REOVIRUSES, ROTAVIRUSES, ORBIVIRUSES, COLTIVIRUSES, CALICIVIRUSES AND

ASTROVIRUSES
- Reovirus: Medium-sized viruses with a double-stranded, segmented RNA genome
- Family includes, HUMAN ROTAVIRUSES: the most important cause of infantile
gastroenteritis around the world
- Acute gastroenteritis, a very common disease with significant public health impact
- In developing countries, it is estimated to cause as many as 1.5 million deaths of
preschool children annually
- Rotavirus is responsible for about 600,000 deaths
- In US, acute gastroenteritis is 2nd only to acute respiratory infections as a cause of
disease in families.
- Calicivirus: small viruses with a single stranded RNA genome.
- The family contains, NOROVIRUSES: major cause of nonbacterial epidemic
gastroenteritis worldwide
- ASTROVIRUSES, also cause gastroenteritis

VIRION Icosahedral, 60-80 nm in

diameter, double capsid shell
GENOME Double-stranded RNA, 10-12
discrete segments, total
genome size of 16-27 kbp
PROTEINS Nine structural proteins; core
contains several enzymes
ENVELOPE None (transient
pseudoenvelope is present
during rotavirus particle
morphogenesis)
REPLICATION Cytoplasm; virions not
completely uncoated
OUTSTANDING Genetic reassortment occurs
CHARACTERISTICS readily
Rotaviruses are the major cause
of infantile diarrhea
Reoviruses are good models for
molecular studies of viral
pathogenesis

REOVIRUSES AND ROTAVIRUSES

Structure and Composition
- VIRIONS : measure 60-80nm in diameter and possess 2 concentric capsid cells,
each with is icosahedral
- Rotavirus have a triple layered structure
- There is no envelope
- Single-shelled virus particles that lack the outer capsid are 50-60nm in
diameter
- Inner core of particles is 33-40nm in diameter
- Double-shelled particle is the complete infectious form of the virus
 - Reovirus genome consists of double-stranded RNA in 10-12 discrete segments
- Total genome size: 16-27 kbp, depending on genus
- Rotavirus genome contains 11 genome segments
- Orthoreoviruses and Orbiviruses each possess 10 segments
- Coltiviruses 12 segments
- Individual RNA segments vary in size from 680 (Rotavirus) to 3900 bp
(orthoreovirus)
- Virion core contains several enzymes needed for transcription and capping of
viral RNAs
- Rotaviruses stable to heat 50C, to a 3.0-9.0 range of pH, Lipid solvents, such as
ether and chloroform, inactivated by 95% ethanol, phenol, and chlorine
- Limited treatment with proteolytic enzymes increases infectivity

Classification
- Family Reoviridae is divided into 15 genera
- 4 of the genera are able to infect humans and animals : Orthoreoviruses,
Rotaviruses, Coltiviruses, Orbivirus
- Genera are divided into 2 subfamilies:
- Spinareovirinae contains viruses with large spikes at the 12 ventricles on the
particle (eg. Orthoreovirus)
- Sedoreovirinae appear more smooth, lacking the large surface projections (eg.
Rotavirus)
- There are atleast 5 species or groups of Rotaviruses (A-E) plus 2 tentative species
(F and G)
- 3 species (A,B,C) infect humans
- Strains of human and animal origin may fall in the same serotype
- Other rotavirus groups and serotypes are found only in animals
- 3 different serotypes of reovirus are recognized , along with about 100 different
orbivirus serotypes and 2 coltivirus serotypes

Reovirus Replication
- Viral particles attach to specific receptors on the cell surface
- Cell attachment protein for reovirus is the viral hemagglutinin (1 protein), a
minor component of the outer capsid
- After attachment and penetration, uncoating of virus particles occurs in
lysosomes in the cell cytoplasm
- Only the outer shell of the virus is removed, and core-associated RNA
transcriptase is activated
- This transcriptase transcribes mRNA molecules from the minus strand of each
genome double-stranded RNA segment contained in the intact core
- There are short terminal sequences at both ends of the RNA segments that are
conserved among all isolates of a given subgroup
- These conserved sequences may be recognition signals for the viral transcriptase
- The functional mRNA molecules correspond in size of the genome segments
- Most RNA segments encode a single protein, although a few encode 2 proteins
- Reovirus cores contain all enzymes necessary for transcribing, capping, and
extruding the mRNAs from the core, leaving the double stranded RNA genome
segments inside
 - After being extruded from the core, mRNAs are translated into primary gene
products
- Some full length transcripts are encapsidated to form immature virus particles
- Viral replicase is responsible for synthesizing negative-sense strands to form the
double-stranded genome segments
- this replication to form progeny double-stranded RNA occurs in partially completed
core structures
- The mechanism that ensure assembly of the correct complement of genome
segments into developing viral core are unknown
- However, genome resortment, occurs readily in cells coinfected with different
viruses of the same subgroup, giving rise to virus particles containing RNA
segments from the different parental strains
- Viral polypeptides probably self-assemble to form the inner and outer capsid shells
- Reovirus produce inclusion bodies in the cytoplasm in w/c virus particles are
found
- These viral factories are closely associated with tubular structures (microtubules
and intermediate fimalents)
- Rotavirus morphogenesis involves budding of single-shelled particles into the
rough endoplasmic reticulum
- The pseudoenvelopes so acquired are then removed and the outer capsid are
added
- This unusual pathway is used because the major outer capsid protein of
rotaviruses is glycolysated
- Cell lysis results in release of progeny virions

ROTAVIRUSES
- Major cause of diarrheal illness in human infants and young animals, including
calves and piglets.
- Infection in adult humans and animals are also common
- Among the rotaviruses are the agents of human infantile diarrhea: Nebraska calf
diarrhea, epizootic diarrhea of infant mice, and SAII virus of monkeys
- Rotaviruses resemble reoviruses in terms of morphology and strategy of replication

