viruses

1. SPECIAL LECTURE
MAJ MAHIMA LALL
LT COL SATISH KUMAR

2. RECENT ADVANCES IN
NEUROTROPIC VIRUSES

3. BIRDS EYE VIEW
 Defining neurotropism
 List of neurotropic viruses
 Recent advances in epidemiology
 Recent advances in immunobiology & pathogenesis
New and emerging neurotropic viruses
 Recent advances in laboratory diagnosis
 Recent advances in vaccines
 Recent advances in management- antivirals

4. Definition of Neurotropic...
 Selective tropism
towards nervous tissue
 affecting or having an
affinity for the
nervous tissue
 dramatic differences
in lifestyle but
establish CNS
infections

5. Viral CNS infections may present as...
latent
encephalopathy myelitis
meningitis
encephalitis
neurotropic
virus

6. Triad of presentation
Viral CNS Disease
Fever Headache Altered Mentation

7. Neurotropic viruses
 Herpes group
 HSV 1&2
 VZV
 CMV
 Human herpesvirus6
 Epstein-Barr virus
 Picornaviridae
 Enteroviruses
 Polioviruses
 Coxsackieviruses
 Echoviruses
 Arboviruses
 Lyssavirus
 Rabies virus
 Arenaviridae
 Lymphocytic choriomeningitis virus
 Paramyxovirus
 Measles virus
 Mumps virus
 Nipah virus
 Hendra virus
 Togaviridae
 Rubella virus
 Papovaviridae
 JC virus
 Retroviruses
 HIV
 Human T lymphotropic virus type 1

8. Epidemiology
Seasonal variation for encephalitides caused by mosquito-borne
viruses, enteroviruses, mumps virus, and herpesviruses.
Summer/Early Fall
Arboviruses
Enteroviruses
Fall/Winter
LCMV
Winter/Spring
Mumps
Nonseasonal
HSV
HIV

9. Common Less common Rare
Acute Meningitis
Enterovirus HSV-1 Adenovirus
Arbovirus LCMV CMV, Influenza A&B
HIV VZV EBV, mumps, rubella
HSV-2
Acute Encephalitis
Arboviruses CMV Adenoviruses, CTFV,
Enteroviruses EBV, HIV LCMV, Parainfluenza,
HSV-1 Mumps Rabies, rotavirus
Occurrence of disease

10. PATHOGENESIS

11. Blood-brain barrier
Encephalitis
Brain-CSF barrier
Meningitis
Growing across, infecting cells that comprise
barrier
Passive transport in intracellular vacuoles
How do viruses go across?

12. Role of Microglia in Central Nervous
System Infections
 "immunologically
underprivileged" organ

an evolving concept of
microglia
 ability to "call in the
troops“
 double edged sword
 Secretory function
 Protective role

13. LABORATORY DIAGNOSIS

14. Conventional versus Recent methods
 CSF analysis
 CSF culture: viral
isolation
 Electron microscopy
 Antigen detection
 IHC
 IF
 Ag capture ELISA
 Antibody detection
 Hemagglutination-
inhibition
 Neutralization tests
 CFT
 ELISA for IgG/IgM
 MAC ELISA
 Dot-blot immunoassay
 Immunochromato-graphic
tests (ICT)
 Nucleic acid detection &
Quantitation

15. VIRAL ISOLATION- general concepts
 Relevant to diagnosis if patient presents while still
viremic
 Neurotropic viruses can be isolated from blood taken
within first week of illness
 Viral recovery from blood is successful only before
neutralising antibody response develops
 Not all viruses are cultivable on cell lines
 Takes 2-8 days in majority of cases
 CSF, Serum, plasma, blood, tissue
scabs, faeces, urine
 Autopsy – Brain

16. CSF processing in CNS infections
 CSF for viral isolation: 2ml of CSF to be
transported promptly to lab & refrigerated
 CSF samples for viral isolation should never be
stored in a -20C freezer since viruses are unstable
at this temperature
 freezers have “frostfree” warm up cycles that are
detrimental to viral stability

17. CSF picture in viral CNS disease
 Lymphocytic pleocytosis
 CSF pleocytosis (>5 lymphocytes/mm3
) present in 95% of
cases of acute viral encephalitis
 Normal glucose
 Normal/mildly raised proteins
 Cell count exceeds 500/mm3
in 10% of cases of acute viral
encephalitis

