Chair of Microbiology, Virology, and Immunology

Paramyxoviruses: Mumps virus, Measles

virus.

Orthomyxovirus (Influenza) FamilyThe name myxovirus was originally applied to influenza viruses. It meant virus with an affinity for mucins. Now there are 2 main groups – the orthomyxoviruses and the paramyxoviruses

Differences between orthomyxoviruses and paramyxoviruses

Feature Orthomyxoviruses Paramyxoviruses

Viruses and diseases

Influenza A,B,C Mumps, measles, respiratory syncytial, parainfluenza

Genome Single-stranded RNA in 8 pieces, MW 2-4x106

Single-stranded RNA in single piece, MW 5-8x106

Inner ribonucleo-protein helix

9-nm diameter 18-nm diameter

Influenza “La malatia per l’influenza della stella”

(the disease caused by the influence of the stars)

In French: grippe, from French verb “agripper” (clinging)

INFLUENZA

severe respiratory disease 20-50 million respiratory illnesses each year

in the U.S. 30 million visits to physicians, 200,000

hospitalizations20,000 deaths

new influenza virus strains associated with severe pandemics and high mortality.

ORTHOMYXOVIRUSES (INFLUENZA VIRUSES)

Classification

Type A viruses cause the most cases of influenza in humans and undergo mutations more frequently than the other type viruses

Type B viruses are endemic in USA and associated with local epidemics

Type C viruses rarely cause disease

Influenza type Hemagglutinin subtype

Geographic source

A/Sydney/5/97 (H3N2)

Year of isolation

Isolate number Neuraminidase subtype

Orthomyxoviruses. Nomenclature

Human influenza virusInfluenza A/Bangkok/1/79(H3N2)Influenza A/Singapore/1/57(H2N2)Influenza B/Ann Arbor/1/86

Influenza virus A

Orthomyxoviruses: medium-sized, enveloped, (-) sense that vary in shape from spherical to helical. Their genome is segmented into eight pieces

Influenza Virus Structure

Flu Viruses Currently infecting... Humans: H1N1, H1N2, and H3N2 Avian Flu Virus: H5N1

Influenza viral genome (-) ssRNA 8 segments (pieces) One gene per segment

nucleoproteinmatrix proteins NS (nonstructural proteins, that are not incorporated into viral particles) gene encodes two different non-structural proteins subunits of RNA polymerase spikes (about 500)Flu viruses are named by the type of surface proteins Hemagglutinin - trimer (HA)

Helps virus enter cell Type A infects humans, birds and pigs Type A has ~ 20 different sub types

Neuraminidase - tetramer (NA) Helps virus exit cell 9 subtypes

ORTHOMYXOVIRUSES

M1 protein

helical nucleocapsid (RNA plus NP protein)

HA - hemagglutinin

polymerase complex

lipid bilayer membrane

NA - neuraminidase

type A, B, C : NP, M1 protein sub-types: HA or NA protein

Haemagglutinin (HA)Encoded by RNA segment # 4

Can agglutinate red blood cells - hence the nomenclature

Cleavage by host-cell protease is required (resulting in HA1 and HA2) for infection to occur

Hemagglutinin glycoprotein is the viral attachment protein and fusion protein, and it elicits neutralizing, protective antibody responses

Neuraminadase (NA) Encoded by RNA segment # 6

Removes neuraminic (sialic) acid from cell and permits dissemination of viruses

Important in releasing mature virus from cells

Stimulates production of protective antibodies

Subtype Hemagglutinin (N) Neuraminidase (H)Human Swine Horse Bird Human Swine Horse Bird

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Influenzavirus BVirions envelopedAbout 500 spikesNucleocapsid enclosed

within lipoprotein membrane

Virions contain 8 segments of linear negative-sense single stranded RNA

Total genome length is 13588 nt

The largest segment 2341 nt

Infect much man and birds. Cause human disease but generally not as severe as A types. Believed to be epidemiologically important - reassortment with type A leads to epidemics.

Influenzavirus C Virions enveloped Many spikes Nucleocapsid enclosed

within lipoprotein membrane

Virions contain 7 segments of linear negative-sense single stranded RNA

Total genome length is 12900 nt

Glycoprotein -hemagglutinin esterase fu

sion (HEF) esterase -> receptor

destroying enzyme

Antigen

Soluble antigens: include ribonucleoprotein and M protein which are much stable in antigenicity.

Surface antigens: include HA and NA which are much variable in antigenicity.

Influenza viruses are divided into 3 groups determined by the ribonucleoprotein (RNP) antigen and M antigen

TYPE A

++++

yes

yes

yes

shift, drift

yes

sensitive

sensitive

2

severity of illness

animal reservoir

human pandemics

human epidemics

antigenic changes

segmented genome

amantadine, rimantidine

zanamivir

surface glycoproteins

TYPE B

++

no

no

yes

drift

yes

no effect

sensitive

2

TYPE C

+

no

no

no (sporadic)

drift

yes

no effect

(1)

Features of viral genera

Animal Susceptibility and Growth of Virus

Human strains of the virus can infect different animals; ferrets are most susceptible. Serial passage in mice increases its virulence, producing extensive pulmonary consolidation and death

The developing chick embryo readily supports the growth of virus, but there are no gross lesions.

Insert figure 25.1Influenza cycle

Influenza virus reproduction

1. Attachment to the epithalial cells of the host through hemagglutinin.

2. Endocytosis3. Uncoating - > This exposes the contents of the virus to the

cytosol. 4.The RNA enter the nucleus of the cell where fresh copies are

made. 5. These copies return to the cytosol where some serve as mRNA

molecules to be translated into the proteins of fresh virus particles. 6. Progeny virions are formed and released by budding from the

plasma membrane of the cell (aided by the neuraminidase) thus spreading the infection to new cells.

Single-cell reproductive cycle

Need to make mRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

AAA(+ve) sense mRNA

(-ve) sense genomic RNA

Need to make mRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

RNA polymerase must bepackaged in virion.

AAA(+ve) sense mRNA

(-ve) sense genomic RNAIf used, RNA modifying enzymes arepackaged in virion.

Influenza Type A Viruses: antigenic Shift 1889-1977

Year Subtype Common Name Type of variation

1889 H2N2

1900 H3N8

1918-1957 H1N1 Spanish flu antigenic drift

1957 H2N2 Asian flu antigenic shift

1957-1968 H2N2 Asian flu antigenic drift

1968 H3N2 Hong Kong flu antigenic shift

1968-1990 H3N2 Hong Kong flu antigenic drift

1977-1989 H1N1 Russian flu reappearance of viruses from 1918,1950

antigenic drift

Ukraine to-day А “Brisben-1“(H1N1), “Brisben-2“(H1N2), “Brisben- 1007" (Н3N2), B “Florida”

1918 Influenza epidemic

> 20 million died of the flu during WW I

A new influenza vaccine must be developed yearly

HumanHuman virusvirus

ReassortantReassortantvirusvirus

Non-humanNon-humanvirusvirus

Mechanisms of Influenza Virus Antigenic “Shift”Mechanisms of Influenza Virus Antigenic “Shift”

15 HAs15 HAs9 NAs9 NAs

DIRECTDIRECT

Antigenic changes of Influenza A

Viruses can undergo frequent changes due to recombination, reassortment, insertions and point mutationsAntigenic driftAntigenic shift occurs every 8-10 yrsMinor antigenic changes favor persistence of the

viruses in the population and allow recombination that can eventually lead to severe epidemics and/or pandemics

ANTIGENIC DRIFT

GRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN SUBTYPE

GRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN SUBTYPE

H3N2

1968

HONG KONG

H3N2

1968

HONG KONG

H3N2

1975

VICTORIA

H3N2

1975

VICTORIA

H3N2H3N2

19931993

BEJINGBEJING

H3N2H3N2

19931993

BEJINGBEJING

H3N2

2004

FUJIAN

H3N2

2004

FUJIAN

Gradual accumulation of mutations that allow the hemagglutinin to escape neutralizing antibodies Epidemic strains thought to have changes in three or more antigenic sites

Antigenic drift

Antigenic differences can result from changes in one amino acid

Can involve any antigenic protein

Can occurs every year

RNA replication is error prone New HA types are created frequently Requires new vaccine every “season”

Antigenic shift Occurs every 8-10 yrs Major antigenic change of either H or N antigens or both H and N Occurs by gene reassortment after simultaneous infection of a cell with

two different viruses Three different H proteins and 2 major N proteins have evolved

H1N1 Spanish flu H2N2 Avian flu H2N2 Asian flu

NA NA

HA HA

What is an Epidemic? The occurrence of more cases of disease

than expected in a given area or among a specific group of people over a particular period of time*.

The occurrence of more cases of disease than expected in a given area or among a specific group of people over a particular period of time*.

EpidemicEpidemic

What is a Pandemic?An epidemic occurring over a very wide area (several countries or continents) and usually affecting a large proportion of the population.

Examples: Cholera AIDS Pandemic Influenza

PandemicPandemic

Where does influenza come from?Type A constantly circulates in natural

reservoirs Birds are the natural reservoir of all

subtypes of Influenza A viruses Migratory waterfowl Chickens, turkeys, ducks, geese

Humans Pigs Horses Other

Type A constantly circulates in natural reservoirs

Birds are the natural reservoir of all subtypes of Influenza A viruses Migratory waterfowl Chickens, turkeys, ducks, geese

Humans Pigs Horses Other

Why do we not have influenza B pandemics?

so far no shifts have been recorded

no animal reservoir known

Epidemiology Source of infection: patients and

carriers. AEROSOL

100,000 TO 1,000,000 VIRIONS PER DROPLET

Common: large droplets (sneezing, coughing, contact with saliva)

Probably common: contact Direct Fomite

Rare: airborne over long distance 18-72 HR INCUBATION

SYMPTOMSFEVERHEADACHEMYALGIACOUGHRHINITISOCULAR SYMPTOMSCHILLS and/or SWEATS

Infection may be very mild, even asymptomatic, moderate or very severe

Clinical ResponsesAcute Symptoms last one week

Abrupt onset of fever, myalgia, headache and non-productive cough

Fatigue and weakness can last 2-3 weeks.Infected individual predisposed to bacterial

infections – Staphylococcus, Streptococcus, Hempohilus

Other complications - Reyes SyndromeImmunity dependent upon localized anti-viral

secretory IgA ( strain specific)Develop long lasting circulating anti-viral IgG

NORMAL TRACHEAL MUCOSA

3 DAYS POST-INFECTION 7 DAYS POST-INFECTION

Immunity to influenza

Antibody to HA - >protective Antibody to NA - > decrease severeity Serum antibody - > years Secretory antibody - > months

Laboratory DiagnosisVIROLOGICAL

Respiratory secretions (direct aspirate , gargle , nasalwashings)

Virus isolation and growth in embryonated eggs Cell culture in primary monkey kidney or madindarby

canine kidney cells Hemagglutination (inhibition) Hemadsorption (inhibition) IFA/ ELISA Direct immunofluorescence

Serodiagnosis

Four-fold or greater increase in hemagglutination inhibition antibody titers between acute and convalescent specimens Hemagglutination inhibition Hemadsorption inhibition ELISA Complement fixation test NT

Laboratory Diagnosis

Masks and Hand Washing

Hand washing Generally perceived to be useful No studies specifically performed for

influenza Easy to recommend

Masks Effectiveness not shown for influenza However, could reduce transmission

associated with large droplets

Prophylaxis

To be Continued…

Types of Vaccine Killed Whole Virus

inactivated virus vaccine grown in embryonated eggs; 70-90% effective in healthy persons <65 years of age, 30-70% in persons ≥65 years

Live VirusAttenuated strains were widely used in Russia but not elsewhere.

Virus SubunitHA extracted from recombinant virus forms the basis of today's vaccines.

SyntheticMuch research is being done to try and find a neutralising epitope that is more stable, and can therefore be used for a universal vaccine.

Trivalent Influenza virus vaccines 1999-2000 A/Sydney/05/97 (H3N2) A/Beijing/262/95 (H1N1) B/Yamanashi/166/98

2000-2001 A/Moscow/10/99(H3N2)-like A/New Caledonia/20/99 (H1N1)-like B/Beijing/184/93-like

To day A/Brisben/59/2007 (H1N1) A/Brisben/10/2007 (H3N2) B/Florida/4/2006

Prevention and Treatment

RIMANTADINE (blocks the M2 ion channel) (M2)type A only, needs to be given early

AMANTADINE (blocks the M2 ion channel) (M2)type A only, needs to be given early

ZANAMIVIR (neuraminidase inhibitors) (NA)types A and B, needs to be given early

OSELTAMIVIR (neuraminidase inhibitors) (NA) types A and B, needs to be given early

70-90% effective in preventing illness

Antivirals:Adamantanes and Neuraminidase Inhibitors

NA

inh

ibit

ors

HA

blo

cke

rs

Avian Influenza Poultry Outbreaks, Asia, 2003-06

Hong Kong 1997: 18 cases of influenza in humans caused by a highly pathogenic avian influenza virus (H5N1); 30% fatality rate

Has spread from E Asia, SE Asia and Pacific to Eurasia, Near East, Europe and to Africa H5N1 has been identified in migratory water birds and /or

poultry in 55 countries as of 5/29/06 H9N2 subtype also detected among infected poultry It has infected humans in 10 countries.

Hong Kong 1997: 18 cases of influenza in humans caused by a highly pathogenic avian influenza virus (H5N1); 30% fatality rate

Has spread from E Asia, SE Asia and Pacific to Eurasia, Near East, Europe and to Africa H5N1 has been identified in migratory water birds and /or

poultry in 55 countries as of 5/29/06 H9N2 subtype also detected among infected poultry It has infected humans in 10 countries.

In the future: reassortment between H9N2 or H5N1 avian viruses and H1N1 or H3N2 human viruses???

Avian Flu Avian influenza, or “bird flu”,

is a contagious disease of animals caused by viruses that normally infect only birds and, less commonly, pigs. Avian influenza viruses are highly species-specific, but have, on rare occasions, crossed the species barrier to infect humans.

Pandemic viruses appear as the result of antigenic shift, which causes new combinations of proteins on the surface of the virus. If the new virus spreads easily from person to person a pandemic can result.

1. Nonpathogenic H5 influenza virus: Wild fowl domestic ducks and geese, domestic chickens.

2. H5 virus became highly pathogenic in chickens domestic ducks and geese. 3. Highly Pathogenic H5 virus reassorted its genome with those of other influenza viruses in

aquatic birds, spread to poultry farms, humans, and occasionally to pigs.

Emergence of New Influenza Subtypes: H5N1Antigenic shift due to genome reassortment within intermediate hosts drives flu epidemics and pandemics

Solid lines: transmission demonstrated; Dotted lines: transmission postulated but not demonstrated.

Avian influenza

Wild birds are the reservoir. Circulation of low pathogenic avian flu in

domestic poultry leads to mutations to highly pathogenic forms over time.

Co-infection with swine or humans infected with human influenza can result in genetic reassortment and highly pathogenic strains.

Avian influenza

Why do new strains of influenza and bird flu arise in Asia?

In 2003, an outbreak of “chicken flu” necessitated killing tens of millions of birds

H5N1 in wild birds, poultry & humans – 5/19/06

Family Paramyxoviridae Subfamily Paramyxovirinae:

Genera: Morbillivirus – measles virus, Respirovirus (earlier Paramyxovirus) – parainfluenza virus serotypes 1 and 3 Rubulavirus - parainfluenza virus serotypes 2, 4а, 4b, mumps virus

Henivirus – Australian Hendra-virus (diseases of human and horses), Malasian Nipah-virus (diseases of

human and swine)

Subfamily Pneumovirinae Genera: Pneumovirus – RS-virus Metapneumovirus – human metapmeumovirus (diseases in children)

PARAMYXOVIRUS FAMILYPARAMYXOVIRUS FAMILYproperties of attachment proteinproperties of attachment protein

GENUS GLYCOPROTEINS TYPICAL MEMBERS

Paramyxovirus genus

HN, F HPIV1, HPIV3

Rubulavirus Genus HN, F HPIV2, HPIV4 mumps virus

Morbillivirus genus H, F measles virus

Pneumovirus genus G, F respiratory syncytial virus

Virion Large virion consists of a

negative RNA genome in a helical nucleocapsid surrounded by an enevlope containing a viral attachment protein

HN of paramyxovirus and mumps virus has hemagglutinin and neuraminidase.

H of measles virus has hemagglutinin activity

G of RSV lacks these activities

M protein

helical nucleocapsid (RNA minusNP protein)

HN/H/G glycoprotein SPIKES

polymerase complex

lipid bilayer membrane

F glycoprotein SPIKES

PARAMYXOVIRUSESpleomorphic

Insert figure 25.5Effects of paramyxoviruses

MUMPS (Epidemic Parotitis)Mumps is an acute contagious disease characterized by a nonsuppurative enlargement of one or both of the parotid glands, although other organs may also be involved.

Properties of the Virus

Mumps virus is a typical paramyxovirus.

It has typical hemagglutination, neuraminidase, and hemolysin activities. Hemagglutination can be inhibited by specific antisera to mumps virus, and this inhibition can be used to measure antibody responses. Similarly, the nucleocapsid of the virus particle forms the major component of the "S" (soluble) complement-fixing antigen.

Mumps virus Droplets spread the infection via saliva and secretions from

the respiratory tract. Incubation period of 2-3 weeks Malaise and fever is followed within a day by painful

enlargement of one or both of the parotid (salivary) glands A possible complication in males after puberty is orchitis -

painful swelling of one or both testicles. Inflammation of the ovary and pancreas can also occur. Disease is usually self-limiting within a few days Aseptic meningitis (usually resolving without problems) or

postexposure encephalitis (can prove fatal) are the most serious complications associated with mumps.

Mumps pathogenesis

Epidemic parotitisOrchitis

Mumps virus CPE

Prevention and treatment

Treatment: none (passive immunization has been used).

Prevention: one invariant serotype therefore vaccines are viable - both formalin-inactivated and live attenuated exist, the latter now being widely used- see MMR.

PARAINFLUENZA VIRUS INFECTIONS

The parainfluenza viruses are paramyxoviruses with morphologic and biologic properties typical of the genus. They grow welt in primary monkey or human epithelial cell culture but poorly or not at all in the embryonated egg. They produce a minimal cytopathic effect in cell culture but are recognized by the hemadsorption method. Laboratory diagnosis may be made by the HI, CF, and Nt tests.

Important Characteristics

Typing: Four types (1-4) : distinguished antigenically, by cytopathic effect, and pathogenically

Haemagglutinin and fusion F protein is found in the envelope

Pathogenesis and Immunity Cause acute respiratory

infections of man ranging from relatively mild influenza-like illness to bronchitis, croup (narrowing of airways which can result in respiratory distress) and pneumonia; common infection of children.

Transmitted by aerosols.

Lab Diagnosis

Nasopharynx specimen is culture in a surrogate cell line in AGMK. Infected cell are detected by hemeadsorption or DFA

DFA also can be done rapidly to identify the agent in direct specimen

Serotypes 1-3 are comfirmed by hemeagglutination inhibition using standardized antisera

MEASLES (Rubeola)

Measles is an acute, highly infectious disease characterized by a maculopapular rash, fever, and respiratory symptoms.

Properties of the Virus. Measles virus is a typical paramyxovirus, related to canine distemper and bovine rinderpest. All 3 lack neuraminidase activity. Measles agglutinates monkey erythrocytes at 37 °C but does not elute, and it interacts with a distinct cell receptor. Measles virus also causes hemolysis, and this activity can be separated from that of the hemagglutinin. In culture, produces characteristic intranuclear inclusion bodies and syncytial giant cells.

Pathogenesis and Immunity Childhood infection almost universal, protection resulting from this is probably

lifelong. Both man and wild monkeys are commonly infected Transmission and initial stages of disease similar to mumps, but this virus can

also infect via the eye and multiply in the conjunctivae. Viraemia following primary local multiplication results in widespread distribution to many organs.

After a 10-12 day incubation period Disease: Fever, Respiratory tract syndrom (dry cough, rhinorrhea, sore throat) conjunctivitis (virus may be excreted during this phase!), followed a few days

later by the characteristic red, maculopapular rash, Koplik's spots Towards the end of the disease, there is extensive, generalized virus infection

in lymphoid tissues and skin

Measles

most serious complication is subacute sclerosing panencephalitis (SSPE), a progressive neurological degeneration of the cerebral cortex, white matter and brain stem

1 case in a million infections involves a defective virus spreading through the

brain by cell fusion and destroys cellsmental disorders

leads to coma and death in months or years attenuated viral vaccine MMR

Subacute sclerosing panencephalitis

Prevention

Both live and killed vaccines exist. Vaccination with the live attenuated vaccine has been practised since the 1960's with a dramatic decline in the incidence of the disease .

Trivalent live attenuated vaccine (MMR) usually given - all of these viruses best avoided during pregnancy!

The particle is slightly smaller (80-120 nm) than other paramyxoviruses, and the nucleocapsid measures 11-15 nm. Although RS is one of the most labile of viruses, it can be stabilized by molar MgSC>4 (like measles and other paramyxoviruses). RS virus does not hemagglutinate.

A soluble complement-fixing antigen can be separated from the virus particle.

This labile paramyxovirus produces a characteristic syncytial effect, the fusion of cells in human cell culture. It is the single most serious cause of bronchiolitis and pneumonitis in infants.

RESPIRATORY SYNCYTIAL (RS) VIRUS

Important Characteristics

RSV is highly infectious, transmission by respiratory secretions.

Primary multiplication occurs in epithelial cells of URT producing a mild illness. In ~50% children less than 8 months old, virus subsequently spreads into the L.R.T. causing bronchitis, pneumonia and croup.

Has been suggested as a possible factor in cot death and asthma.

RSV- syncytium formation

Immunofluoresent stain

RSV is a viral disease. Respiratory Syncytial

Virus (RSV)is a very serious virus often found in children and infants under the age of three.

Adults are at very low risk of catching RSV.

RSV Bronchiolitis - clinical features

This disease is transmitted by: coughing sneezing sharing wash cloths towels and other things

with someone with RSV. Most people with RSV get it in fall and winter.

Babies and elders are most at risk of catching RSV.

Although adults do catch it, it appears to them as being a common cold

This disease is extremely serious when it comes to children and infants under the age of 3 and elders.

This disease can result in death.

Symptoms for this disease are: sneezing runny nose sore throat low fever common cold symptoms just

more severe.

The main thing a doctor will tell you if you ask about treatments for RSV he will most likely say to let it run it’s course.Things you can do for the person that has RSV are:

comfort that person. Things you can do if you have it are: Drink plenty of fluids. Get lots of restAntiviral Agents Ribavirin (Virazole), a synthetic guanosine analogue, given as an

aerosolSupportive Fluids, oxygen, respiratory support, bronchodilators.

Wash hands before you eat and after you use the bathroom. Don’t share towels or wash cloths with others. Eat healthy. Keep clean. Stay clear of people who are ill Disinfection of surfaces Gloves, masks, goggles, gowns Isolation, and cohort nursing Immunization Active Immunization Formalin inactivated vaccine resulted in enhanced disease Subunit vaccines being studied

Passive immunization (immunoprophylaxis) Pooled hyperimmune globulin (RespiGam) Monoclonal antibody to F protein- Palivizumab (Synagis)