VIRUS SIZE EPIDEMIOLOGY IMPORTANT AS A

CAUSE OF
HOSPITALIZATIO
N
Rotaviruses
Group A 60-80 Single most important Yes
cause (viral or
bacterial) of endemic
severe diarrheal illness
in infants and young
children worldwide (in
cooler months in
temperate climates)
 Group B 60-80 Outbreaks of diarrheal No
illness in adults and
Children in China
Group C 60-80 Sporadic cases and No
occasional outbreaks of
diarrheal illness in
children
Enteric 70-90 Second most important Yes
Adenovirus viral agent of endemic
diarrheal illness of
infants and young
children worldwide
Caliciviruses
Noroviruses 27-40 Important cause of No
outbreaks of vomiting
and diarrheal illness in
older children and
adults in families,
communities, and
institutions; frequently
associated with
ingestion of food
Sapoviruses 27-40 Sporadic cases and
occasional outbreaks of
diarrheal illness in
infants, young children, No
and elderly adults
Astroviruses 28-30 Sporadic cases and
occasional outbreaks of No
diarrheal illness in
infants, young children,
and elderly adults

Classification and Antigenic Properties

- Classified into 5 species (A-E)
- 2 tentative species (F and G), based on antigenic epitopes on the internal
structural proteinVP6
- Can be detected by : Immunoflourescence, ELISA, and Immune electron
microscopy (IEM)
- Group A rotavirus are the most frequent human pathogens
- Outer capsid proteins VP4 and VP7 carry epitopes important in neutralizing activity
- VP7 glycoprotein being the predominant antigen
- 5 serotypes are responsible for the majority of human disease
- Multiple serotypes have been identified among human and animal rotaviruses
 - Some human and animal rotavirus share serotype specificity.
- Ex. Monkey virus SAII is antigenically very similar to human serotype 3
- Molecular epidemiologic studies have analyzed isolates based on differences in the
migration of the II genome segment after electrophoresis of the RNA in
polyacrylamide gels.
- These differences in electropherotypes can be used to differentiate group A viruses
from other groups, but they cannot be used to predict serotypes

Animal Susceptibility
- Most isolates have been recovered from newborn animals with diarrhea
- Cross species infections can occur in experimental inoculations, but is not clear if
they occur in nature
- Swine rotavirus infects both newborn and weanling piglets
- Newborns often exhibit subclinical infection, perhaps reflecting the presence of
maternal Ab
- Overt diseases is more common in weanling animals

Propagation in Cell Culture

- Rotaviruses are fastidious agents to culture
- Most group A human rotavirus can be cultivated if pretreated with the proteolytic
enzyme trypsin and if low levels of trypsin are included in the tissue culture
medium
- this cleaves an outer capsid protein and facilitates uncoating
- very few non-group A rotavirus strains have been cultivated

Pathogenesis
- rotavirus infect cells in villi of small intestine (gastric and colonic mucosa are
spared)
- they multiply in the cytoplasm of enterocytes and damage their transport
mechanism
- one of the rotavirus-encoded proteins, NSP4, is a viral enterotoxin and induces
secretion by triggering a signal transduction pathway
- damaged cells may slough into the lumen of the intestine and release large
quantities of virus, w/c appear in stool
- viral excretion usually lasts from 2-12 days in otherwise healthy px
- prolonged in those with poor nutrients
- Diarrhea caused by rotavirusmay be due to impaired sodium and glucose
absorption as damaged cells on villi are replaced by nonabsorbing immature crypt
cells
- It may take 3-8 weeks for normal function to be restored

Clinical Findings and Lab Diagnosis

- Incubation period: 1-3 days
- Typical symptoms: watery diarrhea, fever, abdominal pain, and vomiting leading to
dehydration
- In infants and children, severe loss of electrolytes and fluids may be fatal unless
treated
- Px with milder cases have symptoms for 3-8 days and then recover completely
- However, viral excretion in stool persists up to 50 days after onset of diarrhea
 - Asymptomatic infx with seroconversion occur
- In children with immunodeficiencies, causes severe and prolonged disease
- Adult contacts may be infected, as evidnced by seroconversion, but rarely exhibit
symptoms, te virus in infrequently detected in their stools
- Common source of infection is contacts with pediatric cases
- However, epidemics of severe disease have occurred in adults, esp in closed
populations, as in geriatric ward
- Group B rotaviruses have been implicated in large ourbreaks of severe
gastroenteritis in adults in china
- Lab diagnosis: demonstration of virus in stool collected early in the illness and on a
rise in antibody titer
- Virus in stool is demonstrated by EIAs or EIM
- The EIA test is more sensitive that the EIM
- Genotyping of rotavirus nucleic acid from the stool specimens by the PCR
- Serologic tests can be used to detect an Ab titer riseee, particularly ELISA

Epidemiology and Immunity

- Rotaviruses are the single most important worldwide cause of gastroenteritis in
young children
- Estimates range from 3 to 5 billion for annual diarrheal episodes in children
younger than 5 yrs in Africa, Asia, and Latin America
- Resulting in as many as 1 million deaths
- Developed countries have higher morbidity rate but low mortality rate
- Typically, up to 50% of cases of acute gastroenteritisof hospitalized children
throughout the world is caused by rotavirus
- Usually predominates during winter season
- Symptomatic infxn are most common in children between 6 months and 2 years
and transmission appears to be by the fecal-oral route
- Nosocomial infections are frequent
- Rotavirus are ubiquitous
- By age 3 yrs, 90% of children have serum Ab to one or more types
- This high prevalence of Ab is maintained in adults, suggesting subclinical
reinfections by the virus
- Rotavirus reinfections are common
- Young children can suffer up to 5 reinfections by 2 years of age
- Asymptomatic infections are more common with successive reinfections
- Local immune factor, such as secretory IgA or interferon, may be important in
protection against rotavirus
- Asymptomatic infxn are common in infants before age 6 months, the time during
w/c protective maternal Ab acquired passively by newborns should be present
- Such neonatal infxn does not prevent reinfection, but it does not protect against
the development of severe disease during reinfection

Treatment and Control

- Treatment for gastroenteritis is supportive to correct the loss water and
electrolytes that may lead to dehydration, acidosis, shock, and death
 - Management: replacement o fluids and restoration of electrolyte balance either
intravenously or orally as feasible
- Infrequent mortality from infantile diarrhea in developed countries s the result of
the routine use of effective replacement therapy
- - Significant control measures: fecal-oral route transmission, wastewater treatment
and sanitation
- Oral love attenuated rhesus-based rotavirus vaccine : vaccine for infants, licensed
in the US in 1998
- Withdrawn a year later because of reports of intussusception (bowel blockages) as
an uncommon but serious side effect associated with the vaccine
- 2006. Oral live attenuated pentavalent human-bovine reassortant rotavirus
vaccine
- 2008, oral live attenuated monovalent human rotaviruss vaccine
- Both vaccines are safe and effective and neither is associated with intussusception