18. SEROLOGY
 Serum serologic studies less useful for viruses ~ HSV, VZV,
CMV, EBV
 Seroprevalence in general population high/previous infection
 Acute infection: Seroconversion between acute-phase and
convalescent sera
 Demonstration of IgM antibodies esp in CSF indicator of
CNS infection
 Retrospective diagnosis estd. than urgent diagnosis
 non-specific rise due to polyclonal activation
 Demonstration of intrathecal synthesis of antibody
Serum Ab: CSF Ab, integrity of BBB confirmed by other
antibody (or albumin) being excluded from CSF

19. HERPES VIRUSES

20. HERPES VIRUSES
 Large, DNA containing ,
enveloped viruses
 Four structural elements
 outer envelope
 tegument
 nucleocapsid
 core: genome
 Latency
 Direct destruction
 “friendly fire”

22. Herpes Simplex Virus Encephalitis
 Commonest cause of Sporadic encephalitis
 Headache, behavioural changes, focal seizure or paralysis
 Temporal lobe involvement most common
 CSF shows lymphocytic pleocytosis
 HSV envelope glycoproteins gB, gD, & gH-gL for binding and
entry into cells
 134.5 gene, modulates neurovirulence
 Vmw65, HSV transactivating protein
determines cell death or latency
 Direct virus-mediated damage and
indirect immune mediated

23. HSVE
 High mortality & morbidity
 high clinical suspicion
 at any time of year
 at any age
 in young, but more 50-70yr
 70% cases people have
serological evidence & h/o
previous herpes simplex

24. Laboratory techniques for diagnosis of
HSV infection in CNS
Test Turnaround
time
Interpretation Advantage Disadvantage
Antigen
detection
1-3 h May indicate
infection if
correlated with
symptoms
Rapid
Poor
sensitivity and
specificity
Cell culture 2-7 days Indicates active
infection
Isolate
available
early specimen
collection
Serology 4-6 h probably active
infection
automation
retrospective
PCR 1-2 days Indicates active
infection
High
sensitivity
and
specificity
contamination

25. HSV PCR
 HSV virus culture positive in < 5%
 PCR detects HSV DNA in majority of cases
 Brain biopsy: Gold standard rarely done now
 CSF PCR: sensitivity-96%, specificity-99%
 Negative PCR does not rule out HSE
 CSF PCR may be negative when performed during first 72hrs
 Effect of antiviral therapy on PCR positivity: sharp decline
hence prognostic significance
 Differentiate HSV-1 & HSV-2 by type specific primers

26. Newer methods for diagnosis
 TaqMan PCR is an excellent alternative to conventional nested
PCR assays for the rapid detection of HSV-1, HSV-2, and
VZV in clinical samples.
 Journal of Clinical Microbiology, April 2003, p. 1565-1568, Vol. 41, Rapid Detection of
Herpes Simplex Virus and Varicella-Zoster Virus Infections by Real-Time PCR

27. Multiplex and consensus Primer PCR
 Pan-herpesvirus assay*
 PCR assays detect multiple pathogens in a single
CSF sample
 Herpesviruses with enteroviruses: HSV-1, HSV-2,
EV, and VZV in a single CSF sample
 Assays for multiple polyomaviruses: JCV & BK
 Assays for arboviral diseases
*Laboratory diagnosis of common viral infections of the central nervous system by
using a single multiplex PCR screening assay; JCM, May 1999,p.1352-1355.

28. Newer vaccine strategies
 Peptide vaccines- immunodominant CD8 CTL epitope
 Subunit vaccines
 Fractionated-virus vaccine
 Discontinuously Replicating viruses (DISC)
 Replication-competent Live-virus vaccine

29. CMV ENCEPHALITIS
 CMV Meningoencephalitis disease
of congenitally infected
newborns
 immunocompromised indls
 organ transplant recipients
 cancer patients on chemotherapy
 in immunocompromised, infection
is reactivation of latent virus
 ventriculoencephalitis,
periventricular disease, retinitis
Owl’s eye appearance

30. CMV lab diagnosis
 CSF analysis
 Isolation of virus in cell culture
 Electron microscopy
 Direct detection &/or quantification of viral
proteins or nucleic acids
 Serology
Cytomegalovirus pp65 antigen
detected in polymorphonuclear cells

