk in this respect
(Ref: 1. Guirakhoo F et al, Hum Vaccin 2006. 2. Sabchareon A et al, Am J Trop Med
Hyg 2002. 3. Sabchareon A et al, Pediatr Infect Dis J 2004.)

56. Progress toward a Dengue Vaccine
LEADING

57. Progress toward a Dengue Vaccine

58. Progress toward a Dengue Vaccine
 The leading candidate vaccine in clinical trials at
present is the ChimeriVax dengue vaccine.
 Using a new technology, the premembrane and
envelope genes of yellow fever 17D virus are
replaced with those of each wild-type dengue
virus serotype. ChimeriVax dengue vaccine viruses
are then prepared by electroporation of Vero
cells with RNA transcripts prepared from viral
cDNA.
(Ref: 1. Guirakhoo F, Kitchener S et al. Hum Vaccin 2006
2. Webster DP, Farra J. Lancet Infect Dis 2009.)

59. Kyasanur forest disease
 Kyasanur forest disease is a tick-borne viral
hemorrhagic fever endemic to South Asia
 The disease was first reported from Kyasanur
Forest of Karnataka in India
 The disease was first manifested as an
epizootic outbreak among monkeys killing
several of them in the year 1957. Hence the
disease is also known as Monkey Disease.

60. Kyasanur forest disease (…contd)
 The reservoir hosts for the disease are
porcupines, rats and mice. The vector for disease
transmission is Haemaphysalis spinigera, a forest
tick. Humans contract infection from the bite of
nymphs of the tick
 The disease has a high mortality rate of 10%
 The clinical manifestations of the disease in
humans are:
 High fever
 Headache
 Hemorrhages from nasal cavity and throat
 Vomiting

61. Crimean Congo Hemorrhagic Fever:
India affected
 Index case: Ameena Momin (Case A), 32yr old woman from Korat
village in Sanand, 20 kms from Ahmedabad was admitted to
Sterling Hospital on December 29 and later shifted to Shalby
Hospital on January 1, expired on January 3rd 2011.
 Secondary cases:
 42 yr old Dr Gaganjeet Sharma (Case B) treating the index
case at Shalby, died January 13th.
 25 yr old nurse Asha John (Case C), attending the index case,
died January 18th.
 The husband of the index case (case D) also admitted to the
same hospital on Jan 16, was positive for CCHF virus was
treated with oral ribavarin and discharged after 10 days.
(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagic
fever in India. Lancet July, 2011)

62. Crimean Congo Hemorrhagic Fever:
India affected
(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagic
fever in India. Lancet July, 2011)

63. Crimean Congo Hemorrhagic Fever:
India affected
DIAGNOSIS
 Only Nucleic acid Amplification Tests (eg. PCR, rt-PCR) are
the most reliable method, along with RNA sequencing in case
of outbreaks in previously unknown geographical areas
 High serum LDH, High serum Ferritin, and
thrombocytopenia may lead to a strong suspicion
 Negative assays for locally prevalent diseases, that may
lead to such fever (eg. Malaria, Leptospirosis, Dengue,
Kyasanur Forest Disease for India) shall also lead to strong
suspicion.

64. Crimean Congo Hemorrhagic Fever:
India affected
IMPORTANCE:
 Many severely ill patients with CCHF require admission to
intensive care facilities. To avoid infections in hospital
settings, stringent infection control practices, proper air
handling in intensive-care units, isolation of patients, and
correct handling of clinical specimens are essential. Tick
bites and contact with infected animals are the main modes
of infection to people.
 Disinfection of domestic animals and their accommodation
can help reduce the risk of human infection. Febrile patients
with haemorrhagic symptoms, who are negative for dengue
virus, should be considered as possible cases of CCHF for
the purpose of hospital infection control and isolation of
patients in India

65. Novel Bunyavirus identified in
China (SFTS)
Why do we discuss it here in context of
India?
 RNA from SFTS bunyavirus was detected in roughly 5
percent of ticks of the species Haemaphysalis
longicornis recovered from animals in the area in which
affected patients lived, and the authors propose that this
tick may be a vector for SFTS bunyavirus.
 This tick is found throughout the Asia-Pacific region
including India
 Considering globalization, population migration and lack
of SFTS surveillance, INDIA is at a high risk.
Ref: Yu XJ, Liang MF, Zhang SY, et al: Fever with thrombocytopenia associated with a
novel bunyavirus in China. N Engl J Med 2011; 364(16):1523-1532.

66. Novel bunyavirus identified in
China (…contd)
 Surveillance for infectious disease in China has advanced in
recent years.
 In 2009 and 2010, surveillance detected the emergence of a new
viral pathogen that causes a clinical syndrome including fever
and thrombocytopenia that has been termed severe fever with
thrombocytopenia syndrome (SFTS).
 SFTS is characterized by
 Gastrointestinal symptoms
 Leukopenia
 Fever
 Thrombocytopenia
 30% mortality rate.

67. Novel bunyavirus identified in
China(…contd)
 In June 2009, a patient in Xinyang City in Henan
Province (central China) presented with SFTS
 Blood sample was obtained from this patient 1 week after
onset of symptoms.
 Multiple cell lines that were susceptible to both viral and
rickettsial agents were inoculated with the patient’s blood.
 During the period June 2009 to March 2010, additional
cases of SFTS were identified in central and northeast
China, and blood samples were obtained from these
patients as well.

68. Novel bunyavirus identified in
China(…contd)
 In these cases, serum or white blood cells were inoculated
onto Vero cells.
 The virus isolated from the first patient was analyzed by
RFLP assay, whereas samples from the second group of
patients were analyzed using PCR.
 EM and neutralization assays were also performed.

69. Novel bunyavirus identified in
China(…contd)
 The investigators isolated a novel pathogen, which they
named SFTS bunyavirus.
 Analysis of viral RNA showed that the virus was a member
of the Bunyaviridae family in the genus Phlebovirus.
 EM confirmed bunyavirus morphology. Based on
identification of viral DNA or specific antibodies, or both,
171 patients with SFTS were shown to have infection with
SFTS bunyavirus.
 Immune response specific to the virus was shown in 100
percent (35 of 35) of matched serum samples obtained
during acute infection and convalescence.

70. All that are round and spiculated are
not Dengue
Thanks for your attention