REOVIRUSES
- Not known to cause human disease

Classification and Antigenic Properties

- Ubiquitous
- With a very wide range of mammalian, avian and reptilian hosts
- 3 distinct but related types of reovirus have been recovered and demonstrable by
neutralization and hemagglutination inhibition tests
- Reovirus contain a hemagglutinin for human O or bovine erythrocytes
Epidemiology
- Reovirus can cause many inapparent infxn because most people have serum Ab by
early adulthood
- Ab are also present in other species
- All 3 types have been recovered from healthy children, from young children during
outbreaks of minor febrile illness, from children with enteritis or mild repiratory
disease, and from chimpanzees with epidemic rhinitis
- Human studies failed to demonstrate a clear cause and effect relationship of
reovirus to human illness
- In inoculated volunteers, reovirus is recovered far more readily from feces than
from nose or throat

Pathogenesis
- Defined recombinants from 2 reovirus with differeing pathogenic phenotypes are
used to infect mice
- Segregation analysis is then used to associate particular features of pathogenesis
with specific viral genes and gen products
 - The pathogenic properties are primarily determined by the protein species found
on the outer capsid of the virion

ORBIVIRUSES AND COLTIVIRUSES

- Orbiviruses are a genus within the reovirus family
- Commonly infect insects and any transmitted by insects to vertebrates
- About 100 serotypes are known
- None of these virus causes serious clinical disease in humans, but may cause mild
fevers
- Serious animal pathogens include blue tongue virus of sheep and African horse
sickness virus
- Ab to orbiviruses are found in many vertebrates including humans
- The genome consists of 10 segments of double-stranded RNA
- Total genome size of 18kbp
- The replicative cycle is similar to that of reovirus
- Orbivirus are sensitive to low pH in contrast with general stability within Reovirus
- Coltiviruses: another species of other reoviridae
- Consists of 112 segments of double-stranded RNA
- 29 kbp
- Colorado tick fever virus, transmitted by ticks is able to infect humans.

CALICIVIRUSES
- Members of the family Caliciviridae are important agents of gastroenteritis in
human.
- The most significant members are the Noroviruses, the prototype strain being
Norwalk virus.
- Caliciviridae is divided into five genera: Norovirus, Sapovirus, Nebovirus,
Lagovirus and Vesivirus.
- Important Properties of Caliciviruses
Virion: Icosahedral, 27-40 nm in diameter, cup-like depressions on capsid suface.
Genome: Single stranded RNA, linear, positive sense nonsegmented; 7.4-8.3 kb in
size; contains genome-linked protein (VPg)
Proteins: Polypeptides cleave from a precursor polyprotein; capsidis composed of
a single protein.
Envelope: None
Replication: Cytoplasm
- Clinical Findings and Laboratory Diagnosis
- Noroviruses are the most important cause of epidemic viral gastroenteritis
in adults.
- Norwalk viral gastroenteritis has an incubation period of 24-48 hours. The
onset is rapid, and the clinical course is brief, lasting 12-60 hours.
- Symptoms include diarrhea, headache and malaise.
- Reverse trancriptase PCR is the most widely used technique for
detection of human caliciviruses in clinical specimens (feces, vomitus).
- Electron Microscpoy is frequently used to detect stool samples.
- ELISA immunoassays can detect antibody responses.
- Epidemiology and Immunity
- Human calcivirus have worldwide distribution.
 - Noroviruses are the most common cause of nonbacterial gastroenteritis in
United States, causing an estimated 21 million cases annually.
- The viruses are most often associated with epidemic outbreaks of
waterborne, foodborne and shellfish associated gastroenteritis.
- Most outbreaks involve foodborne or person to person transmission via
fomites or aerosolization of contaminated body fluid (vomitus, fecal
material)
- Characteristics of norovirus include low infectious dose (as few as 10 virus
particles). Relative stability in environment, and multiple modes of
transmission. It survives 10 ppm chlorine and heating to 60C; itcanbe
maintained in steamed oysters.
- No in vitro neutralization assay is available to study immunity.
- Treatment and Control
Treatment is symptomatic.
Effective handwashing is probably the most important method to prevent
norovirus infection and transmission
Careful processing of food and education of food handlers are important
because many food borne outbreaks occurs
Purification of drinking water and swimming pool water should decrease
norovirus outbreaks.
There is no vaccine.
-

ASTROVIRUS

- Astrovirus are about 28-30 nm in diameter and exhibit a distinctive starlike

morphology in electron microscope.
- They contain single strandes, positive-sense RNA, 6.4- 7.4 kb in size.
- The family Astroviridae contains two genera; all human viruses are classified in
the Mamastrovirus genus. Atleast eight serotypes of human viruses are
recognized by IEM and Neutralization.

- Astroviruses cause diarrheal illness and maybe shed in extraordinarily large

quantities in feces.
- The viruses are transmitted by fecal oral route through contaminated food or water
, person-person contact, or contaminated surfaces.
- They are recognized as pathogens for infants and children, elderly institutionalized
patients and immunocompromised persons. They may be shed for prolonged
periods by immunocompromised host.
- Animal Astrovirus are found in variety of mammals and birds and have recently
been identified in several places.