31. CMV PCR
 CSF PCR in CMV CNS infections in immunodeficient hosts
Sensitivity: 82-100%, Specificity: 86-100%
 Concern- can PCR distinguish between active disease and
asymptomatic infection
 Detection of CMV transcripts (human CMV mRNA)
 Transcription of late human CMV genes occurs only after
viral replication
 pp67 late-gene transcript assay superior sensitivity
 Quantitative CMV PCR gives CMV DNA copy number: better
index of disease severity
 CMV PCR of CSF used to identify mutations in UL97 region:
ganciclovir resistance

32. Varicella-zoster virus
 Varicella-zoster involves sensory ganglion cells
 VZV is rarely cultured from CSF
 CSF PCR positivity for VZV DNA in meningoencephalitis~60%
 Intrathecal synthesis of VZV-specific antibody or presence
of anti-VZV immunoglobulin M (IgM) Ab in CSF is specific for
encephalitis
 Zoster reactivation can present with meningoencephalitis
 CSF PCR is positive for VZV DNA in majority of cases

33. ENTEROVIRUSES

35. Enterovirus Cell Membrane Receptors
Enterovirus Serotype Receptor Protein
 Polioviruses 1-3 Poliovirus receptor (PVR)
 Coxsackieviruses A13, A18, A21 Intercellular adhesion
molecule 1 (ICAM-1)
 Coxsackieviruses B1-B6 Coxsackie adenovirus
receptor (CAR)
 Coxsackieviruses B1, B3, B5 Decay accelerating
factor (DAF)
 Echoviruses 1, 8 VLA-2 (α2ß1)
 Echoviruses 6, 7, 11, 12, 13, 20, 21, 29, 33 DAF
 Enterovirus 70 DAF

36. Enterovirus lab diagnosis
 Enteroviruses commonest cause of aseptic meningitis
 Encephalitis caused is severe and fatal
 Sensitivity of virus culture: 65-75%~ insensitive
 Sensitivity & specificity of PCR > 95%
 CSF PCR >> serum PCR
 Urine PCR: 62-77%
 NASBA is additional molecular method

37. Polio Virus
 Poliovirus attacks anterior horn cells
 Poliovirus receptor (PVR) present at motor endplate
 Binds to PVR-expressing motor endplate, endocytosis,
retrograde transport to ant horn cells
 RT-PCR for RNA in CSF
 Isolation of virus from rectal and throat swabs ??
relevance
 Serologic tests for IgM & IgG have limited use:
heterotypic, anamnestic responses

38. Beginnings of a polio free era...

39. Pulse Polio Immunization
National immunization days
vaccinating all children below age of 5yrs
Oral polio vaccine

40. Is Polio immunization program a failure?
 Epicentre of current circulation of the
virus is in western Uttar pradesh, 70%
of cases are minority population
 Intensified efforts- fatigue
 Outbreaks of vaccine-derived
polioviruses
 Challenged the program

41. Polio vaccines
Inactivated Poliovirus Vaccine (IPV)
 SALK Vaccine
 consists of formalin inactivated virus
 all 3 poliovirus serotypes
 produces serum antibodies only
 prevents paralytic polio
 does not induce local immunity thus not
prevent local infection of gut
 Enhanced potency IPV
 continuous cell line, 40-,8-,32- Da antigen units
 microcarrier systems for vaccine production

42.  Oral Poliovirus Vaccine (OPV)
 SABIN Vaccine
 live attenuated virus: all 3 serotypes 106.0
105.0
105.5
TCID50
 Oral administration
 Produces local immunity: induction of IgA response
 Systemic immunity
 Prevents viral shedding
 Rarely causes paralytic poliomyelitis, around 1 in 3 million doses

43. One theory about how HIV began in humans states that HIV was spread to people
by an oral polio vaccine (a vaccine given by mouth) used in Africa in 1950s.
most recent polio case in country had onset ofparalysis on 13 February 2007, in Andhra Pradesh. 3
other cases are from Uttar Pradesh and Bihar.Sub-national Immunization Days taking place in Uttar
Pradesh, Bihar and key infected areas.
What’s new in news?
In January and February 2007, India conducted two
National Immunization Days (NIDs), targeting more
than 182 million children under 5 years of age, during
each campaign
Staggered Supplementary Immunization Activities are being conducted
throughout UP and Bihar in April and May

44. COXSACKIE
 90% of cases of community acquired aseptic
meningitis: Group B coxsackie and echoviruses
 Serotypes causing aseptic meningitis
 group B coxsackie serotype 2 -5
 echovirus serotype 4, 6, 9, 11, 16 & 30
 Serotypes causing encephalitis
 coxsackie types A9, B2 & B5
 echovirus types 6 & 9
 Virus isolation & culture sensitivity:30-35%
 PCR sensitivity 66%-90%

45. Chronic meningoencephalitis in agammaglobulinemic
and other immunocompromised patients
 Fatal CNS infections in patients with defects in B-
lymphocyte function~ X-linked agammaglobulinemia
 Most cases causes by Echoviruses

46. PARAMYXOVIRUSES

47. MUMPS
 Most common extra salivary gland manifestation of mumps: CNS
involvement
 CSF pleocytosis
 Meningitis (common)
 Encephalitis (rare)
 Clinical meningitis occurs in 1% to 10% of persons with mumps
parotitis
 Only 40-50% of patients with mumps meningitis have parotitis
 Men>>Women
 Mumps meningitis +parotitis = spring,- parotitis = summer
 Mumps meningitis benign, complete recovery
 Mumps encephalitis complications & sequelae

48. Lab diagnosis of mumps
 Direct demonstration on Electron microscopy:
“Herring bone” or zipper like RNA in CSF is diagnostic
 Serology results uninterpretable due to cross reactions “original
antigenic sin”
 IgM ELISA Fourfold rise between acute & convalescent sera
 Isolation of virus in CSF: monkey kidney/HEp2 cells
 Complement fixation test using soluble (S) & viral (V) antigens
 PCR :viral RNA detected from CSF, Throat swabs etc.
 Vaccine: Jeryl Lynn or Urabe strains

49. Rubeola (measles)
Acute measles encephalitis
 Viral invasion of CNS is a
common feature of measles
 Only 1 in 1000-2000 develop
measles encephalitis
 Moderate to severe with
neurologic sequelae
 Hypersensitivity to virus in
brain tissue
 F and H glycoproteins
responsible for fusion to host
cell
 CD46 and SLAM signaling
lymphocyte activation molecule

50. Lab diagnosis
 Immunoflourescent staining of respiratory secretion for
measles antigen
 Warthin-Finkeldey giant cells in giemsa stained nasal
secretions
 Tissue culture techniques-human or monkey kidney cells
 PCR-detection of viral RNA
 Specific neutralization
 Haemagglutination inhibition test
 ELISA-detection of specific antibody
 IgM-within 2days of appearance of rash
 IgG-after 10 days

51. Subacute sclerosing panencephalitis (SSPE)
 Molecular methods for diagnosis not of proven
sensitivity/specificity
 Real time PCR used to monitor viral load in SSPE during
ribavarin & interferone therapy
 Unusually high measles antibody both in serum and in CSF
 Measles virus RNA decreases with therapy paralleling
decrease in measles virus antibody
 RT-PCR amplification of 3’ terminus of nucleocapsid gene
used for typing of strain variation

52. Treatment & prophylaxis
 Ribavarin
 Vit A prophylaxis
 Human gammaglobulin given within 6 days of exposure can prevent
or modify the disease
 Live attenuated vaccine (Edmonston-Zagreb strain),single s.c dose
 Post-vaccinial or post-infectious encephalitis
 Rare complication 2 – 3 weeks after exposure to certain viruses
(e.g. measles) or vaccines; strong autoimmune component

53. NEW & EMERGING
NEUROTROPIC VIRUSES
Epizoonotic encephalitis with direct animal to
human transmission

55. NIPAH VIRUS DISEASE
 Family – Paramyxoviridae
 Natural reservoir – Fruit Bats
 Isolation: Malaysia & Singapore (1999)
 Direct contact with pigs
 Multiorgan vasculitis with predilection for CNS
 Nipah virus encephalitis, persistent convulsions
& personality changes
 Diagnosis – MRI, serology, isolation, RT-PCR
 Treatment – Ribavirin
 High case fatality

56. LYSSAVIRUS

57. RHABDOVIRIDAE
 Family- Rhabdoviridae
 Genus- Lyssa virus
 Strain- Rabies virus
 Rabies viruses are
enveloped, bullet-shaped
with helical nucleocapsid
 Single stranded, negative
sense RNA viruses

