Virus / Flaviviridae / West Nile Virus / Detailed Viral Descriptions:
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< > Literature Reports of LIFE CYCLE AND TRANSMISSION for West Nile Virus:

OVERVIEW

Editorial Summary (Editorial Overview Text Replicated on Overall Virus page - West Nile Virus)

SOURCES OF VIRUS

  • Following inoculation of WN virus by a mosquito (Culicidae - Mosquitoes (Family)) (normal route of infection) into a vertebrate host, and an incubation period, a period of viraemia develops. Many bird species develop a level of viraemia sufficient to infect mosquitoes feeding on them during the period of viraemia (usually one to four days), although different bird species vary considerably in the level and length of viraemia and therefore their suitability as reservoir hosts for infection of mosquitoes. Most mammal species (Mammalia - Mammals (Class)) generally do not develop viraemia of a sufficient titre for effective transmission to mosquitoes (i.e. they are 'dead-end' hosts) however some species may develop viraemia sufficient to transmit infection to mosquitoes. Birds (Aves - Birds (Class)) are therefore recognised as the major source of WN virus for infection of mosquitoes and thereby of other vertebrate hosts, including humans (Homo sapiens - Human) and horses (Equus caballus - Domestic horse). At least some species of amphibians and reptiles may also be suitable hosts for infection of mosquitoes which feed on them.
  • As virus may be found in skin and other organs of birds after the viraemic stage of infection it is possible that infected birds may continue to act as a source of virus for mosquitoes/ticks or for other animals (by ingestion) for a longer time.
  • A variety of bird species have been shown to shed virus orally and/or cloacally following infection.
  • It has been shown that infected mice and sparrowscontain sufficient virus to infect predators which eat them.

MECHANISMS OF SPREAD

  • The principle vectors of WN virus are mosquitoes which feed on an infected host and after a period of time, during which the mosquito can travel some distance, transfer the virus to a new host when they next feed.
  • Findings of different subtypes of WN Virus in the same location and the same subtypes in different locations are consistent with spread by migrating birds.
  • In Europe, the principle cycle of WN virus circulation is a rural, sylvatic, cycle, with transmission between wild, usually wetland birds and ornithophilic (bird-loving) mosquitoes. A second, urban, cycle may also occur (as in the outbreak in Bucharest, 1996-97), between synanthropic or domestic birds and mosquitoes (mainly Culex pipiens/molestus) (Culex pipiens complex - Northern and Southern house mosquitoes) feeding on birds and on humans.
  • A bird-tick (Argasidae - Soft ticks (Family) or Ixodidae - Hard ticks (Family)) cycle may occur in some dry warm habitats which do not contain mosquitoes.
  • It has been suggested that the rate and pattern of spread in North America can be explained by dispersive movements of Passer domesticus - House sparrow.
  • Vertical (transovarial and transtadial) transmission may occur from one generation of mosquitoes to the next generation.
  • Transmission in humans by means of organ transplants and blood transfusions has been documented.
  • Intrauterine infection (transplacental transmission) and transmission via breast milk have been documented in humans.
  • Transmission in the laboratory due to accidental inoculation of humans by contaminated sharps has been documented. 
  • Transmission to birds via ingestion of virus solution, infected mosquito or infected vertebrate host has been demonstrated experimentally.
  • Transmission to cats by ingestion of infected mice has been demonstrated.
  • Bird-to-bird transmission has been demonstrated in the absence of mosquitoes to act as vectors.
  • Virus has been found in the ovaries of birds which have survived infection, presenting the possibility of transovarial transmission in birds.

ROUTES OF INFECTION

  • The virus usually enters the definitive host in a low dose through peripheral inoculation as the mosquito (or tick) feeds.
  • Bird to bird transmission has also been recorded where the virus may enter the definitive host by the oral or intra-tracheal route or possibly through skin damage. 
    • The precise route of bird to bird transmission is unclear although virus has been found in oral / cloacal swabs experimentally.

(J64.19.w1, J71.54.w1, J84.5.w2, J84.7.w22, J84.7.w22, J84.9.w2, J91.62.w1, J91.65S3.w1, J110.39.w4, J110.39.w5, J127.46.w1, J133.951.w9, J133.951.w37, J214.267.w9, J223.78.w1, J269.94S.w1, B240.14.w14, B241.49.w49, B243.31.w1, B244.w1, B324.33.w33, P38.2001.w1, W8.Nov01.WNV8, W27.29Sept02.wnv1, W27.04Oct02.wnv1, W27.19Dec02.wnv1, W181.28Jan04.WNV6, W380.Dec02.wnv1, N7.51.w2, N7.51.w3, N7.51.w4, N7.51.w5, N7.51.w7, N7.51.w8) 

SPREAD WITHIN THE VERTEBRATE HOST

  • Initial replication is thought to occur in dendritic cells at the site of inoculation, followed by movement to lymph nodes and then to the vascular system. In the vascular system the virus spreads to peripheral organs (e.g. spleen, live, heart, kidneys, lungs). The exact method by which virus enters the CNS is not clear.