ARTHROPOD-BORNE AND RODENT BORNE VIRAL

DISEASES
Arthropod-borne viruses (arboviruses) and rodent borne viruses represent complex
transmission cycles involving arthropods or rodents.
 Classified among Arenaviridae, Bunyaviridae, Flaviviridae, Reoviridae and
Togaviridae families.
Filoviridae African Hemorrhagic fever viruses
Arbovirus transmitted by bloodsucking arthropods from one vertebrae host to
another.
- The vector acquires a life long infection through the ingestion of blood from a
viremic vertebrae
- They multiple in the tissues of arthropod without evidence of disease and
damage
- Some arbovirus are maintained in nature by transvorian transmission in
arthropods.
- Major arbovirus diseases worldwide
Yellow Fever
Dengue
Japanese B Encephalitis
St. Louis Encephalitis
Western Equine Encephalitis
Eastern Equine Encephalitis
Tick Borne Encephalitis
West Nile Fever
Sandfly Fever
- Most important Arbovirus in United States
La Crosse encephalitis
West Nile Fever
St Louis Encephalitis
Eastern Equine Encephalitis and Western Equine Encephalitis

Rodent Borne Viral diseases are maintained in nature by direct intraspecies or

interspecies transmission from rodent to rodent without arthropod vectors.
- Transmission: Contact with body fluids or excretions
Major Rodent Borne Diseases
Hantavirus infections
Lassa fever
South American Hemorrhagic fevers
Most important rodent-borne in the US:
Hantavirus pulmonary syndrome
Colorado tick fever
**African hemorrhagic fevers: Marburg and Ebola (unknown reservoirs but
are suspected to be rodents or bats)

HUMAN ARBOVIRUS INFECTIONS

-humans are the accidentl hosts who play no important role in maintenance or
transmission cycle of the virus
-exceptions: Urban yellow fever and dengue
- some of the natural cycles are simple and involve infection of a non human
vertebrae host (mammal or bird)
-transmitted by a species of mosquito or tick (eg, jungle yellow fever, Colorado tick
fever)
 -Tick Borne Encephalits- can occure adter ingestion of raw milk from goats and
cows infected by grazing in tick infested pastures where tick rodent cycle is
occurring
-Diseases produced by arboviruses may be divided into three clinical syndromes:
fevers of an undifferentiated type with or without maculopapular rash and
usually benign
encephalitis (inflammation of the brain) often with a high case fatality rate
hemorrhagic fevers also frequently severe and fatal
- some are arbitrary and may be associated with more than one syndrome
(dengue)
- the degree of viral multiplication nad its predominant site of localization in
tissues determine the clinical syndrome
- individual arboviruses can produce a minor febrile illness in some patients and
encephalitis or a hemorrhagic diathesis in others
- each continent tends to have its own arbovirus pattern and names are usually
suggestive including:
Venezuelan equine encephalitis
Japanese B encephalitis
Murray Valley (Australia)
- Many encephalitis are alphavirus and flavivirus infections spread by mosquitoes
- The group of California encephalitis diseases is caused by Bunyavirus

Togavirus and Flavivirus Encephalitis

Classification and Properties
Togaviridae family, Alphavirus genus consists of about 30 viruses 70 nm in
diameter that possess single-stranded, positive-sense RNA genome
Envelope contains two glycoproteins
Mosquito or blood-feeding arthropod transmitted between vertebrates
Worldwide distribution
All alphaviruses are antigenetically related
Inactivated by acid pH, heat, lipid solvents, detergents, bleach, phenol, 70%
alcohol, and formaldehyde
Most have hemagglutinating ability
Rubella virus, separate genus in the Togaviridae family, no arthropod vector and is
not an arbovirus
Arboviruses are in the Flavivirus genus in the Flaviviridae family
Initially as group B arboviruses in togavirus family but has differences in viral
genome organization
Flaviviridae family consists of 70 viruses 40-60 nm in diameter that have single-
stranded, positive-sense RNA genome
The viral envelope contains two glycoproteins.
Mosquito, tick, rodent, bat vectors
All flaviviruses are antigenically related
Similar inactivation and hemagglutinating ability to alphavirus
Hepatitis C has no arthropod vector and not an arbovirus
Replication of Togaviruses and Flaviviruses
 Alphavirus RNA genome is positive sense
Genomic length and subgenomic (26S) mRNAs produced during transcription
Genomic-length transcript produces a precursor polyprotein encoding the
nonstructural proteins (ie, replicase , transferase) needed for viral RNA replication
Subgenomic mRNA encodes structural proteins
The proteins are elaborated by posttranslational cleavage
Alphaviruses replicate in cytoplasm and mature by budding nucleocapsids through
plasma membrane
Sequence data indicates western equine encephalitis virus is a genetic
recombinant of eastern equine encephalitis and Sindbis viruses
Flavivirus RNA genome is positive sense
A large precursor protein is produced from genome-length mRNAs during viral
replication; cleaved by viral and host protoeases to yield both structural and
nonstructural viral proteins
Flaviviruses replicate in the cytoplasm
Particle assembly occurs in intracellular vesicles

Proliferation of intracellular membranes characteristic of flavivirus-infected cells

Antigenic Properties
All aplhaviruses genetically related
Common antigenic determinants show cross-reactions in immunodiagnostic
techniques
Hemagglutination-inhibition, ELISA, and immunofluorescence tests define eight
antigenic complexes/serogroups of alphaviruses, four typified by western equine
encephalitis, eastern equine encephalitis, Venezuelan equine encephalitis, and
Semliki Forest virus
ID of specific virus by neutralization test
All flavivirus share antigenic sites
At least eight antigenic complexes have been identified by neutralization tests for
alphaviruses and 10 serocomplexes for flaviviruses
Envelope (E) protein = viral hemagglutinin, contains the group-, serocomplex-, and
type-specific determinants
 Sequence comparisons of E glycoprotein gene show that viruses within a
serocomplex share over 70% amino acid sequences but amino acid homology
across serocomplexes is <50%

Pathogenesis and Pathology

Primary viral multiplication occurs either in myeloid and lymphoid cells or in
vascular endothelium
Multiplication in CNS depends on virus ability to pass blood-brain barrier and infect
nerve cells
Inapparent infection usual in birds and mammals
Viremia for several days, arthropod acquire virus in this period (First dissemination
step)
Mice used in study
Usually virus is controlled before neuroinvasion occurs
Invasion factors include level of viremia, the genetic background of host, innate
and adaptive immune responses, and virulence of virus strain
Infants and elderly adults most susceptible to CNS infections
Diphasic equine encephalitides in horses first phase (minor illness) and second
phase (major illness)
Clinical Findings
4-21 days incubation period
Inapparent infections common
Some develop mild flu-like illness while others develop encephalitis
Sudden onset with severe headache, chills and fever, nausea and vomiting,
generalized pains, and malaise
Within 24-48 hours, marked drowsiness develops and patient may become
stuporous
Mental confusion, tremors, convulsions, and coma develop in severe cases
Fever lasts 4-10 days
Varying mortality rate
Japanese B encephalitis, mortality rate in older age groups may be as high as 80%
Sequelae may be mild to severe and include mental deterioration, personality
changes, paralysis, aphasia, and cerebellar signs