60. EPIDEMIOLOGY
 In India, there are 15-30,000 rabies deaths per year
 Reservoirs for rabies virus - Dogs, cats, squirrels, bats,
foxes & raccoons
 Infection is by bite of an animal, or via aerosols from bat
droppings
 Newly discovered rabies related viruses- Australian bat
lyssavirus (ABLV) & European bat lyssavirus (EBLV)

61. Pathogenesis:Viral invasion of cells
 Specific binding occurs at
neuro-muscular junctions,
where virus colocalises with
acetyl choline receptors
 neural cell adhesion molecule
 p75 neurotropin receptor
(p75NTR)
 virus enters cells by
adsorptive endocytosis
 Fast axonal transport
system-retrograde

62. Retrograde axonal
transport
 Following inoculation, virus replicates locally, gains access to
peripheral nerves & travels in a retrograde direction in
axoplasm to the CNS
 Massive replication on membranes within neurons
 Direct transmission of virus from cell to cell occurs across
synaptic junctions
 Centrifugal spread from CNS in autonomic nerves/nerve
trunks, deposits virus in skeletal & cardiac muscle, lungs,
kidney, adrenal medulla, retina, cornea and salivary glands
 Viral replication with budding from plasma membrane takes
place in salivary glands

63. Effect of virus on neuronal function

64. AIM TEST
Skin punch biopsy Antigen detection, IFA test on frozen
section
viral RNA RT-PCR
Saliva, tears, CSF Virus isolation Tissue culture
Suckling mouse
inoculation
Viral RNA RT-PCR
Serum Antibody detection
CSF Antibody detection
Antemortem diagnosis of Rabies

65. Postmortem diagnosis
AIM TEST
Brain Antigen detection IFA test on
impression smear
Needle necropsy Viral RNA RT-PCR
Brainstem,
cerebellum
Virus isolation Tissue culture
Suckling mouse
inoculation
Retrospective
diagnosis
Antigen detection in
formalin-fixed
tissue
Enzyme methods

66. Rapid microscopic examination for
Negri bodies
 Negri bodies may be
identified in certain
regions of brain, such as
hippocampus, these
inclusion bodies are
diagnostic of rabies
 Cytoplasmic
 Ammon’s horn
 Basophilic inner granules

67. RT-PCR in rapid diagnosis of Rabies
 CSF, saliva & tissue
 Adv: decomposed brain
 N gene is the most
conserved in lyssa viruses
 RT-PCR allows specific
determination of
geographic & host species
origin of a particular virus
 RT-PCR with TaqMan
genotype-specific probes
can distinguish among six
rabies viruses & rabies
related viruses
P. Crepin, L. Audry
Intravitam Diagnosis of Human Rabies by PCR Using Saliva and Cerebrospinal Fluid
J. Clin. Microbiol. 1998 36: 1117-1121

68. Rapid fluorescent focus inhibition test (RFFIT)
 Virus neutralization test
 Fixed amount of live rabies virus
& dilutions of test serum are
added to cell monolayers &
incubated for 20h
 If neutralizing antibodies to rabies virus glycoprotein are
present in the patient’s serum, few or no infected foci are seen
when the direct fluorescent antibody (DAF) is applied to cell
monolayer
 Rabies virus specific antibodies may be found in serum as a
result of previous vaccination
 In contrast occurrence of rabies virus Ab in CSF is diagnostic ,
since Ab from vaccination do not cross intact BBB

69. RABIES VACCINES
 Neural
 Semple
 Non Neural
 Egg -Duck, chick embryo
 Tissue culture
 human diploid
 primary (chick embryo)
 continuous (Vero)

70. CELL CULTURE VACCINES
 Human diploid cell (HDC): Pitman-moore
strain
 Purified chick embryo cell (PCEC):
Flury-Low Egg Passage strain
 Purified vero cell vaccine (PVC);
 All 3 available in India
 Same dosage schedule for adults and
children
 Post exp prophylaxis-0,3,7,14,30, (90);
protection for 5 yrs

71. Injection sites for intradermal
vaccination
Current Trends in Vaccination
against RABIES
2 site regimen (Thai Regimen)2 site regimen (Thai Regimen)
8 site regimen (Oxford Regimen)8 site regimen (Oxford Regimen)
Multisite intradermal regimens
recommended by WHO