CELL INFECTION AND VIRUS REPLICATION

  • The virus attaches to the cell via a receptor, enters the cell and the nucleocapsid is released.
  • The viral RNA is translated into three structural and seven non-structural proteins.
  • The positive sense viral RNA is translated into negative sense RNA and this in turn acts as a template for synthesis of more viral positive sense RNA.
  • Viral RNA and the C protein interact to form the nucleocapsid precursor; the envelope is thought to be acquired by budding into the endoplasmic reticulum.
  • Immature virions are thought to be transported via the secretory pathway to the cell surface where release of mature virions occurs.

(J214.267.w9, J285.3.w1, J286.44.w1, B240.14.w14, B324.32.w32, B324.33.w33, P48.1.w8) 

Transmission Mechanisms that have been reported
  •  

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Sources of Virus

Source Information

Birds:

Mammals:

  • In general for a mammal to act as an amplification host its viraemia must exceed 5.0 log-10 pfu/ml blood serum; at lower levels mosquitoes may be infected but the risk of transmission to mosquitoes taking a blood meal is lower; less mosquitoes become infected while feeding. (P48.1.w16)
  • Infections of mammals have generally resulted in either no detectable viraemia or in only low level viraemia. (B241.49.w49) 
  • Chronic infection of tissues has been reported in monkeys. (B324.33.w33)
  • Experimental infection in Eulemur fulvus (Lemuridae - Large lemurs (Family)) produced a viraemia of four to six days duration, at a level sufficient to infect Aedes aegypti  (Aedes aegypti - Yellow fever mosquito) mosquitoes. (J91.34.w1)
  • Neither humans (Homo sapiens - Human) nor horses (Equus caballus - Domestic horse) are considered to play a role in the transmission cycle; they are incidental hosts. (B243.31.w1)
  • Among mammals, "only horses and lemurs have moderate viraemia and seem to support West Nile virus circulation locally." (J84.5.w2)
  • Equines develop only low magnitude viraemias of short duration when infected with WN virus; they are unlikely to act as amplifying hosts. (P39.4.w16)
  • Horses developed peak viraemias of only 2 PFU/ml blood serum. Aedes albopictus - Asian tiger mosquito fed on infected horses did not become infected. (P48.1.w16)
  • In brain and spinal cord of infected, clinically affected, horses, virus titres of 104.0 to 106.8 PFU/gram tissue have been recorded. (P39.4.w16)
  • Horses experimentally infected by bite of infected mosquitoes developed only very low titre viraemia (highest viraemia titre approximately 460 Vero cell plaque forming units (PFUs) per ml). Viraemias were not sufficient to infect Aedes albopictus (Aedes albopictus - Asian tiger mosquito) mosquitoes fed on eight of the horses on days 3, 4 and 5 post infection. (J133.951.w37)
  • Dogs experimentally infected developed low level viraemia and were considered unlikely to act as competent hosts. (P39.4.w16,  P48.1.w16)
  • Cats experimentally infected developed viraemia approaching the threshold required for competence; occasional mosquitoes may become infected by feeding on infected cats. (P39.4.w16, P48.1.w16)
  • Infected mice acted as an adequate source of virus to infect cats (J84.10.w1) and an owl Bubo virginianus - Great horned owl.(J84.9.w2)
  • Levels of viraemia seen in cats infected experimentally either by mosquito bite or by consumption of infected mice were considered sufficient for infection of mosquitoes, although at a lower efficiency than with many bird species. (J84.10.w1)
  • Pigs experimentally infected developed low level viraemia and were considered unlikely to act as competent hosts. (P48.1.w16)
  • WN virus has been detected in human breast milk by TaqMan PCR . (W27.04Oct02.wnv1, N7.51.w1)

Reptiles:

  • A study of alligators with WNV infection in Florida found that viral loads were sufficiently high to infect mosquitoes. (W27.07Aug03.wnv1)
  • Viral loads in plasma were measured as 103.6 to 106.5 log10 pfu/ml. Liver had the highest viral loads of those tissues which were sampled, reaching a maximum of 108.9log10 pfu/0.5cm3, while in brain and spinal cord viral loads were much lower, maximum 106.6 log10 pfu/0.5cm3. (P39.4.w16)

Amphibians:

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Mechanisms of Spread of Virus between Host Animals

Source Information General data: 
  • "Bird migration appears to be the major mechanism of WNV dissemination. Birds could spread the virus either in the viraemic state or by serving as hosts for infected ticks." (B244.w1)
  • Transmission between geographical areas by migrating birds was thought likely when virus isolates from birds in Siberia, Russia, in summer 2002 were found to have a high level of homology with an isolate from Volgograd (Caspian Sea region, Russia) in 1999. (W27.16May03.wnv1)
  • Comparison of isolates from various locations in Africa and in France, from a variety of hosts and vectors and different years, revealed closely related subtypes in distant geographical locations. The data was considered to be consistent with dissemination of the virus by migrating birds. (J223.78.w1)
  • "Studies in Egypt, Israel and South Africa , have implicated Cx. univittatus [Culex univittatus Culex (Genus)] as the main species transmitting WN virus in all three countries based on field isolation rates." (B241.49.w49)
  • "Although many vertebrate species show evidence of exposure to WN virus in nature, wild birds have been most consistently implicated as important hosts in the transmission cycle of this virus." (B241.49.w49). 
  • In Europe, the principle cycle of WN virus circulation is a rural, sylvatic, cycle, with transmission between wild, usually wetland birds and ornithophilic mosquitoes. A second, urban, cycle may also occur (as in the outbreak in Bucharest, 1996-97), between synanthropic or domestic birds and mosquitoes (mainly Culex pipiens/molestus) (Culex pipiens complex - Northern and Southern house mosquitoes) feeding on birds and on humans. (J84.5.w2)
  • Based on the rate of spread (about 300km maximum during three months of activity in 1999 and 400km during four months of known activity in 2000), the directions of spread (north and east as well as south and west) and the relative suitability of different bird species to act as competent hosts and transport the virus, it has been suggested that spread of WN virus through the New World from the starting point in New York can be explained by the non-migratory but dispersive movements of Passer domesticus - House sparrow. (J269.94S.w1)
  • Mosquitoes are generally recognised to be the major vectors of WN virus. (B244.w1)
  • Transmission may also occur via other arthropods, including ticks (Argasidae - Soft ticks (Family) or Ixodidae - Hard ticks (Family)) and e.g. Oeciacus hirundinis - swallow bug. (J64.19.w1)
  • WN virus has been isolated from ticks on several occasions, but their involvement in the transmission cycle of the virus has not been defined. (B241.49.w49)
  • Enzootic/maintenance vectors are competent vector species, principally feeding on birds and not necessarily involved in transmission to humans or horses. (P39.4.w14)
  • Epizootic/epidemic vectors (bridge vectors) are competent vector species which are more general feeders. These species may not be able to maintain infection in nature in the absence of enzootic vector species. (P39.4.w14)
  • In New York, Culex pipiens appears to be an important enzootic vector for WN virus. (J84.7.w15)
  • A bird-tick cycle may occur in some dry warm habitats which do not contain mosquitoes. (J84.5.w2)
  • In Egypt, low-level transmission by mosquito (possibly Culex pipiens) may occur even in the winter. WN virus has been isolated from ticks (Argas hermanni) (Argas (Genus)) collected in February; ticks may also play a role in overwintering of virus (B241.49.w49)
  • Overwintering of infected adult mosquitoes, transovarial transmission and migration of birds may all allow recurrence of infection in an area following a winter without local transmission of infection. (B241.49.w49)
  • Overwintering of arboviruses within arthropod vectors in temperate climates may involve overwintering of adult Culex pipiens (Culex pipiens complex - Northern and Southern house mosquitoes) mosquitoes, infected eggs of Aedes spp. and other arthropod vectors such as ticks. (P32.1.w4)
  • Work carried out in Queens, New York City, USA found that Culex pipiens and Culex restuans fed mainly on birds ( a variety of species) with only 2.8 and 13% of meals, respectively, taken from mammals; it was considered that the feeding habits demonstrated by the study "support their implication as enzootic vectors" of WN virus. It was further noted that the results did not rule out a possible role for Culex pipiens in transmitting WN virus to mammals (including humans). The species Culex salinarius and Coquilletidia pertubans fed mainly on mammals (81.2% and 82.6% respectively) but also took (fewer) blood meal from birds; these two species were considered from this data to be potential bridge vectors of WN virus. (J110.39.w5)
  • Data from field and laboratory studies indicates that in the USA Culex spp. probably act as maintenance vectors and that certain Aedes spp. and Ochlerotatus spp. probably act as bridge vectors transmitting WN virus from the Culex spp./avian cycle to humans and horses. (P39.4.w14)

Biological transmission and vector competence of mosquitoes:

Vertical transmission in mosquitoes:

  • Vertical transmission is known to be an important overwintering mechanism for survival of some mosquito- and tick-borne flaviviruses. Flaviviruses are capable of entering the fully developed egg through the micropyle at the time of fertilisation/oviposition. B243.31.w1, B324.33.w33)
  • Vertical transmission was demonstrated following intrathoracic inoculation of Culex pipiens mosquitoes with WN virus. Virus was isolated by cell culture from 2/1,417 F1 adult progeny after rearing at 18C (minimum filial infection rate of about 1.4 per 1000) and from 4/1,873 F1 adult progeny reared at 26C (minimum filial infection rate of 2.1 per 1000). In contrast to Culex pipiens, following infection of Aedes albopictus mosquitoes by intrathoracic inoculation, virus was not detected in any of more than 13,000 F1 progeny. (J110.39.w4)
  • WN virus was isolated from male Culex univittatus mosquitoes collected in Kenya in 1998. This first field isolation of WN virus from male mosquitoes indicates natural vertical transmission of the virus in this species of mosquito. (J91.62.w1)
  • Vertical transmission was demonstrated following intrathoracic inoculation of Culex pipiens quinquefasciatus and Culex tarsalis mosquitoes. (P39.4.w13)
  • Vertical and transtadial transmission of WN virus may act as an overwintering mechanism for the virus and could enhance the amplification rate. (P39.4.w13)

Bird-to-bird transmission:

  • Transmission from experimentally inoculated crows (Corvus brachyrhynchos - American Crow) to "in contact" crows housed with the infected birds has been demonstrated, in the absence of any potential mosquito vectors. Transmission was thought to be oral but the precise route of transmission (oral contact through preening or feather picking, through contact with food or water, via faeces or from shared perches) has not been determined. (J133.951.w9, W8.Nov01.WNV8, P38.2001.w1)
  • No transmission occurred from inoculated crows to other crows housed in the same room but not in direct contact. (J133.951.w9, W8.Nov01.WNV8)
  • A single instance of apparent transmission from an experimentally inoculated chicken (Gallus gallus domesticus - Domestic chicken (Phasianidae - Grouse, Turkeys, Pheasants, Partridges, etc. (Family))) to an "in contact" bird has been recorded, with the development of a transient low-level viraemia in the control bird. (J84.7.w22)
  • Bird-to-bird transmission (bird inoculated by mosquito to uninoculated cage mate) was demonstrated in Corvus brachyrhynchos - American Crow, Cyanocitta cristata - Blue jay, Pica pica - Black-billed magpie and Larus delawarensis - Ring-billed gull. (J84.9.w2)
  • Bird-to-bird transmission was demonstrated for geese; geese held in-contact with geese which had been subcutaneously inoculated with WN virus became infected despite being in a mosquito-proof room. Two of 20 individuals in-contact with the 10 infected three-week-old geese died while the virus was recovered from a further three individuals. The data was taken to suggest strongly that horizontal transmission could occur in commercial flocks and it was suggested that cannibalism and feather-picking of sick geese could aggravate transmission. (J6.32.w1)
  • Possible bird-to-bird transmission and apparent bird-to-human transmission, occurring at the same time as an outbreak of avian pox, associated with a high incidence (96% seropositive) of infection in female turkeys on one breeder farm and clinical illness and detection of IgM antibodies in several workers on the farm. (N7.52.w5)

Oral transmission:

Transovarial transmission in birds:

  • Following experimental infection via mosquito bite of 25 bird species, in birds which had survived infection and were euthanased at 14 days post infection, virus was detected in a variety of organs including in ovaries, suggesting the possibility of transovarial transmission. (J84.9.w2)

Transfusion and transplant (iatrogenic) transmission:

  • Iatrogenic human-to-human transmission was recorded for the first time in 2002. Transmission occurred through blood transfusions and organ transplants.
    • During the period 28 August to 2nd October 2002 15 patients from ten states were reported to the CDC with confirmed WN virus meningoencephalitis or meningitis, the illness having started within one month of the patients receiving blood or blood components. In some cases it was confirmed that blood used for such donations was infected with WN virus. (W380.Dec02.wnv1)
    • By 26th October 2002, 47 persons had been reported to CDC with possible transfusion-related WNV infection (confirmed or probable WNV infection with onset within a month after receiving blood components) and by 28th October investigation has shown such transmission in six persons and ruled out transmission by transfusion in 14 individuals, with investigations on the other 27 cases ongoing. (N7.51.w5)
    • Following 60 investigations, August 2002 to January 2003, 20 cases of transmission via blood or blood product transfusion had been confirmed, with 14 infectious donors identified and 21 investigations still ongoing; there was no transmission evidence in 19 cases. Virus was transmitted in red blood cells, plasma and platelets, with virus isolated from one stored unit of plasma. Five of the 14 infected donors and seven of the 20 confirmed recipient cases were asymptomatic. (P39.4.w2)
    • