• Dengue viruses (DENV) is a mosquito borne viruses, belongs to the family Flaviviridae, the genus Flavivirus.
• Dengue viruses consist of five serotype named dengue virus types 1, 2, 3, 4 and 5 (DENV-1, -2, -3, -4 and -5), that are responsible for diseases.
• These serotypes are closely related but antigenically distinct.
• DENV-5 is a new serotype which has recently been described from Malaysia.
• Hotta and Kimura were the first to isolate the virus in 1943, by intracranial inoculation of serum from an acutely ill patient into suckling mice.

Morphology of Dengue virus:

• Dengue virus is spherical enveloped viruses with single-protein capsids, two membrane proteins, envelope and membrane.
• It has a single-stranded Positive-sense RNA genome of ~10,700 nucleotides, surrounded by a nucleocapsid and covered by a lipid envelope that contains the viral glycoproteins.
• The RNA genome lacks poly A tail at 3’end and contains a single open reading frame (ORF) flanked by two untranslated regions (5’ and 3’UTRs).
• The 5’ and 3’ terminal RNA sequences of the genome form large stem loop structures known as stem loop A (SLA) and 3’ stem loop (3’ SL) respectively, both essential for viral replication.
• The single ORF encodes a precursor polyprotein, which is co- and post-translational cleavage resulting in the formation of three structural proteins, Capsid (C), membrane (M), and envelope (E), and seven non-structural proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5. NS2B and NS3 form the proteases, and NS3 also has helicase and RNA triphosphate activity; NS5 is the RNA dependent RNA polymerase and also has methyltransferase activity.
• It has four serotypes, DEN-1, DEN- 2, DEN-3 and DEN-4, which are capable of causing dengue fever (DF).

Pathology and clinical features of Dengue:

• Dengue is an acute infectious vector borne disease caused by dengue viruses and transmitted to human by the Aedes species, primarily by Aedes aegypti.
• The prevalence of dengue viral infection has been estimated that over 2.5 billion people live in the areas of risk with an incidence of 50-100 million cases per year and several thousands of deaths are estimated to occur annually worldwide, resulting in 500,000 cases of DHF/DSS and 25,000 deaths.
• Dengue virus induces clinical illness ranging from asymptomatic or mild febrile illness, i.e., Dengue Fever (DF) to severe disease forms, i.e., Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).
• Infection with any one of the four DENV serotypes has the potential to involve all human organ systems and cause a wide variety of clinical manifestations ranging from mild febrile illness to severe and fatal disease.
• Symptomatic dengue disease is separated into two different clinical syndromes, dengue fever (DF), and the more severe dengue hemorrhagic fever (DHF); DF was described as a nonspecific febrile illness with prominent constitutional symptoms, while DHF was defined as a distinct syndrome characterized by increased vascular permeability, altered hemostasis and hemorrhage. DF is also referred to as the ‘break bone’ disease due to its nature of severe joint and muscular pain.
• Following an infectious mosquito bite there is an incubation period of up to 2 weeks (commonly 5-7 days), after which the individual develops symptoms suddenly and the illness typically follows three phases- an initial febrile phase, a critical phase starts 4-5 days from fever onset, followed by a spontaneous recovery phase.
  • In febrile phase, patient experiences sudden onset of high fever (39-40°c) accompanied by nonspecific constitutional symptoms including headache, general malaise, nausea, vomiting, myalgia, and joint pain.
  • In recovery phase, the increased vascular permeability and abnormal hemostasis are transient and usually resolve within 48-72 hours. Spontaneous reabsorption of fluid starts around 6-8 day of illness and progress rapidly, usually concurrent with improvement in the patient’s symptoms. Loss of hair has been reported during convalescence.

Primary infection:

• Primary infection with dengue virus results in a self-limiting disease characterized by mild to high fever lasting 3 to 7 days, severe headache with pain behind the eyes, muscle and joint pain, and a rash.
• Complications often occur within two days after the fall in temperature. Epistaxis, bleeding of gums, passage of black stools, rashes (petechiae, maculopapular, bruises, etc.) and sub-conjunctival hemorrhage are indicators of increasing disease severity.
• A maculopapular rash usually appears 3 – 4 days after the onset of fever. Secondary infection with a different dengue virus serotype is the more common form of the disease in many parts of Southeast Asia and South America.
• The major clinical symptoms can include high fever, hemorrhagic events, and circulatory failure, and the fatality rate can be as high as 30%. Early diagnosis of dengue shock syndrome is particularly important, as patients may die within 12 to 24 hr if appropriate treatment is not administered.

The major host factors that influence clinical manifestations are:
• Age: constitutional symptoms become more prominent with increasing age, that adults complain of headache, retro-orbital pain, and severe myalgia and arthralgia more frequently than children.
• Gender: Females have a lower threshold for vascular leakage than males. Although dengue is diagnosed more frequently in male than female patients, female patients have a higher risk of developing DSS and of dying from this complication than male patients [34].
• Pregnancy and transplacental infection: With the increasing burden of dengue seen among young adults, exposure to infection during pregnancy is becoming more frequent.

• Clinical findings alone are not sufficient to make an accurate diagnosis of DENV as several other infectious diseases may present with similar findings, which requires the need for laboratory testing for the dengue confirmation.
• Leucopenia, thrombocytopenia, increased hematocrit and liver enzyme levels are common laboratory findings suggestive of DENV infection.
• Serological tests, such as rapid diagnostic test (RDT), Enzyme-linked immunosorbent assay (ELISA) are widely used.
• In Nepal, serological testing has been used as an important tool for diagnosis of suspected DENV infection during outbreaks.

The post Dengue: Introduction appeared first on Online Biology Notes.

• Rabies virus is also known as street virus.
• It causes rabies which is an acute infection of CNS and is always fatal in untreated cases.
• The virus is transmitted to human from the bite of rabid animals especially from the saliva.

Properties of Rabies virus:

• It is a Rhabdovirus which is large rod or bullet shaped.
• It belongs to Rhabdoviridae family and lyssa virus genus.
• Shape and size:
  • These are bullet shaped virus measuring 7 X 180nm diameter (50-95 nm diameter, 130-139nm length).
  • The virion is composed of RNA, protein, lipid, and carbohydrate.
• Genome:
  • The viral genome consists of non-segmented -ve sense single stranded RNA.
• Nucleocapsid:
  • The nucleocapsid is spiral or helical composed of RNA, phosphorylated nucleoprotein and large RNA dependent RNA polymerase I.
• Envelope:
  • The lipid bilayer envelope derived from the host cell membrane contains virus encoded glycoprotein (G-spikes).
  • These glycoproteins are found to bind specifically to cellular receptor and confirm neurotrophism in infected cells.
  • These are the major factors for neuro-invasiveness and pathogenicity on the inner surface of viral envelope, closely associated with G-protein is a second membrane known as matrix protein which is thought to play role in viral budding.

Life cycle of Rabies virus

• Virus replication can broadly be categorized into following:
  • Attachment, penetration and uncoating
  • Transcription, translation and replication of viral genome.
  • Maturation and release

Step I: Attachment, penetration and uncoating:

• Rabies virus attaches to cell surface via glycoprotein.
• Attachment of virus by glycoprotein takes place at neuromuscular junction possessing acetylcholine receptor, phosphatidyl serine receptor, neuronal cell adhesion molecule or P75 neurotropin receptor.
• After the attachment virus enters the host cell by endocytosis and fuses with the endosome thereby releasing ribonucleoprotein complex (RNP) into the cytoplasm.
• The fusion of virus with endosome takes place as the pH decreases below 6.2

Step II: Transcription, translation and reproduction:

• The negative sense ssRNA is transcribed by virion associated RNA polymerase to positive sense mRNA which codes for structural protein and polymerase complex.
• The positive sense RNA serves as template for the synthesis of viral genome and +mRNA that gives rise to different viral proteins.

Step III: Maturation and release by budding:

• Virus matures by budding from the cytoplasmic membrane.
• In this process the newly replicated RNA associates with viral polymerase protein to form coiled and condensed RNP core in the cytoplasm.
• RNP then assembles with matrix protein of cell surface and finally acquires the envelope by budding through the cell surface.

Pathogenesis of Rabies:

• Rabies virus is excreted in saliva of rabid animal so, human acquire virus by the bite of rabid animals.
• Virus multiplies in muscle or connective tissue at the site of inoculation and then enter peripheral nerves via motor end plates at neuromuscular junction and spread upto the CNS.
• Virus, however, can enter directly into CNS without local multiplication.
• It multiplies in the grey matter in brain and propagates through efferent nerves to salivary gland and other tissues like kidney, heart, cornea, retina, pancreas.
• The highest titre of virus is seen in submaxillary gland.
• Rabies virus produces a specific eosinophilic cytoplasmic inclusion with basophilic granules called the Negri bodies.
• The Negri body is filled with viral nucleocapsid and forms the pathognomonic basis of rabies diagnosis.
• The absence of negri body however doesn’t rule out the absence of rabies virus.
• Susceptibility to rabies infection and incubation period may depend on host age, genetic makeup, immunity, viral strain involved, inoculum size and the distance virus has to travel from the point of entry to CNS.