Laboratory Diagnosis
A. Recovery of Virus and Direct Detection
Biosafety precautions
Blood viremia only in early infection, before onset of symptoms
CSF and tissue specimens
Able to grow on common cell lines, Vero, BHK, HeLa, and MRC-5
Intracerebral inoculation of suckling mice for isolation
Antigen detection and PCR assays are available
Virus-specific monoclonal antibodies in IF assays has facilitated rapid virus ID

B. Serology
 Neutralizing and hemagglutination-inhibiting antibodies detectable within
few days after onset of illness and can endure for years
HI test simplest diagnostic test identifies only the group
Most sensitive serologic assays detect virus specific IgM in serum or CSF by
ELISA
Fourfold titer for diagnosis
First sample after onset, second sample 2-3 weeks later
Cross reactivity must be considered
Antibodies to other members may appear
Difficult diagnosis in epidemic caused by one member of serologic group
occurs in an area where another group is endemic
Immunity
Immunity permanent after single infection
Humoral and cellular immunity important
In endemic areas, population may build up immunity due to inapparent infections;
proportion of persons with Ab to virus increases with age
Common antigen immunization is helpful to other virus within group (no Japanese
B encephalitis in areas endemic for West Nile fever)
Epidemiology
Almost entire human population may become infected by arbovirus (asymptomatic).
Most occur in summer months when arthropods are active in northern hemisphere
A. Eastern and Western Equine Encephalitis
Eastern Equine is the most severe of the arboviral encephalitides
Inapparent infections unusual
Western Equine low level transmissions
Birds and Culex tarsalis mosquito involved in maintenance cycle
B. St. Louis Encephalitis
Most important cause of epidemic encephalitis in North America
Seroprevalence rates are generally low
Presence of infected mosquitoes required
Socioeconomic and cultural factors affect the degree of exposure
C. West Nile Fever
Caused by a member of the Japanese B encephalitis antigenic complex of
flaviviruses
Occurs in Europe, Middle East, Africa, former Soviet Union, Southwest Asia,
United States
Sequence analysis showed Middle Eastern origin
Has been detected in all 48 contiguous states in the US
Leading cause of arboviral encephalitis in the US
Fatal encephalitis more common in older people
Produces viremia and an acute, mild febrile disease with lymphadenopathy
and rash
Transitory meningeal involvement may occur during the acute stage
Only one antigenic type of virus exists and immunity is presumably
permanent
No human vaccine
 D. Japanese B Encephalitis
Leading cause of viral encephalitis in Asia
Mostly among children and elderly adults
Mortality rate can exceed 30%
High percentage of survivors are left with neurologic and psychiatric
sequelae
First and second trimesters of pregnancy infections have reportedly led to
fetal death
Seroprevalence studies indicate nearly universal exposure by adulthood
No treatment
Vaccines are available in Asia
Inactivated Vero cell culture-derived vaccine licensed in US

E. Chikunguya Virus
Member of the Semliki Forest antigenic complex
Clinically, infection resembles dengue fever
Characterized by high fever and severe joint pain
No vaccine
F. Tick-Borne Encephalitis
Flavivirus, important cause of encephalitis in Europe, Russia and northern
China
Chiefly occurs in early summer
Ixodes persulcatus and Ixodes ricinus are carriers in forested areas
Three subtypes cause human disease: European, Far-Eastern (most virulent),
and Siberian
Many species of animals can be infected
No specific treatment
PPEs useful
Vaccines produced in Austria, Germany and Russia based on European and
Far-Eastern strains of the virus
Treatment and Control
No specific Treatment
Biologic control impractical
Most effective is Arthropod Control
Personal Measures for protection
Effective killed-virus vaccines for horses developed against eastern, western, and
Venezuelan equine encephalitis
Attenuated live-virus vaccine for Venezuelan equine encephalitis available for
curtailing epidemics among horses
Vaccines not for human use
Experimental inactivated human vaccines on investigational basis for laboratory
workers
Killed-virus and attenuated live-virus Japanese B encephalitis vaccines for humans
are in use in Asia countries
Vaccine available in US for travel in endemic countries
Zika Virus
Classification and Properties
member of family Flaviviridae and genus Flavivirus
transmitted by daytime Aedes mosquitoes such as A. aegypti and A. albopictus
name comes from Zika Forest of Uganda first isolated on 1947
enveloped and icosahedral
nonsegmented, single-stranded, positive-sense RNA genome
closely related to Spondweni virus
Viral Replication
positive-sense RNA genome can be directly translated into viral proteins
structural proteins encapsulate the virus
replicated RNA strand is held within a nucleocapsid formed from 12-kDa protein
blocks; the capsid is contained within a host-derived membrane modified with two
viral glycoproteins
Replication of viral genome would first require creation of an anti-sense nucleotide
strand
Clinical Findings
Zika fever resembles dengue fever
Symptoms last less than seven days
Symptoms include fever, red eyes, joint pain, headache, and a maculopapular rash
Linked with Guillain-Barr syndrome
Documentation reveals that Zika virus may be spread from mother-to-child in the
womb but is not yet confirmed
Fetal syndromes include microcephaly from destruction of different parts of the
brain, calcifications in the eye and microphthalmia
Laboratory Diagnosis
Difficult to diagnose due to overlaps of other arboviruses or arthropod borne
viruses
Can be identified by reverse transcriptase PCR (RT-PCR) in acutely ill patients
Viremia period is short
RT-PCR testing done on serum collected 1 to 3 days of symptom onset or on saliva
of urine samples collected during the first 3 to 5 days
Zika virus detected more frequently in saliva than serum
Longest Period of detectable virus is 11 days and does not establish latency
Specific IgM and IgG antibodies can be used and is detectable within 3 days onset
of illness
Serological cross-reactions with closely related flaviviruses such as dengue and
West Nile Fever as well as vaccines are possible
CDC recommend screening for pregnant women even when not having symptoms
Travelling pregnant women should be tested between two and twelve weeks after
their return from travel
Ordering and interpretation of tests are difficult
 Women in affected areas are recommended for testing at the first prenatal visit
with a doctor as well as in the mid-second trimester
Additional testing should be done if there are any signs of Zika virus disease
Women with positive results should have fetal monitoring by ultrasound every
three to four weeks to monitor their anatomy and growth
Infants with suspected congenital Zika virus disease, CDC recommends testing
with both serologic and molecular assays such as RT-PCR, IgM ELISA and plague
reduction neutralization test (PRNT)
Newborns with exposed mother, positive blood tests, microcephaly or intracranial
calcifications should have further testing through physical investigation for
neurologic abnormalities, dysmorphic features, splenomegaly, hepatomegaly, and
rash or other skin lesions
Cranial ultrasound, hearing evaluation, and eye examination are other
recommended tests
Immunity
There is currently no vaccine

Epidemiology
First known case was in a sentinel rhesus monkey in the Zika Forest in Uganda in
1947
First human cases were reported in Nigeria in 1954
Antibodies to Zika in healthy people in India were found but could also be due to
cross-reaction with other flaviviruses
Zika virus moved to Southeast Asia by 1945
There have been outbreaks in Oceania in 2013-2014 and there has been an
outbreak in the Americas recently in late 2015 and is still likely to spread this 2016

Treatment and Control

There is currently no specific treatment for Zika virus infection
Care is supportive with treatment of pain, fever, and itching
Advice to pregnant women is to avoid any risk of infection as far as possible, as
once infected there is little that can be done beyond supportive treatment

Bunyavirus Encephalitis

Bunyaviridae Family contains more than 300 viruses

Mostly transmitted by arthropods
 Spherical measuring 80-120nm, single stranded, negative-sense or ambisense,
triple segmented RNA genome 11-19 kb (total size)
Envelope has two glycoproteins
Produce mosquito-borne encephalitides of humans and animals others cause
Hemorrhagic fever
Transovial transmission occurs in some mosquitoes (some are transmitted by
sandflies)
HANTAVIRUS PULMONARY SYNDROME
- caused by a virus transmitted by rodents
BUNYAVIRUS
- are sensitive to inactivation by heat, detergents, formaldehydes and low pH ;
some are hemagglutinating
CALIFORNIA ENCEPHALITIS VIRUS COMPLEX
- Comprises 14 antigenetically related viruses in the Orthobunyavirus genus of
the family.
Includes:
La Crosse Virus a significant human pathogen in the US.
- major cause of encephalitis and aseptic meningitis in children, particularly in
the upper Midwest.
- They are transmitted by various woodland mosquitoes primarily Aedes
triseriatus
- Principal vertebrae host are small mammals such as squirrels, chipmunks
and rabbits.
- Human infection is tangential
- Overwinterring can occur in eggs of the mosquito vector and virus is
transmitted transovarially
- adult mosquitoes that develop from infected eggs can transmit the virus by
bite
- Onset of California encephalitis viral infections is abrupt typically with severe
headache, fever and in some cases vomiting and convulsions
- Illness last 10-14 days convalescence may be prolonged
- Neurologic sequelae are rare
- Serologic confirmation by HI, ELISA or neutralization tests is done on acute
and convalescent specimen.

SANDFLY FEVER
- is a mild, insect borne disease
- also called Phlebotomus fever is caused by a Bunyavirus in the Phlebovirus genus
- the disease is transmitted by a female sandfky, Phlebotomus papatasii ( a midge
only a few milliliters in size.
- Transovial transmission occurs
- During tropics they are prevalent
- During cooler climates, only warm season
- Endemically, infection is common in childhood
- Non-immune adults, large outbreak occurs and ocassionally mistaken as malaria
- In humans, bites of sandfly results in small itching papules on the skin (persist up
to 5 days)
- Disease begins abruptly after Incubation Period of 3-6 days
- Virus is found in the blood briefly near the time of the onset of symptoms
- Symptoms:
 headache, malaise, nausea, fever, photophobia, stiffness of the neck
and back, abdominal pain and leukopenia
- No specific treatment
- Most common just above the ground
- Their small size is the reason why they can pass through ordinary screens and
mosquito nets
- Insects feed primarily feed at night
- Prevention:
use of insect repellants during the night and residual insecticides
around living quarters

RIFT VALLEY FEVER

- the agent of this disease,, a bunyavirus of the Phlebovirus genus, is a mosquito
borne zoonotic virus pathogenic primarily for domestic livestock
- Humans are secondarily infected during the course of epizootics in domesticated
animals
- Infection among laboratory workers are common
- Epizootics occur after heavy rains that allow hatches of the primary vector and
reservoir (Aedes spp mosquitoes)
- Viremia in animals leads to infection of other vectors with collateral transmission to
humans.
- Transmission to humans is by contact with infected animal blood and body fluid
and mosquito bites
- Disease in humans is usually mild febrile illness that is short lived, recovery is
almost complete
- Complications:
retinitis, encephalitis and hemorrhagic fever, permanent loss of vison
may occur

COLORADO TICK FEVER

- classified in the genus Coltivirus
- African horse sickness and bluetongue viruses are in the genus Orbivirus
- Rotaviruses and orthoreoviruses have no arthropod vectors
- Also called Mountain fever or Tick fever, is transmitted by tick
- Virus appears to be antigenitically distinct from other known viruses, and only
antigenic type is recognized
- Is a mild febrile disease without rash
- Incubation period is 4-6 days
- Disease has a sudden onset with fever and myalgia
- Symptoms:
headache, muscle and joint pains, lethargy and nausea and vomiting
- Temperature is usually diphasic
- After first bout of 2 days, patient may feel well symptoms reappear and last 3-4
days
- Disease in humans is self limited
- Virus may be isolated from whole blood by inoculation of cell cultures
- Viremia may persist for 4 weeks or longer
- RT-PCR assays can detect viral RNA in red blood cells and in plasma
- Specific neutralizing antibodies appear in the second week of illness that can be
detected by plaque reduction tests
 - Serologic assays: ELISA, Fluorescent Antibody Testing
- Single infection produced lasting immunity
- Disease is limited to areas where the wood tick Demacentor andersoni is
distributed
- Cases occur in young men, group with greatest exposure to ticks
- D. andersoni collected in nature can carry the virus
- Tick is a true reservoir and transmitted transovarially by adult female ticks
- Natural infection occurs in rodents which acts as hosts for immature stages of the
ticks
- No specific therapy
- Prevention:
avoiding tick-infested areas by using protective clothing or repellent
chemicals

RODENT-BORNE HEMORRHAGIC FEVERS

- the zoonotic rodent-borne hemorrhagic fever includes Asian (eg Hantan and Seoul
viruses) South American (eg, Junin and Machupo viruses) and African (Lassa virus)
- Hantavirus aslo causes hantavirus pulmonary syndrome in the Americas (eg, Sin
Nombre virus)
- The natural reservoir of Marburg and Ebola viruses (African Hemorrhagic fever) not
know but are suspected to be rodents or bats
- Causative agents are classified
BUNYAVIRUS
ARENAVIRUS
FILOVIRUS

BUNYAVIRUS DISEASES
- Hantavirus are classified in the Hantavirus genus of the Bunyaviridae family
- Virus cause two serious and often fatal human diseases:
Hemorrhagic fever with renal syndrome
Hantavirus Pulmonary Syndrome
- Several distinct hantaviruses each associated with specific rodent host
- The virus in rodents are life long and without deleterious effects
- Transmission among rodents seems to occur horizontally and transmission to
humans occurs by inhaling aerosols of rodent excreta (urine, feces, saliva)
- Presence of hantavirus associated diseases is determined by the geographic
distribution of the rodent reservoirs

HEMORRHAGIC FEVER WITH RENAL SYNDROME

- Is an acute viral infection that causes and interstitial nephritis that can lead
to acute renal insufficiency and renal failure in severe forms of diseases
- Hantaan and Dobrova viruses can cause severe diseases that occurs in Asia
particularly in CHINA, RUSSIA AND KOREA and in Europe (BALKANS)
- Generalized hemorrhage and shock may occur
- Moderate form of HFRS cause by Seoul virus occurs throughout Eurasia
- NEPHROPATHIA EPIDEMICA- caused by Puumala virus and is prevalent in
Scandinavia, nephritis resolves without hemorrhagic complications and
fatalities are rare
- Apodemus agrarius- Hantan virus was not isolated in Korea from a rodent
- Urban rats are persistently infected with Hantaviruses
 - Brown Norway rats are infected with Seoul virus
- Hantavirus infections have occurred in persons whose ocupations place them
in contact with rats (eg, longeshoremen)
- Treated by supportive therapy
- Prevention depends on rodent control and protection from exposure to rodent
droppings and contaminated material

HANTAVIRUS PULMONARY SYNDROME -

- found to be caused by a novel Hantavirus (Sim Nombre virus)
- first hantavirus agent recognized to cause disease in North America and the
first to cause primarily and adult respiratory distress syndrome
- deer mouse (Peromyscus maniculatus) is the primary rodent reservoir for Sin
Nombre virus
- deer mice are widespread
- other Hantavirus know to cause HPS in the US:
New York virus
Black Creek Canal virus
Bayuo virus
- Each having different rodent host
- Andes virus is one oc the causative hantavirus and is found in Argentina and
Chile
- Choclo virus Panama
- Infections with hantavirus is not common and subclinical infections appear
to be unusual, particularly with Sin Nombre virus
- It is generally severe
- Case fatality is higher
- Disease begins with fever, headache and myalgia followed by rapidly
progressive pulmonary edema often leading to severe respiratory
compromise
- No signs of hemorrhage
- Hantaviral antigens are detected in endothelial cells and macrophage in
lungs, heart, spleen and lymph nodes
- Pathogenesis of HPS involves functional impairment of the vascular
endothelium
- Person to person transmission
- Laboratory diagnosis depends on :
detection of viral nucleic acid by RT-PCR
detection of viral antigens in fixed tissues by immunohistochemistry
detection of specific antibodies using recombinant proteins
- ELISA test to detect IgM antibodies may be used to diagnose acute
infections
- Fourfold rise in IgG titer between acute and convalescent sera is diagnostic
- IgG long lasting
- Isolation of Hantavirus is difficult and requires the use of containment
facilities
- Current therapy: Maintenance of adequate oxygenation and support of
hemodynamic functioning
- Ribavirin antiviral drug is of some benefit as therapy in HPS
- Prevention:
Rodent control
 Avoidance of contact with rodents and rodent droppings
Care must be taken to avoid inhaling aerosolized dried excreta when
cleaning rodent-infested structures

ARENAVIRUS DISEASES
typified by pleomorphic particles
surrounded by an envelope with large, club-shaped peplomers;
50300 nm in diameter (mean, 110130 nm)
Arenaviruses are divided into
Old World viruses (eg, Lassa virus) and New World viruses
establish chronic infections in rodents
each virus is generally associated with a single rodent species

Humans are infected when they come in contact with rodent excreta.

Lassa Fever

first recognized cases of Lassa fever occurred in 1969 among Americans stationed in
the Nigerian village of Lassa.

highly virulentthe mortality rate is about 15% for patients hospitalized with Lassa
fever.

Overall, about 1% of Lassa virus infections are fatal.

Lassa virus is active in all western African countries situated between Senegal and
Republic of Congo.

incubation period: 13 weeks from time of exposure

disease is characterized by very high fever, mouth ulcers, severe muscle aches, skin
rash with hemorrhages,
pneumonia, and heart and kidney damage.

Deafness is a common complication, affecting about 25% of patients during recovery;

hearing loss is often permanent.

Lassa virus infections cause fetal death in more than 75% of pregnant women. During
the third trimester, maternal
mortality is increased (30%), and fetal mortality is very high (>90%). Benign febrile
cases do occur.

Diagnosis :

detection of IgM and IgG antibodies by ELISA

.
Immunohistochemistry can be used to detect viral antigens in postmortem tissue
specimens.

Viral sequences can be detected using RT-PCR assays in research laboratories

House rat (Mastomys natalensis) - principal rodent reservoir of Lassa virus.

>virus can be transmitted by human-to-human contact.

Ribavirin is the drug of choice for Lassa fever

No vaccine exists, although a vaccinia virus recombinant that expresses the

glycoprotein gene of Lassa virus is able to induce protective immunity both in guinea
pigs and in monkeys.

South American Hemorrhagic Fevers

considered to be members of the Tacaribe complex

serious human pathogens are the closely related Junin, Machupo, Guanarito, and Sabia
viruses
Bleeding is more common in Argentine (Junin) and other South American hemorrhagic
fevers than in Lassa fever.

Junin hemorrhagic fever (Argentine hemorrhagic fever)

major public health problem in certain agricultural areas of Argentina; disease has a
marked seasonal variation
exclusively among workers inmaize and wheat fields who are exposed to the
reservoir rodent, Calomys musculinus
produces both humoral and cell-mediated immunodepression
effective live-attenuated Junin virus vaccine is used to vaccinate high-risk
individuals in South America.

Machupo hemorrhagic fever (Bolivian hemorrhagic fever)

first outbreak was identified in Bolivia in 1962.

Calomys callosus, host of Machupo virus

Guanarito virus (the agent of Venezuelan hemorrhagic fever)

Mosquito is the answer
identified in 1990
mortality rate of about 33%
emergence was tied to clearance of forest land for small farm use

Sabia virus was isolated in 1990 from a fatal

case of hemorrhagic fever in Brazil.

Both Guanarito virus and Sabia virus induce a clinical disease resembling that of
Argentine hemorrhagic fever and probably have similar mortality rates

Lymphocytic Choriomeningitis (LCM) virus

Discovered in 1933 and is widespread in Europe and the Americas

natural vector: wild house mouse, Mus musculus

5%of mice throughout the United States carry the virus

occasionally transmitted to humans, presumably via mouse droppings

LCM in humans: acute disease manifested by aseptic meningitis or a mild systemic

influenza-like illness.

incubation period is usually 12 weeks, and the illness lasts

13 weeks.

can be transmitted vertically from mother to fetus, and infection of the fetus early in
pregnancy can lead to serious defects

Infections are usually diagnosed retrospectively by serology using ELISA for IgM
and IgG antibodies

Other diagnostic approaches: immunohistochemical staining of tissues for viral

antigens, RT-PCR for viral nucleic acid, and viral culture using Vero cells.

FILOVIRUS DISEASES

Classification and Properties of Filoviruses

Filoviruses

pleomorphic particles, appearing as long filamentous threads or as odd-shaped

forms

diameter: 80nm

two known filoviruses: Marburg virus (665 nm) and Ebola virus (805 nm) - are
antigenically distinct and are classified in separate genera

large filovirus genome - single-stranded, nonsegmented, negative-sense RNA 19 kb in

size and contains seven
genes

unusual coding strategy with the Ebola viruses: unusual coding strategy with the
Ebola viruses and requires transcriptional editing or translational frame-shifting to be
expressed. > Virions are released via budding from the
plasma membrane.

Filoviruses are highly virulent and require maximum containment facilities (Biosafety
Level 4) for laboratory work.
infectivity is destroyed by heating for 30 minutes at 60C, by ultraviolet and -
irradiation, by lipid solvents, and by bleach and phenolic disinfectants.
Natural hosts and vectors, if any, are unknown but are suspected to be bats or possibly
rodents

African Hemorrhagic Fevers (Marburg and Ebola Viruses)

highly virulent in humans and nonhuman primates, with infections usually ending in
death

incubation period : 39 days for Marburg disease ; 221 days for Ebola

cause similar acute diseases characterized by fever, headache, sore throat, and muscle
pain followed by abdominal pain, vomiting, diarrhea, and rash, with both internal and
external bleeding, often leading to shock and death

Filoviruses have a tropism for cells of the macrophage system, dendritic cells, interstitial
fibroblasts,and endothelial cells

Highest mortality rates (2590%) of all the viral hemorrhagic fevers

Marburg virus disease

recognized in 1967 among laboratory workers exposed to tissues of African green
monkeys
(Cercopithecus aethiops) imported into Germany and Yugoslavia
Marburg virus can infect guinea pigs, mice, hamsters, monkeys, and various cell
culture systems

Ebola virus
discovered in 1976 when two severe epidemics of hemorrhagic fever occurred in
Sudan and Zaire (now the Democratic Republic of the Congo
subtypes of Ebola virus (Zaire, Sudan) are highly virulent
mean time to death from the onset of symptoms is 78 days

In 1989, infections caused by a filovirus closely related to Ebola virus were

detected in cynomolgus monkeys (Macaca fascicularis) imported into the United States
from the Philippines and held in a quarantine facility in Virginia. None of the workers
became sick, indicating that the virus (Reston strain) possesses low pathogenicity for
humans.
high mortality rate among pigs in the Philippines in 2008 led to the
discovery of Ebola Reston virus in animals other than primates.

A genetically distinct Ebola-like virus was described in 2011 in dead

insectivorous bats in Europe (Spain)

Filovirus infections appear to be immunosuppressive

Fatal cases often show impaired humoral immune responses

Viral antigens in serum can be detected by ELISA, providing a rapid screening test of
human samples.

Fresh virus isolates can be cultured in cell lines such as Vero and MA-104 monkey cell
lines

It is probable that Marburg and Ebola viruses have a reservoir host, perhaps a bat or a
rodent, and become transmitted to humans only accidentally.

Monkeys are not considered to be reservoir hosts because most infected animals die too
rapidly to sustain virus survival.

Human infections are highly communicable to human contacts, generally by direct

contact with blood or body fluids.

Typically, outbreaks of Ebola virus infection are associated with the introduction of virus
into the
community by one infected person followed by dissemination by person-to-person
spread, often within medical facilities.

***Since the natural reservoirs of Marburg and Ebola viruses are still unknown,
no control activities can be
organized. Use of isolation facilities in hospital settings remains the most effective
means of controlling Ebola disease outbreaks.

no specific antiviral therapies available

Treatment is directed at maintaining renal function and electrolyte balance and

combating hemorrhage and shock.

There is no vaccine, but candidate vaccines are under development.