72. NEWER ANTIRABIES VACCINES
 Subunit vaccine
 Glycoprotein
 DNA Plasmid vaccine
 Vaccines from plants
 Tobacco
 Musk melon

73. Whats new in news?
CDC to Recognize the First World Rabies Day!
WORLD RABIES DAY, SEPTEMBER 8, 2007

74. POLYOMAVIRUS

75. JCV & Progressive Multifocal
Leucoencephalopathy

76. Lab diagnosis
 JC virus causes a progressive, fatal disease characterized by
multifocal demyelination of white matter, enlarged
oligodendrocytes with nuclear inclusion & bizarre astrocytes

immune suppression or chronic disease, e.g. lymphoma, AIDS
 Detection of JCV agnoprotein by use of specific polyclonal
antibody (Ab): marker for PML diagnosis & infection
 JCV specific ELISA
 Detection of JCV DNA in brain biopsy tissue/CSF by in situ
hybridization or PCR
 CSF PCR sensitivity 50-75%,
specificity 100%
 urine & blood viral loads not
indicative of CNS infection

77. RETROVIRUS

78. HIV- Is it neurotropic?
 Progressive HIV-1 infection... neurological and
psychiatric abnormalities
 Criteria of neurotropism : virology-based definition
necessitates infection of neurons
 Microglia are the principal target for HIV-1
 HIV-1 is neurotropic in ways similar to other
lentiviruses that cause disease of CNS
 HIV-1 better described as being neurovirulent or
neuroinvasive rather than neurotropic

79. Neurological Complications of HIV/AIDS
 HIV associated dementia
 Meningitis
 Encephalitis
 Peripheral neuropathy
 Radiculopathy
 Guillian-Barre syndrome
 Cognitive impairement

80. Pathogenesis
 Monocyte-associated virus is predominant route of HIV-1 traffic
into brain
 Microglia are principal target for HIV-1
 Neurotoxic mediators released from microglia
 HIV-1 enters microglial cells via CD4 receptors and chemokine
coreceptors such as CCR3, CCR5, and CXCR4
 Multinucleated giant cells, microglial nodules in HIV encephalitis

81. Possible factors in pathogenesis of HIV
associated CNS disease
VIRAL PROTEINS
Blocking of transmitters
interference with neurotrophic factors & cell function
Toxicity of viral polypeptides
CYTOKINES
Direct toxicity
Neuroimmune mediated CNS dysfunction/injury
Stimulation of astrocytes
BLOOD BRAIN BARRIER DISRUPTION
Exposure of parenchymal cells to circulating cytokines & systemic factors
Ingress of HIV-1 infected cells & free virus
AUTOIMMUNITY
Immune response to HIV antigens, molecular mimicry
INCREASED CNS HIV-1 VIRAL LOAD
Neurovirulent strains

82. Pathogenesis

83. HTLV-1
 HTLV-1 associated myelopathy-tropical spastic paraparesis (HAM-TSP)
 Pathogenesis: autoimmune model, cytotoxic model
 Increased inflammatory cells in HAM-TSP spinal cord suggests an
immunological basis
 Spastic paraparesis, bladder dysfunction, sensory and motor
 Elevated antibody titres to HTLV-1 in blood & CSF
 CSF PCR for proviral DNA & serological analysis for intrathecal
synthesis of HTLV-1 specific antibodies- diagnostic
 No therapy
 Vaccine develpoment
Lobulated lymphocytes
“Flower cells”

84. Atypical agents – prions
 BSE – bovine spongiform encephalopathy
 CJD – Creutzfeldt-Jakob disease
 vCJD – variant CJD
 GSSD – Gerstmann-Straussler-
Scheinker disease
 TSE – transmissible spongiform
encephalopathy

85. How to go about a case of encephalitis...

88. References
 Textbook of clinical neurology Human immunodeficiency virus
& AIDS. ch 44,viral infections ch 41.
 Principles & practice of infectious diseases ch 120 Neurologic
diseases caused by HIV-1 & opportunistic infections
 Medical microbiology by David greenwood
 Harrison’s Principles of internal medicine,16Edn,vol1
 Molecular methods for diagnosis of viral encephalitis, CMR,
oct 2004,p903-925.
 Rabies and other lyssavirus diseases, The lancet,vol363,Mar
2004
 Microglia: immune effector cells of the brain, CMR, vol 17,
2004