Clinical manifestation of rabies:

• Incubation period in humans is 1-3 months but may be as short as 1 weeks to as long as many years.
• It is usually shorter in children than in adults.
• The clinical spectrum of rabies can be divided into three phases.

1. Prodomal phase:

• It lasts for 2-10days and is characterized by any of the following non-specific symptoms, malaise, anorexia, headache, photophobia, nausea and vomiting.
• Unusual sensation at the wound site is also observed.

2. Acute neurological phase:

• It lasts for 2-7 days and is characterized by the presence of nervous system disorder like nervousness, apprehension, hallucination, lacrimation, pupillary dilation, and increased salivation, hydrophobia and painful throat muscle while swabbing.

3. Coma and death:

• Neurological phase is followed by coma and death.
• Major cause of death is respiratory paralysis.

Lab diagnosis of Rabies:

Specimens:

• Acute mortem – hair follicles, saliva
• Post mortem- salivary gland, brain stem, hippocampus, cerebellum.
• Rabies virus can cause severe disease and is dangerous for many person in contact.
• Because of this risk from contact and handling, The British Advisory Community on danger pathogen together with WHO has classified Rabies virus in hazard group III pathogen and should be handled in biosafety level 2.
• In Rabies endemic area animals captured should be sent for laboratory confirmation of Rabies but without any delay post exposure treatment of the bitten person should be done.
• Domesticated dogs and cats, particularly if previously vaccinated against Rabies should be observed in isolation for upto 10-14 days.
• If they survive for that time, it is unlikely they were incubating rabies virus at the time of incident.
• If they succumb or die anti-rabies treatment of bitten person should be strated.

Microscopy/ Histological examination:

• This involves the examination of tissue infected with rabies virus rapidly and accurately using direct immunofluorescence which uses anti-rabies monoclonal antibodies.
• Impression preparation of brain and cornea tissue is often used.
• Similarly, a definite pathological diagnosis is based on the finding of Negri bodies in the brain or spinal cord.
• Negri bodies are found in impression preparation or histological section.
• These are sharply demarcated more or less spherical and 2-10 μm in diameter, consisting basophilic granules yield in an eosinophilic matrix.
• Negri bodies are infiltrated with viral antigens and can be demonstrated by immune-fluorescence technique.

References:

1. https://www.cdc.gov/rabies/about.html

Rabies virus: Properties, life cycle, pathogenesis, diseases and diagnosis

The post Rabies virus: Properties, life cycle, pathogenesis, diseases and diagnosis appeared first on Online Biology Notes.

• Cytomegalovirus (CMV) is a genus of viruses belonging to the order Herpesvirales, in the family Herpesviridae.
• CMV is the largest member of the human herpes virus family.Its natural host is human and monkeys.
• There is little genetic homology between human CMV and CMV of other species.
• Cytomegalovirus is a common virus infecting people of all ages rarely causing any clear illness.
• Out of 150 children, 1 is born with congenital cytomegalovirus infection.
• 80% of adults get infected by CMV before 40 years of age.

Structure:

• The complete virion is 150 to 200 nm in diameter and icosahedral in shape and consists of an inner core, a capsid, and an envelope.
• The inner core of the virus is 64-nm which encloses linear double-stranded DNA molecule.
• The capsid is 110 nm in diameter, composed of 162 capsomers units.  
• The envelope contains lipoproteins and at least 33 structural proteins, some of which are glycosylated.
• The glycoprotein determine the strain of CMV, are used for cellular entry of the virus, and are the targets of virus-neutralizing antibody.
• CMV has genome of 236 kbp and more than 200 open reading frames (ORFs) encoding more than 80 viral proteins, including glycoproteins, phosphor-proteins and other transcription/replication proteins
• Genome analysis has indicated that mammalian CMV have co-speciated with their respective hosts over the last 80 million years. This prolonged period of co-evolution has resulted in a high level of co-adaptation between the virus and its host

CMV replication cycle

• Replication of CMV, after the virus penetrates the cell occurs in three relatively distinct phases:

i. Immediate-early (IE) phase:

• This phase begins with the immediate early transcription of the IE (alpha) genes during the first 4 hours after viral entry.
• This immediate early transcription event is dependent only on cellular factors and does not require de novo viral protein synthesis.
• Non-structural proteins appear in the nucleus within 4 hours after infection. These proteins are essential for the regulation of the expression of the early- and late-phase genes, and also for manipulating various cellular processes.

ii. Early (E) phase:

• In the early phase, the transcription of the E (beta) genes occurs, which depends upon the transcriptional product of IE gene.
• In this phase all the essential protein required for virus replication are produced, including DNA polymerase and helicase-primase.

Late (L) phase:

• This phase include transcription of L (gamma) genes which occurs approximately 24 hours after infection. The transcriptional products includes structural and maturation proteins.
• Assembly and packaging of structural proteins occurs in the nucleus and finally maturation in Golgi-derived vacuole from where the virus release.
• The replication continues for several days until cell lysis occurs.

Mode of transmission:

• CMV is one of the most successful human pathogens, since it can be transmitted both vertically and horizontally
• Infection takes place through both sexual and nonsexual contact.
• CMV infection can be transmitted through saliva, urine, stool or breast milk as well.
• It can also spread through body fluids-semen or vaginal fluids.
• It also gets transmitted during blood transfusion if the blood transfused is infected and during infected organ transplant.
• Pregnant infected women directly pass CMV to their unborn child.

Pathogenesis:

• CMV is a complex virus that appear to employ multiple strategies to evade the host immune system.
• When CMV enters the human body, it infects and penetrate virtually all types of cells, including monocytes, macrophages, neutrophils, neurons and hepatocytes.
• CMV also infect epithelial and endothelial cells it occurs through endocytosis.
• Primary CMV infection usually occurs during the first decades of life.
• Primary infection is followed by a latent infection that can persist throughout the life of the host.
• The primary infection results in the most severe disease especially when the host immunity is compromised.
• During latency, CMV cannot be eliminated by host defence but the immune system keeps the virus under close surveillance, giving it little chance to reactivate and cause symptomatic disease
• Reactivation of CMV infection from latency occurs in conditions, such as allograft rejection, sepsis, administration of Anti-leucocyte antibody (ALA) therapies. These clinical immune compromising conditions result in the release of cytokines and other pro-inflammatory mediators that play a role in the reactivation of virus from latency.
• Tumor necrosis factor (TNF)-α is the primary cytokine responsible for reactivation of CMV from latency. TNF-α binds to the TNF receptor on latently infected cells and activates protein kinase C and nuclear factor κB (NF-κB). In turn, NF-κB acts on the immediate early promotor of the virus to activate virus replication.
• If the host cellular immune response is functioning properly, virus will be eliminated and host will recover.
• If the host T-cell response is impaired, virus multiplies causing inflammatory reactions.
• If the host is profoundly immune-compromised, virus multiplies exclusively causing tissue invasive disease and possibly death.

Host immunity

• The innate immune responses are the first line of defence against CMV. It is primarlly responsible for host defence during perinatal period, because adaptive immune response is well developed.
• The acquired immune response to CMV includes both humoral and cell mediated immunity. After development of acquired immune response, CMV enters latency.
• Humoral immune response leads to activation of B cell and production of antibodies-IgM, IgG, and IgA. But these antibodies do not offer protection against CMV because the virus replicates intracellularly.
• Cellular immune response is mediated by CMV-specific CD4⁺ and CD8⁺ lymphocytes that controls virus replication and provokes long-term protection from CMV disease.

CMV infection and diseases:

• There are 3 classes of CMV infection-

i. Primary or Acquired infection:

• It is the condition where a person gets infected by CMV for the first time. No any prominent symptoms are seen but some individuals show symptoms as such of mononucleosis.
• In most case, the symptoms are not prominent or get unnoticed. But if seen, they include:
  • Fever
  • Night sweats
  • Loss of appetite
  • Weight loss
  • Fatigue
  • Inflammation of glands
  • Pain in muscle and joints
  • Sore throat

ii. Reactivation/ Recurrence infection:

• Once a person gets CMV it stays in their body for lifetime as dormant/latent but during the time of weakened immune system, it can reactivate again. This condition is reactivation of CMV.
• The regions to be likely affected are lungs, eyes and the digestive system
• Symptoms include:
  • Fever
  • Dyspnea (shortness of breath)
  • Pneumonia
  • Mouth ulcers
  • Difficulty in seeing or blurred vision
  • Liver inflammation
  • Hepatitis
  • Gastrointestinal bleedings and ulcerations
  • Diarrhea

iii. Congenital infection:

• Congenital CMV is when the CMV infected pregnant woman transmits it to the unborn child.
• About 90% child don’t show any symptoms but others will suffer from hearing disability during the first six months of time.
• Symptoms include:
  • Jaundice
  • Splenomegaly
  • Enlarged liver
  • Pneumonia
  • Skin rashes or red spots under skin
• 75% of congenital CMV babies may face effect on brain. It may lead to conditions as such:
  • Autism
  • Seizures
  • decreased head size
  • epilepsy
  • Impaired or blurred vision
  • Difficulty in co-ordination
  • Deafness

Complications:

• Complications are seen in few cases depending on the time range of infection and overall health conditions.
• In case of healthy adults, CMV causes mononucleosis rarely.
• Other rare conditions of complications are liver, brain, digestive system and nervous system related disorders.
• In case of people with weak immunity, they might have risk of retinitis, colitis, esophagitis, and hepatitis. Complications can further include encephalitis, pneumonia etc.
• Newborns with congenital CMV can face complications as blurred vision, hearing loss, nervous system related disorders and seizures.

Laboratory diagnosis:

• Specimens:
  • Blood, serum, body fluids, urine, biopsy sample etc
  • In case of newborns, either saliva or urine tests are done.
• Polymerase chain reaction  (PCR):
  • PCR assay has been used virus to detect replicating viruses.
  • PCR assay can provide information about viral load
• Isolation of virus:
  • Human fibroblasts cell are used for virus isolation
• Serology:
  • Different serological assay can be used to detect antibodies against cytomegalovirus.
  • Detection of IgG antibodies indicates past infection and detection of viral IgM antibodies suggests a current infection.

Treatment:

• CMV infections cannot be cured but the symptoms can be controlled through medications.
• Ganciclovir and foscarnet are the antiviral drugs to halt the progress of infection in case of CMV retinitis. These are released intravenously and treatment proceeds for long period of time.
• CMV infections observed during organ transplant can be treated with cytomegalovirus immune globulin intravenous (CMV IGIV). It is intravenous immune globulin rich with antibodies that acts against CMV.
• For individuals with HIV/AIDS, the antiretroviral drugs help to prevent against CMV infections.
  
  

Cytomegalovirus (CMV)- Replication, Transmission, Pathogenesis, Diseases, diagnosis and treatment

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• Hepatitis B is the most widespread and most important type of viral hepatitis.  Hepatitis B virus (HBV) infects the liver and to less extent kidney and pancreas.
• It belongs to Hepadna viridiae family
• Mammalian hepadna virus consists of outermost surface antigen (HBsAg) which has 240 units.
• It is ds DNA virus. However one of the strand is shorter than another strand
• It has circular DNA

Structure of Hepatitis B virus:

• HBV is a small, enveloped DNA virus measuring 42 nm in diameter. It has an outer envelope and an inner core which is 27 nm in diameter enclosing the viral genome and a DNA polymerase.
• Three types of morphological form can be seen;
  • Small 20-22nm circular or spherical form: this is the most abundant form and it comprises of viral surface proteins.
  • 20 x 200 nm filamentous form known as Australia antigen
  • Double walled 42nm spherical form called Dane particle: this particle is the complete hepatitis B virus.
• Envelope consists of at least three membrane spanning proteins;
  • Large Hepatitis B virus surface (LHBS) protein: LHBS consists of S-domain as well as pre-S-domain (pre-S1 and pre-S2), both internally and externally. LHBS is important for infection.
  • Medium hepatitis B virus surface (MHBS) protein: MHBS is made up of S-domain and pre-S2 domain
  •  Small hepatitis B virus (SHBS) protein: SHBS consists of only S-domain. It is the main constituent of virus envelope and is important for virion assembly

Antigen present in Hepatitis B virus:

• There are different types of group specific surface antigen (HBs Ag). The most common group of surface antigen of all Hepatitis B virus is ‘a’ with two other antigenic soft determinants d/y or w/r. combination of these antigens resulting in four different strains- (adw, adr, ayr or ayw). Among these strains adw is most common.
• There are two glycosylation sites resulting in at least 6 different types of surface proteins. They are GP-42, GP-39, GP-36, GP-33, GP-27, and GP-24)
• Surface antigen (HBs Ag) encircles 25-28nm core.
• The core antigen is made up of nucleo-capsid which consists of DNA and capsid (HBc Ag)
• Surface antigen interacts with the core antigen with the help of heat shock protein 70 (hsc70).
• The core encapsidate the viral DNA and the enzyme DNA polymerase.
• DNA polymerase consists of 4-domains
  • Priming domain
  • Tetner (spacer) domain
  • Reverse transcriptase domain to transcribe RNA into DNA
  • RNase H domain whose role is to cleave RNA from DNA_RNA duplex.
• HBx Ag: The function of this protein is still unknown.

Genome:

• Genome is a circular ds DNA composed of 3200 nucleotide. One of the strand (+strand) is incomplete, hence the DNA appears partially ds and partially ss.
• DNA polymerase is associated with + strand which has both DNA dependent DNA polymerase and RNA dependent reverse transcriptase functions.
• Genome of Hepatitis B virus encodes four open reading frame (ORF).
• First ORF is Pre-S-S (pre-surface-surface):
  • This ORF encodes three antigens obtained by differential initiation of translation at three different initiation codons.
  • The three antigens are- HBs Ag, pre S2 (unknown function) and pre S1 (helps in virus attachment, assembly and release)
• Pre-core-core ORF (Pre-C-C):
  • It codes for HBc Ag and HBe Ag. These are also overlapping and obtained by differential mutation of overlapping translation at two AUG codon
  • When C-region is translated, core protein (HBc Ag) is formed and when translation begins from pre-C region to C-region, HBe Ag is formed.  
  • HBe Ag has no role in assembly
• Open reading frame P (ORF-P):
  • It codes for viral DNA polymerase
• Open reading frame X (ORF-X):
  • It codes for HBx Ag which acts as transcriptional trans-activator.

Replication of Hepatitis B virus:

• Hepatitis B virus is para-retro virus. The virus penetrates into host cell by binding to sodium taulocholate cotransprotein (NTTP) receptor.
• The virus penetrates the cell by endocytosis.
• The viral nucleic acid which is released by membrane fusion is then carried to the nucleus of host by chaperons.
• The partially ds DNA is then converted into covalently closed circular DNA (cc-DNA) which is a complete ds circular DNA.
• The viral DNA is then transcribed by RNA polymerase producing 4 mRNA (3.5kbp, 2.4kbp, 2.1kbp and 0.7kbp). One of which is intact and longer than virus DNA itself.
• The longest mRNA encodes for HBc Ag, HBeAg, and polymerase
• The other two mRNA encode for surface glycoproteins and the smallest mRNA (0.7kbp) encodes for X-protein (HBx) which is involved in virus replication and also helps in spread of virus.
• The mRNA are transported to the cytoplasm for translation and the proteins are produced.
• The surface proteins are localized in membrane Endoplasmic reticulum or golgi, from where virus eventually buds out after completion of replication
• The largest mRNA (3.5kbp) encapsidated along with viral polymerase with HBc Ag and HBeAg
• The polymerase synthesize single stranded DNA (-ve sense) from the RNA by its reverse transcriptase activity.
• The RNase H activity of the polymerase degrade RNA from RNA-DNA hybrid.
• The virus is then bud out form the ER and acquire the envelope containing the surface antigens.
• It is during this time or later +ve sense DNA is copied from the viral –ve sense DNA strand.
• This virion is then subjected to vesicular transport and is released by exocytosis from the membrane.

Mode of transmission of Hepatitis B:

• Hepatitis B is transmitted by the exchange of body fluids-blood, serum, semen, breast milk and in some circumstances saliva.
• Concentration of HBV in various body fluid:
  • High: blood, serum, wound exudates
  • Moderate: semen, vaginal fluids, saliva
  • Low: urine, feces, sweat, tears, breast milk
• Transmission of HBV occurs by following routes:
  • From infected mother to child
  • Transfusion of HBV infected blood or blood products
  • Sexual contact with infected person.

Pathogenesis of hepatitis B virus:

• Hepatitis B virus infection is an immune-pathological disease.
• HBV after entering the blood infects hepatocytes with the expression of viral antigen on the surface of infected cells. Copies of HBV genome integrate into hepatocyte chromosome and remain latent.
• Viral DNA and HBc Ag can be detected in nucleus while HBs Ag in cytoplasm and at hepatocyte membrane.
• The intracellular accumulation of filamentous form of HBs Ag produces the ground glass appearance of affected hepatocyte which is the characteristic feature of HBV infection.
• Adaptive immunity is more involved than the innate immunity in causing tissue destruction. Infact both tissue destruction and virus clearance from the host cell are attributed to cellular immunity (CMI)
• MHC I activated CD8 T-cell and MHC II activated CD4 T-cells are involved in pathology of disease. However most of the destruction is due to CD8 T cells.
• CD8 T cells also induce TNF-α and other cytokines including interferon-Ƴ which further compound the non-specific liver destruction.

Primary infection (acute infection):

• The incubation period of HBV is usually 4-10 weeks
• HBs Ag begins to appear in the serum during the incubation period and continues to increase. Its level can reach as high as 10,000 to 100,000 PEI (paul erlich institute). Its level begins to decline with the prodromal phase, disappear completely with appearance of symptoms and during recovery.
• Anti-HBc IgM antibody begins to rise with the onset of symptoms and this is the marker of acute infection.
• As HBs Ag become detectable, there is also marked increase in viral titer which can peak upto 10⁹ to 10¹⁰ virions/ml.
• HBe Ag also become detectable. By this time 70-80% of liver cells are infected.
• Even if liver injury occurs, alanine aminotransferase level does not rise until and unless liver infection is well established. This reflects the amount of time required for T-cell response to initiate and attack the hepatocytes.
• Once the response begins, viral titers from liver and blood begin to decline.
• With clearance of virus, HBs Ag and HBe Ag level disappear. Subsequently level of anti-HBs Ag begins to rise.
• Even though HBs Ag and HBe Ag disappear with the appearance of anti- HBs Ag and anti-HBe Ag, it has been found HBV DNA persists. This HBV genome may not be enclosed within a virion or the genome is incomplete.

Chronic infection:

• A chronic carrier of HBV is an important event in the pathogenesis.
• Persistent infection of hepatocytes result in presence of HBV and HBs Ag in blood for at least 6 months. Whether an infected host cell will become a chronic carrier state or will be free of infection depends on the cytotoxic T-cell response.

Clinical manifestation:

• Clinical disease in susceptible host can be symptomatic or asymptomatic. Asymptomatic cases are more common in children.
• Infection in adults whether it is symptomatic or asymptomatic, is self-limited which may be due to virus clearance or immune response of individuals towards the virus.

Acute hepatitis B:

• It is characterized by gradual onset of anorexia, malaise and fatigue.
• During Acute (icteric) phase, the liver become tender with the development of jaundice, nausea, vomiting and passing of dark colored urine

Chronic hepatitis B:

• 5% of the adults may develop persistent infection.
• Persistent infection can also be symptomatic or asymptomatic.
• In people with sub-clinical persistent infection, normal level of serum amino-transferase and normal or nearly normal findings on liver biopsy indicates asymptomatic chronic persistent infection.
• However abnormal liver function test and histological findings on biopsies indicate symptomatic chronic persistent infection.
• Depending upon the extent of viremia and host response, chronic infection can lead to liver cirrhosis and hepato-cellular carcinoma (HCC).
• HCC usually occurs in association with cirrhosis. However in some case HCC has been found to occur independently.
• If the extent of virus infectivity is high chronic infection can also lead to glomerulonephritis and polyarthritis nodosa due to deposition of circulating immune complexes.

Lab diagnosis of HBV:

• The initial diagnosis of Hepatitis B is made on the basis of clinical features and laboratory findings on serum bilirubin and enzymes transaminase, ALT and AST
• Specimen: Blood, serum, body secretions
• Microscopy: Immunofluorescence staining:
  • Immunofluorescence staining of infected hepatocytes show HBV core antigen in the nucleus and infectious Dane particle in cytoplasm.
• Molecular diagnosis:
  • Detection of viral DNA by molecular methods such as insitu hybridization and PCR in tissue sample and serum reflects the degree of virus replication in liver.
• Serology:
  • Appear Standard serological test of HBV is detection of HBs Ag.
  • HBs Ag is the first marker to appear in blood after infection. It can be detected even before elevation of transaminase enzymes and onset of clinical symptoms.
  • HBs Ag remains in blood circulation throughout the acute phase of disease.
  • When HBs Ag is no longer detectable, its antibody (anti-HBs) appears and is detectable for longer period.
  • Detection of IgM anti HBs indicates recent infection while detection of IgG anti-HBs indicates past infection.
  • Presence of anti-HBs without other serological virus marker indicates immunity following vaccination.
  • HBc Ag is not detectable in blood because it is enclosed within HBs Ag. But anti-HBc antibody appears in serum 1-2 weeks after appearance of HBs Ag.
  • HBe Ag appears in blood concurrently with HBs Ag or soon afterwards. The disappearance of HBe Ag coincides with the fall of transaminase levels in blood which is followed by anti-HBe Antibody.

Treatment and Vaccines:

• Treatment of patients with hepatitis is supportive and directed at allowing hepatocellular damage to resolve and repair itself.
• Recombinant interferon-α and pegylated interferon-α are currently used in HBV treatment.
• Antiviral drugs: Nucleotide analogs (Lamivudine)
• Orthotopic liver transplant:  It is the treatment for chronic hepatitis end stage liver damage
• Vaccine for HBV is available since 1982. Initially vaccine was prepared by purifying HBs Ag associated with 22 nm particle and inactivating through formalin treatment.
• Recombinant HBs Ag produced by recombinant DNA technology using yeast cell or mammalian cell line culture is used as first recombinant vaccine.
• Passive immunization using specific hepatitis B immune globulin (HBIG) have shown effective protection. However it is not recommended for pre-exposure prophylaxis.

Hepatitis B: replication, transmission, pathogenesis, disease, diagnosis and treatment

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• Mumps is an acute contagious disease of children, characterized by acute enlargement of one or both salivary glands. The disease is caused by mumps virus. More than 1/3^rd of all mumps infections are asymptomatic.  It causes a mild childhood disease but in adults complications including meningitis and orchitis.
• Mumps virus is a typical paramyxovirus possessing both HN and F proteins.
• Mumps virus is labile, rapidly inactivated at room temperature or by exposure to formaldehyde, ether or UV.

Mode of transmission

• Infection is transmitted by direct contact, air- borne droplets or fomites contaminated with saliva and also possibly urine.
• Closer contact is necessary for transmission of mumps
• It is difficult to control transmission of mumps because of the variable incubation periods. There are large number of asymptomatic cases and Virus may be present in saliva before clinical symptoms.

Pathogenesis:

• Mumps virus causes a lytic infection of cells and Humans are the only natural hosts
• Once the virus enter the respiratory tract, the infection begins
• The incubation period is typically about 14-18 days (range from 2 to 4 weeks)
• Primary replication occurs in nasal or upper respiratory tract epithelial cells.
• Once viremia occurs, virus is then disseminated to target tissues such as the CNS and salivary glands (parotid gland). Involvement of the parotid gland is not an obligatory step in the infectious process.
• Salivary glands show desquamation of necrotic epithelial cells lining the duct.
• The virus replicates in the target tissues and then causes a secondary phase of viremia.
• The virus then spread throughout the body- to kidneys, testes, ovary, pancreas and other organs.
• Infection of the CNS especially meninges, causes meningitis or meningoencephalitis.
• The CNS is commonly infected and may be involved in the absence of parotitis.
• Mumps is a systematic viral disease with a propensity to replicate in epithelial cells in various visceral organs.
• Virus shed in the saliva from about 3 days before to 9 days after the onset of salivary gland swelling.

Clinical symptoms:

• The clinical symptoms of mumps reflect the pathogenesis of infection.
• At least 1/3^rd of all mumps infection are asymptomatic
• Majority of infection occurs in children under 2 years of age.
• The most characteristics feature of symptomatic cases is the swelling of salivary gland which occurs in about 50% patients.
• A prodromal period of malaise and anorexia is followed by rapid enlargement of parotid as well as other salivary glands.
• Parotitis:
  • Swelling may be confined to one parotid gland or one gland may enlarge several days after the other gland.
  • Gland enlargement is associated with pain and fever.
• Meningitis and meningoencephalitis:
  • CNS involvement is common in 10-30 % cases. Meningoencephalitis usually occurs 5-7 days after the inflammation of salivary gland.
  • Mumps causes aseptic meningitis.
• Orchitis and Oophotitis mumps:
  • The testes and ovaries may be affected especially after puberty.
  • 20-50 % of men who are infected with mumps develop orchitis.
  • Because of the lack of elasticity of tunica albuginea which does not allow the inflamed testes to swell the complications is extremely painful.
  • Oophotitis mumps occurs in about 5 % of women.
• Pancreatitis:
  • It is reported in about 4% of cases.
• Deafness:
  • It is reported in 4 % cases but last for short period of time
• Complications:
  • Arthritis, nephritis, thyroiditis, mastitis, thrombocytopenia, pneumonia, myocarditis, death due to mumps is rare.

Laboratory diagnosis

• Mumps diagnosis is usually clinical, but confirmed by virus isolation and serology.

Specimens:

• Saliva, secretions and CSF and Urine; collected within few days after onset of infection.
• Virus can be isolated from saliva for up to 4-5 days, CSF for about 8-9 days and from urine for about 15 days after the onset of symptoms.

Isolation of the virus:

• The specimen have to be inoculated soon after collection as the virus is labile.
• The prepared specimen is inoculated into monkey kidney cell cultures, human amnion, hela cells or 6-8 days old chick embroys by the amniotic route
• Virus growth can be detected by hemadsorption and identified by Hemadsorption inhibition using specific antiserum.

Serology:

• In this technique 4 fold rise in Antibody titer is the evidence of mumps infection.
• Presence of antibody to V-antigen is indicative of past infection and that of S antigen indicated recent infection.
• Complement Fixation (CF) and Hemagglutination Inhibition (HI) tests are commonly used.  

Nucleic acid detection:

• RT-PCR is very sensitive method.
• PCR assay can identify virus strains and provide useful information in epidemiological studies

Treatment and immunization:

• No specific antiviral drugs are available for mumps infection.
• An effective live attenuated vaccine is available against mumps. The vaccine consists of Jeryl- Lynn strain of mumps virus attenuated by serial passage in eggs and chick fibroblasts
• Mumps vaccine is available in combination with measles and rubella as MMR, which is a live attenuated vaccine. The vaccine is given as a single subcutaneous injection.
• Two doses of MMR is recommended for school going children

Mumps virus: mode of transmission, pathogenesis, clinical disease and immunization

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• The family Orthomyxoviridae contains a single genus Influenza virus with three types-A, B and C. Influenza viruses are classic respiratory viruses. They cause influenza, an acute infections disease of the respiratory tract that occurs in sporadic, epidemic and pandemic forms
• A unique feature of influenza virus is its ability to undergo antigenic variations. The surface glycoprotein (Haemagglutinin and Neuraminidase) show variations and are primarily responsible for antigenic variations. Antigenic variability is highest in influenza virus type A and less in type B, while it has not been demonstrated in types C.

Structure of Influenza virus

• Shape:
  • The influenza virus particle is typically spherical with a diameter of about 80-120 mm but pleomorphism is common.
  • Filamentous forms upto several micrometers in length and readily visible under the dark ground microscope are frequent seen in freshly isolated strains.
• Symmetry:
  • The virus is core consists of ribonucleoprotien in helical symmetry.
  • The nucleoprotein (NP) associates with the viral RNA to form a ribonucleoprotein (RNP) which is a structure of 9 mm in diameter that assumes a helical configuration and forms the viral nucleocapsid.

• Genome and protein:
  • Influenza virus contains negative sense single stranded RNA (-ssRNA) genome which is segmented.
  • Type A and B influenza virus consist of 8 pieces of segmented RNA (while type C influenza virus contains 7 segments), which encode for 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1, PB2).
  • Because of the segmented nature of the genome when a cell is infected by two different viruses of a given type, mixtures of parental gene segments, may be assembled in progeny visions. This phenomenon call genetic re-assortment, may result in sudden changes in viral surface antigens- a property that explains the epidemiological features of influenza and poses significant problems for vaccine development.
  • Also contains a viral RNA-dependent RNA polymerase that transcribes the negative polarity genome into mRNA.
  • Three large proteins (PB1, PB2, and PA) are bound to the viral Ribonucleoprotein and are responsible for RNA transcription and replications.
  • The matrix (M1) protein which forms a shell underneath the viral lipid envelope is important in particle morphogenesis and is a major competent of the virion.
• Envelope and glycoprotein spikes:
  • The nucleocappsid is surrounded by an envelope which has an inner membrane of protein known as matrix or M protein which is virus encoded and outer lipid layer derived from infected host cell membrane during the process of replication by budding.
  • Two virus encoded glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA) are inserted into the envelope and are exposed as spikes about 10mm long on the surface of the surface of the particles.
  • These glycoproteins are triangular and mushroom shaped respectively. They are synthesized in the early period of replication cycle, and get attached to the plasma membrane at specialized patches where budding occurs.
  • These two surface glycoproteins are the important antigens that determine antigenic variation of influenza viruses and host immunity.
  • The HA represents about 25% of viral protein the NA about 5%.
  • The M2 ion channel and the NS2 protein are also present in the envelope but a few copies per particle.
• Haemagglutinin (HA):
  • Haemagglutin derives its name from its ability to agglutinate erythrocytes under certain conditions.
  • Haemagglutinin is a glycoprotein composed of two polypeptides- HA 1 and HA2, responsible for hemadsorption and haemagglutination. These two polypeptides are joined together by disulfide bond.
  • The Haemagglutinin consists of 500 spikes each measuring 12nm in length.
  • The triangular shaped HA is inserted into the virus membrane by its tail end which is hydrophilic in nature. The distal end which contains five antigenic sites (HA1-HA5) is responsible for binding of vision to host cells.
  • Haemagglutinin is one of the major antigen of influenza virus and is responsible for antigenic variation.
  • HA enables the virus to absorb to muco-protein receptors on red cells as well as an respiratory epithelial cells.
  • HA agglutinates certain RBCs which is inhibited by the neutralizing antibodies. This forms the basis of the haemagglutination inhibition test used in the sero-diagnosis of influenza.
• Neurominidase (NA):
  • Neuraminidase is a glycoprotein receptor and is important in determining the subtype of influenza virus isolates.
  • It consists of 100 mushroom-shaped spikes that is a tetramer, composed of four identical monomers. A slender stalk is tapped with a box shaped head.
  • NA functions at the end of the viral replication cycle.
  • Neuraminidase is a sialidase enzyme that removes sialic acid from glycol-conjugates. It causes hydrolysis of N-acetyl neuraminic acid or sialic acid residues present on the glycoprotein receptors on red cells, hence causes elution or detachment of cells absorbed to virion particles.
  • It facilitates release of the virus particles from infected cells surface during the budding processes and helps prevent self-aggregation of virions by removing sialic acid residues from viral glycoprotains.
  • It also degrades the mucus layer, thereby exposing the epithelial membrane of the respiratory tract for infection of the virus.

Types of Influenza viruses

On the basis of antigenic differences in nucleoprotein and the matrix protein (M) the influenza virus is divided into 3 types.

1. Influenza virus A:
   • They are the causative agent for all flu pandemics and are known to infect humans other mammals and birds.
   • Influenza A virus are further classified into sub types based on the properties of their major membrane glycoproteins; Haemagglutinin and Neuraminidase.
   • Till now, 15 HA (H1-H15) sub types and 9 NA1 (N1-N9) sub types have been identified from influenza viruses of birds, animals and humans.
2. Influenza virus B1: They are known to infect humans and seals.
3. Influenza virus C: They are known to infect humans and sometimes pigs.

Nomenclature and classification of Influenza virus:

• WHO in 1971 proposed a new system of classification and was later modified.
• According to this the complete designation of a strains will include the (a) type (b) place of origin (c) serial number and (d) year of isolation followed by (e) antigenic subtypes of the HA and NA in parentheses.

Antigenic variations:

One important characteristics of Influenza virus is that, it regularly change their surface antigen (haemagglutinin and neuraminidase present in the surface). These antigenic changes are of two types; antigenic shift and antigenic drift

• Antigenic shift
  • It is the major and drastic and discontinuous type of change in the antigenic structure, resulting in a novel virus strain which is antigenically unrelated to the predecessor strains.
  • The antigenic shift occurs due to major antigenic changes in HA or NA antigens and is caused by replacement of the gene for Haemagglutinin by one coding for a completely different amino acid sequence due to exchange of whole RNA segment.
  • This is characterized by alteration of virtually all the antigenic sites of the HA.
  • The antigenic shift occurs when two different strains of Influenza virus infect a single host cell.
• Antigenic drift
  • It is the minor and gradual sequential change in antigenic structure.
  • Antigenic drift refers to the minor antigenic changes in either the haemagglutinin or neuraminidase or both.
  • The drift results from point mutations in the HA and NA genes.
  • The antigenic drift is characterized by changes in certain epitopes in the HA, while others are being conserved.

 Mode of transmission of Influenza:

• Influenza virus infection transmits through air borne route as well as through contaminated hands
• It spreads from person to person via respiratory secretions during the acts of sneezing and coughing
• It also transmits by contact with contaminated hands or surfaces.

Pathogenesis of Influenza virus:

• Influenza virus enters the body through upper respiratory tract, where the virus attaches to and infects the mucosal epithelial cells.
• Influenza virus have some defense mechanism to protect itself from removing from cough reflex. Similarly, the influenza virus has also mechanism to protect itself from neutralization by IgA antibody present in mucus lining of upper respiratory tract.
• Viruses attach to sialic acid receptors on epithelial cells by their haemagglutinin spike present on the viral envelope.
• Neuraminidase lowers the viscosity of the mucous film in the upper respiratory tract and promoting the spread of virus containing fluid to lower respiratory tract.
• After attachment on epithelial cell, virus enters into the cell and begins the replication cycle.
• Infection of mucosal cells results in cellular destruction and degradation of the superficial mucosa. The resulting edema and mononuclear cell infiltration of the involved areas are accompanied by symptoms including non-reproductive cough, sore throat and nasal discharge.
• By continuous replication, epithelial layer of respiratory tract is completely removed. Then many opportunistic normal flora penetrate through damaged epithelium causing opportunistic bacterial infections. Many bacteria like Eg. Streptococcus, staphylococcus and Haemophilus influenza are carried into lungs along with mucus. In lungs these bacteria multiply causing secondary bacterial pneumonia.
• Occasionally in patients with underlying heart or lung disease the infection may extensively involve the alveoli, resulting in interstitial pneumonia, sometimes with marked accumulations of edema and lung haemorrhage.
• Influenza virus remains localized in epithelium of upper respiratory tract and do not penetrate into inner tissues. So influenza virus is not found in blood of infected individuals.

Clinical manifestation of Influenza:

1. Uncomplicated Influenza; Flu like symptoms
   • Symptoms of classic influenza include chills, headache, dry cough followed by high fever, generalized muscular aches, malaise and anoxeria.
   • Sneezing, rhinorrhea and nasal obstruction are common.
   • Patients may also report photophobia, nausea, vomiting, diarrhoea.
   • The fever usually last for 3-5 days.
   • Respiratory symptoms typically last another 3-4 days.
   • The cough and weakness may persists for 2-4 weeks after major symptoms subside.
   • Clinical symptoms of influenza in children are similar to those adults although children may have fever and higher incidence of gastro intestinal manifestation such as vomiting.
2. Pneumonia
   • Serious complications of influenza usually occur in the elderly and immune-compromised individual especially those with underlying chronic disease.
   • Pregnancy appears to be risk factor for lethal pulmonary complications in some epidemic.
   • The lethal impact of influenza epidemic is reflected in the excess death due to pneumonia and cardiopulmonary disease.
   • Pneumonia complicating influenza can be viral, secondary bacterial or a combination of two.
   • Increased mucous secretion helps to carry the agents into the Lower respiratory tract. Influenza infection enhances the susceptibility of patients to bacterial infections.
   • The major bacterial pathogen associated are S. aureus, S. pneumonia and H. influenza.
   • Combined viral bacterial pneumonia is approx. 3 times common than primary influenza pneumonia.
3. Reye’s syndrome
   • Reye’s syndrome is an acute encephalopathy of children and adolescent usually between 2-16 years of age.
   • The cause of Reye’s syndrome is unknown but it is a recognized rare complications of Influenza B and A.
4. Guillain- Barre syndrome
   • It is characterized by encephalomyelitis and polyneuritis which is a rare complication of influenza virus infection.

Laboratory diagnosis of Influenza virus

Sample:

• Samples for diagnosis includes- nasal or throat washing, throat swab, gargles which are collected within 3 days after infection or appearance of symptoms.
• If the sample is for isolation of virus, it should be kept at 4°C. if storage time is more than 5 days, sample should be frozen at -70°C.
• Serum sample is needed for serology to detect antibody.

Method of diagnosis of Influenza virus:

1. Direct antigen detection
   • Various antigens of virus can be detected in nasal washing and throat swab. Antigens are components of virus which are left during assembly of virus particles.
   • Rapid diagnosis of influenza may be made by demonstration of the viral antigen directly by ELISA or Florescence antibody test (FAT) or Radio-immuno-assay (RIA), using specific monoclonal antibodies.
2. Isolation and identification of virus
   • Virus isolation is obtained readily from patients during the first two or three days of illness.
   • Throat gargles is the best specimen and is collected in saline broth or a buffered salt solution and is sent immediately to the laboratory or if delayed, stored at -4 degree Celsius. Isolation can be made in eggs or in monkey cell culture.
   • The specimen is inoculated into the amniotic cavity of 11-13 day old eggs.
   • After incubation at 35 degree Celsius for 3 days, the amniotic fluid and allantoic fluids are harvested separately and tested for haemagglutinin. This is carried out by using fowl and guinea of red cells in parallel and incubating at room temperature at 4 degree Celsius.
   • Usually Influenza A agglutinate only guinea pig cells, influenza B agglutinateboth fowl and guinea pig red cells and influenza C agglutinate only fowl cells at 4 degree Celsius.
   • The isolate is identified and typed by Complement fixation (CF) test with antisera to types A, B and C.
   • Subtype identification is made by Hemaggluination inhibition test.
   • Inoculation into monkey kidney or baboon kidney is the preferred method where facilities are available. Inoculated cell cultures are incubated with or without serum and in presence of trypsin, virus growth in tissue cultures is detected by direct demonstration of viral antigen in infected cell cultures by Immunoflurescence or by testing for hemadosorption with human, fowl and guinea pig red cells. In a positive hemadsorption test, red cells adhere to the virus budding from infected cells. Now a days most laboratory uses secondary baboon kidney or Mardin- Darby canine, kidney (MDCK) cells.
3. Serodiagnosis:
   • Antibodies to several viral proteins (hemagglutinin, neuraminidase, nucleoprotein and matrix) are produced during infection with influenza virus.
   • Routine serodiagnosis test in use are hemagglutination inhibition assay (HIA) and ELISA, FAT etc to detect antibodies.
   • To confirm recent infection, two serum samples, one collected during acute phase and other during convalescence phase are needed. Four fold rise in antibody titer in acute phase is indication of recent infection.
4. Molecular diagnosis:
   • Rapid test based on detection of influenza RNA in clinical specimen using RT-PCR is also possible.

Treatment of Influenza virus:

• Two antiviral drugs- Amantadine and Rimantadine are available for treatment of influenza. These drugs are effective for type A but not against type B.
• Zanamivir and Oseltamivir are newer drugs for treatment of influenza and are effective against both influenza type A and B viruses.
• Vaccine: Due to antigenic shift and drift, vaccine production is difficult.

Influenza virus-Structure, Types, Nomenclature, Transmission, Pathogenesis, Diseases, Diagnosis and Treatment

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• Virus are the obligate intra cellular particles, they replicate inside host cell only.
• For a specific virus to replicate within a specific host cell, certain condition must be fulfilled. Some of the criteria that are required to be fulfilled in order to viral replication are;
  • The host cell must be permissive and the virus must be compatible to host cell.
  • The host cell must not degrade the virus.
  • The viral genome must possess the information for multiplying utilizing the normal metabolism of host cell.
  • The virus must be able to use the metabolic capability of host cell to produce new progeny virus particles containing replicated copy of viral genome.
• A cell within which virus replicates is called host cell. Therefore the host may be permissive or non-permissive.
• Those host cell within which virus replicates is called permissive or compatible host cell and those within which virus cannot replicate is called non-permissive or non-compatible host cell.
• The host cell range of a virus is defined by the types of cells within which replication of that particular virus occurs.
• Some virus have broad host range and can replicates within several types of host cell whereas other virus have narrow host range.

Outcome of virus replication:

Virus replication of host cell can have three possible outcomes.

 i. Productive infection:

• It occurs in permissive cell which results in viral replication within it producing progeny viruses that can infect other compatible host cells.
• The complete infectious virus produced in such infection is called virions.

ii. Abortive infection:

• It occurs in non-permissive host cell so that virus replication does not occurs or because virus replication produces viral progeny that are incapable of infecting other host cell.

iii. Restrictive infection:

• It occurs when host cell is transiently permissive so that infective viral progeny are sometime produce and other time the virus persists within cell without production of infective viral progeny.
• It results in occasional release of virus with no cell death.

Stages of virus replication:

• Although the specific detail of virus replication vary from one virus to another, general replication is same for most virus.
• The stages includes;
  • Attachment of virus to outer surface of suitable host cell; a process called Adsorption
  • Penetration of virus into host cell
  • Release of viral genome from capsid; a process called un-coating that sometime occurs simultaneously with penetration)
  • Synthesis of viral proteins
  • Synthesis of viral genome
  • Assembly of viral progeny (virion)
  • Release of progeny virus from host cell.

i. Attachment (Adsorption):

• This is the first step in virus infection in which interaction of virion with a specific receptor site on the surface of host cell occurs.
• The receptors sites are normal cell surface components of host cell such as protein, polysaccharides or lipoprotein-polysaccharide complex to which virus attach.
• For eg. HIV binds to CD4 cell receptor of T-lymphocytes
  • Rhinovirus binds to ICAM-1
  • Epstein Barr virus binds to C3 complement receptor.
• Each host cell contains upto 100,000 receptor sites for a given virus.
• In general viral receptor carryout normal function in cell.
• For eg. In some bacteriophage, receptor are pilli and flagella and in other virus receptor site may be transport binding protein etc.
• Receptor of influenza virus is glycoprotein found in RBC and on other cell of mucus membrane of susceptible host.

ii. Penetration:

After binding of virus, virus is taken up inside the cell which is referred as penetration or engulfment.

• The entry of virus into host cell may involves;
  • Transfer of only genome across cytoplasmic membrane
  • Transport of entire virus across cytoplasmic membrane by endocytosis
  • Fusion of viral envelope with cytoplasmic membrane of host cell.

iii, Uncoating;

• Shortly after penetration, uncoating of virus take place.
• Uncoating is defined as release of viral genome from capsid and is accessible to enzymes required to translate, transcribe and replicate it.
• The uncoating process vary from virus to virus.
• Transcription of viral genome is usually the next step in all virus except in those virus whose genome acts directly as mRNA (eg. Picorna virus).
• RNA viruses that carry minus(-) stranded RNA first transcribe their DNA to plus (+) stranded RNA that function as mRNA.
• The transcription is catalyzed by viral RNA polymerase released during uncoating.

iv. Biosynthesis:

• The biosynthesis process of virus replication can be divided into early event and late events.
• Early event:
  • In most virus, only part of nucleic acid is initially transcribed into mRNA.
  • The early mRNA codes for early proteins (enzymes) required for nucleic acid replication
  • After nucleic acid replication, many copy of progeny nucleic acids formed.
• Late event:
  • Late mRNA is transcribed from progeny genome.
  • Late mRNA codes for structural proteins by the process of translation. The translation process always occurs in cytoplasm of host cell, even if the mRNA synthesized in nucleus, it enter cytoplasm for translation.

v. Assembly:

• When critical number of various viral components have been synthesized, they assembled into mature virus.
• The assembly occurs in nucleus or cytoplasm of host cell depending upon types of virus.
• DNA virus assembled in nucleus except Poxvirus and RNA viruses assembled in cytoplasm except Influenza virus and Reo virus.

vi. Release:

• Release of mature virus from host cell is the final event in virus replication.
• The mechanism of virus release vary with types of virus.
• The naked viruses are generally released by cell lysis.
• The enveloped viruses are released by budding through special area of host cell membrane; during which virion acquire a portion of host cell membrane.
• In some animal and plant virus, host cells are not killed, the virus release through special channels.

For example: Replication of Herpes simplex virus

Virus replication; Outcomes and steps

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

General properties

• General morphology as Paramyxoviruses
• Roughly spherical with size of 120-250 nm in diameter, often pleomorphic.
• Spikes on envelope contains haemagglutinin but no neuraminidase
• The virus causes agglutination of erythrocytes (RBCs) but it is not followed by elution as the virus does not produce any neuraminidase activity.
• Grows well on human or monkey kidney and human ammonion cultures. Isolates can be adapted for growth on continuous cell lines (Hela, Vero) and in the amniotic sac of hen’s eggs.
• CPE consists of multinucleate syncytinum formation with numerous acidophilic nuclear and cytoplasmic inclusion.
• Multinucleate giant cells are also found in the lymphoid tissue of patients.
• The virus is labile and readily inactivated by heat, UV, ether and formaldehyde.
• It can be stabilized by molar mgso4, so that it resist heating at 50 degree Celsius for 1 hour.
• The measles virus is antigenically stable. It shares antigens with the viruses of canine distemper and bovine rinderpest.

Mode of transmission of Measles virus

• Transmission occurs by direct contact with respiratory secretion and aerosols created by coughing and sneezing.
• The virus enters the body through the respiratory tract and conjunctiva.
• Haematogenous and transplacental transmission can occur when measles occurs during pregnancy.

Pathogenesis of Measles virus

• Humans are the only natural host for measles virus although numerous other species, including monkeys, dogs and mice can be experimentally infected.
• The virus gains access to the human body via the respiratory tract, where it multiplies locally.
• The infection than spreads to the regional lymphoid tissue, where further multiplication occurs.
• Primary viraemia disseminates the virus, which then replicates in the reticuloendothelial system.
• Finally a secondary viraemia seeds the epithelial surface of the body, including the skin respiratory tract and conjunctiva, where focal replication occurs. The described events occur during the IP which typically lasts 8-12 days but may last up to 3 weeks in adults.
• During the prodromal phase (2-4 days) and the first 2-5 days of rash, virus is present in tears, nasal and throat secretions, urine and blood.
• The characteristics maculopapular rash appears about day 14 just as circulating antibodies become detectible and the viraemia disappears and the fever falls.
• The rash develops as a result of interactions of immune T cells with the virus infected cells in the small blood vessels and last for about 1 week.
• Involvement of CNS is common in measles. Progressive measles inclusion body encephalitis may develop in patients with defective cell mediated immunity.
• A rare late complications of measles is subacute sclerosing panencephalitis (SSPE); a fatal disease that develops years after the initial measles infections and is caused by virus that remains in the body after acute measles infections.

Clinical manifestation of measles

• Infections in non-immune host are almost always symptomatic.
• Measles is a highly contagious febrile illness.
• After an incubation period of 8-12 days, measles is typically a 7-11 days illness with a prodromal phase of 2-4 days followed by an eruptive phase of 5-8 days.

i. Prodromal phase of measles:

• The prodromal phase is characterized by fever and three (C’s – coryza of nose, conjunctivitis usually associated with photophobia and Cough), Kopliks spots and lymphopenia.
• The cough and coryza reflects an intense inflammatory reaction involving the mucosa of the respiratory tract.
• Kopliks spots are small, bluish white ulceration of the buccal mucosa.
• The fever and cough persists until the rash appears and then subside within 1-2 days. The rash starts on the head and the spreads progressively to the chest, the trunk and down the limbs which appears as light pink, discrete maculopapular that coalesce to form blotches and becomes brownish in 5-10 days. The fading rash resolves with desquamation.

ii. Modified measles:

• Modified measles occurs in partially immune persons such as infants with residual maternal antibody.
• The IP is prolonged, prodromal symptoms are diminished.
• Koplik’s spots are usually absent and rash is mild.

iii. Atypical measles:

• Atypical measles occurs in individuals who received the older inactivated vaccine and later exposed to a wild strain.
• It may also rarely occur in persons vaccinated with attenuated vaccine.
• It is characterized by a prolonged high fever, pneumonitis and the rash.
• The rash characteristically begins peripherally and may be urticarial, macularpapular, hemorrhagic or vesicular.

iv. Complications of measles:

• The most common complications of measles is otitis media occurring in 5-9 % of cases.
• Pneumonia is life threatening complications of measles, caused by secondary bacterial infections.
• Giant cells pneumonia is serious complication in children and adults with deficiencies in CMI.
• Complications involving CNS are the most serious.
• Acute encephalitis occurs in about 1:1000 cases.
• Post infections encephalomyelitis is an autoimmune disease associated with an immune response to myelin base protein.
• The mortality in encephalitis associated with measles is about 10-20 %. The majority of survivors have neurologic sequela.
• Subacute sclerosing panencephalitis (SSPE) is a rare late complications of measles, occurs with an incidence of about 1:300000 cases.
• It is a degenerative disease of the CNS caused by persistent measles infection. The disease begins 5-15 years after a case of measles. It is characterized by progressive mental deterioration, involuntary movements muscle rigidity and coma the condition is associated with the presence of an extremely high measles antibody titers in the blood and CSF.
• Other complications of measles include bronchopneumonia, laryngotracheobronchitis (croup), diarrhoea.
• Measles includes labor in some pregnant women, resulting in spontaneous abortion or premature delivery. The virus may across the placenta and infect the fetus during maternal measles.

Laboratory diagnosis of measles:

• Typical measles is reliably diagnosed on clinical grounds.
• Laboratory diagnosis is necessary in case of modified or atypical measles.

1. Specimens:

• Respiratory specimens, conjunctival specimen, urine, blood and brain tissue.
• Specimens are collected during the prodromal stage and the period following until 2 days after the appearance of the rash.

2. Microscopy:

• Demonstration of multinucleated giant cells measuring up to 100 nm in diameter in Giemsa stained smears is diagnostic of measles.
• Immunofluorescent study of exfoliated respiratory cells in nasopharyngeal secretion virus particles.

3. Antigen detection:

• Measles antigen can be detected directly in epithelial cells in respiratory secretions, urinary sediments, pharyngeal secretions by direct immunofluorescent antibody test

4. Isolation and identification of virus:

• Nasophayngal and conjunctival swabs, blood samples, respiratory secretions and urine collected from a patient during febrile period are appropriate sources of viral isolation.
• Growth can be obtained in primary human or monkey kidney cells.
• Growth occurs slowly with CPE containing both intra-nuclear and intra-cytoplasmic inclusion bodies in 7-10 days.

5. Serology:

• Serologic confirmation of measles depends on a 4 fold rise in antibody tilter between acute phase and convalescent phase sera or demonstration of measles specific antibody in a single specimen drawn between 1 and 2 weeks after the onset of rash.
• ELISA
• Haemagglutinin Inhibition assay
• Neuraminidase test

Treatment and control of measles:

• Ribavirirn either intravenous or in aerosol form is evaluated now a days to treat severely affected adults and immunocompromised individuals with acute measles or SSPE.
• However the drug is yet to be used for regular treatment of cases of measles.
• Vitamin A treatment in developing countries has reduced mortality and morbidity.
• Active immunization
• A highly effective and safe attenuated live measles virus vaccine is available. It now uses Schwartz and Moraten attenuated strain of the original Edmonstron B strain.
• The vaccine is available in monovalent form in combination with live attenuated Rubella vaccine (MR) and live attenuated rubella and mumps vaccine (MMR)- currently used for universal immunization of children.
• Efficacy – 95 % (90-98%)
• Duration of immunity- life long
• Schedule- 2 doses – first at the age of 9 month- second at the age of 2 years
• Passive immunization
• Pooled sera containing antibody against measles virus confess passive immunity to infants and susceptible contacts of measles cases.

Measles morbillivirus

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 Structure and genome of Nipah virus:

• Nipah virus ( NiV) is a RNA virus belongs to family Paramyxoviradae and genus Henipavirus.
• Size: 40-600nm
• Shape: pleuromorphic
• Envelope: present

Genome and proteins of Nipah virus:

• Single stranded negative sense RNA, 18246 bp (Malaysian isolate) and 18252 bp (bngladesh isolate)
• Genome has six transcriptional unit that six structural proteins. They are nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G) and polymerase (L)
• Protein associated with genome: large (L) protein, phospoprotein (P)
• Viral proteins: fusion protein (F) and attachment glycoprotein protein (G)
• Phosphoprotein (P): it role as a polymerase cofactor, enhancing polymerase processivity and allowing the encapsidation of the newly synthesized viral genomes and antigenomes.
• Phosphorotein of Nipah virus has an additional role in immunosuppression: blocking interferon signaling by binding host STAT-1.

Mode of transmission of Nipah virus:

• Nipah virus is a zoonotic virus.
• Flying fox (family Pteropodidae and particularly species of genus Pteropus ) are the natural host for Nipah virus.

1. Direct contact: Human get infection by direct contact with infected animals (pigs and fruit bats) or human
2. Droplet infection: respiratory droplets, nasal or throat secretion of infected animals
3. Eating contaminated fruits and juices with body secretion of infected animals
4. Human to human transmission with direct contact with infected person

Pathogenesis and pathology of Nipah virus:

• Human get infected with Nipah virus by direct contact with the body secretion of infected animals or by ingestion of contaminated food products.
• Fruit bats of family Pteropodidae are the natural host and reservoir of Nipah virus, while they remain uninfected.
• Infected fruit bats sheds virus in their urine or body secretion with infects pigs as well as other domesticated animals.
• Domesticated animals especially pigs are the intermediate host for Nipah virus and human get infection by direct contact with these animals.
• Human infection is also reported by consumption of contaminated fruits or date palm saps.
• Incubation periods in infected pig ranges from 4-14 days. Infected pigs may develops symptoms such as acute respiratory and neurological illness.
• Nipah virus are belived to infect respiratory tract epithelial tissue resulting is shedding of epithelial lining along with nasopharyngeal secretion.
• Patients develops symptomatic respiratory infection in early stage of infection.
• During late stage, virus spread to lungs endothelium resulting in endothelial syncytium and mural necrosis. Nipah virus can then enter the bloodstream and disseminate throughout the host in either free form or by binding host leukocytes. (Nipah virus has been shown to bind to CD3+ leukocytes without entry or replication of the virus)
• Other target organ of Nipah virus the brain, spleen and kidneys.
• Entry of Nipah virus into the CNS is thought to occur through two distinct pathways: anterogradely via the olfactory nerve and/or via the hematogenous route through the choroid plexus and cerebral blood vessels.
• Infection of the CNS in humans is characterized by vasculitis, thrombosis, parenchymal necrosis, and presence of viral inclusion bodies.

Clinical symptoms of Nipah virus infection:

• Clinical illness in human ranges from Asymptomatic to acute or sever symptomatic to fatal encephalitis
• Initially patients develops Influenza like symptoms such as; Fever, Sore throat, Headaches, Vomiting and Myalgia or Muscle pain
• Acute respiratory infection; Difficult in breathing.
• Some patients develop Atypical pneumonia
• Neurological illness results in encephalitis and seizures.
• Case fatility rate ranges from 43% to 100% in sporadic cases.
• Patients surviving acute encephalitis have been reported to show long term neurological conditions such as personality change and seizures.

Lab diagnosis of Nipah virus infection:

• Non-specific diagnosis by sign and symptoms
• Samples: nasal secretion, blood, contaminated fruits or infected animals
• RT-PCR
• ELISA
• Virus culture

Prevention and control of Nipah virus infection:

• Aware and educate people to take preventive measure to reduce contact with virus
• Apply preventive measure to sap collection such that bats cannot contaminate the collected sap.
• Boils the collected sap before consumption.
• Apply preventive measure while handling domesticated animals especially sick animals.
• Avoid direct or unprotected contact with infected person.
• Wear NH95-grade and higher masks
• Follow standard infection control procedure during handling patients and samples

Treatment and therapeutic measure of Nipah virus infection:

• Intensive supportive care
• No vaccine and No drugs

References:

1. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012709
2. https://jidc.org/index.php/journal/article/view/23592639/857
3. https://ac.elscdn.com/S0042682200903404/1s2.0S0042682200903404main.pdf?_tid=972b3087493b4c6e9e2a4757a4ab9bca&acdnat=1527346515_1876c33bf7defb4070f2472705b0dbed
4. http://www.who.int/news-room/fact-sheets/detail/nipah-virus

Nipah virus: Structure and genome, mode of transmission, Pathogenesis, Symptoms, prevention and treatment

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Properties and classification of HAV:

• Hepatitis A virus (HAV) ia a member of Family Picornaviridae. It was previously classified as Enterovirus 72. Now it is assigned to new genus Heparnavirus (Hepatovirus).
• HAV is a RNA virus.( 7.48kb )
• Genome: the genome is single stranded positive sense RNA (+ss RNA). The 3′ end of RNA is polyadenylated and 5′ end consists of viral protein VPg. There are 4 viral proteins ie. VP1, VP2, VP3 and VP4. (* VP1 and VP3 are major antibody binding site)
• Shape: Small spherical
• Capsid: icosahedral
• Size: 27nm
• Envelope: absent (No envelope)

• Replication: In cytoplasm of Host cell
• Resistance: HAV is stable to treatment with 20% ether, acid (pH 1.0 for 2 hours), heat(60C for 1 hours) and temperature of 4C or below.
• Susceptible: HAV is destroyed by Autoclaving, boiling in water for 5 minutes, by dry heat (180 C for 1 hours), by UV light, formaldehyde (1: 4000 at 37C for 72 hours0 and chlorine (10-15 ppm for 30 minutes).
• Hepatitis A virus causes an acute highly contagious hepatitis, also known as infectious hepatitis in children and young adults.
• Classification of Heparnavirus; Hepatitis A virus is the only known species of the genus Hepatovius. There is only one serotype of HAV and 4 genotypes

Mode of transmission:

• HAV is mainly transmitted through Faeco-oral route by eating/drinking contaminated food and water.
• also transmitted by direct person to person contact
• virus is also transmitted by food handlers and children
• Rarely transmitted by blood products, blood transfusion or intravenous drug use.
• HAV is excreted in faeces during incubation period of 3-5 weeks

Pathogenesis of Hepatitis A virus:

• Human is the primary reservoir of Hepatitis A virus. The virus may survive outside the body for months.
• HAV infection is caused by ingestion of contaminated foods or water. The virus first multiplies in the intestinal epithelium and then enter the blood stream and then migrates to liver parenchymal cells.
• HAV attaches to liver cell through Ig-like cellular receptor on the host cell and enter inside the cell by receptor mediated endocytosis.
• Inside the endosome, virus releases VPg protein which creates pores on the endosomal membrane and releases viral genome in the host’s cytoplasm and damage the hepatocytes.
• Virus multiplication  occurs in hepatocytes and Kupffer’s cells causing monuclear infiltrate, ballooning of hepatocytes, degeneration and acidophilic bodies in the hepatocytes.
• Liver damage is not due to HAV but rather due to immunological response by cytotoxic T cells.
• Newly synthesized viruses in liver cells sheds into bile ducts and excreted in faeces.
• HAV occasionally caused transient viremia.
• Rapid infection of virus is faster than development of immune response

Clinical features of Hepatitis A:

• The incubation period is  between 2-1o weeks.
• After about 2 weeks of infection, virus is detectable in the liver blood and faeces.

clinical features includes-

I. Mild illness without Jundice with little liver cell damage

• Loss of appetite
• Nausea
• Gastrointestinal upset
• Enlarged and painful liver

II. Acute hepatitis with jaundice with severe liver cell damage

• More acute symptoms especially nausea
• Loss of appetite
• Fever
• Jaundice
• Headache
• Pain in muscle and joint
• Skin rashes

Laboratory diagnosis of Hepatitis A:

Specimen: faece, blood

1. Liver function test (LFT):

• Increase in both Alanine aminotransferase (ALT) and Aspartate aminotransferase  enzyme by 4-100 times in Hepatitis patient than normal.
• A sharp rise of ALT with short duration (4-20 days) is suggestive of HAV infection.
• Increase serum bilirubin level (5-20 mg/dl)
• Increase serum globulin level
• Decrease in serum albumin level

2. Serology:

• Antibody detection: rapid kit for detection of anti-HAV IgM in serum
• Antigen detection: detection of hemagglutination antigen or virus particle in faeces

3. Virus culture:

• culture of HAV is difficult and takes upto 4 weeks to get result
• In cell line culture, virus does not replicate well and produces variability till cytopathetic effects.

4. Molecular diagnosis:

• Electron microscopy
• RT-PCR

Hepatitis A virus (HAV): properties, classification, mode of transmission, pathogenesis, clinical features and laboratory diagnosis

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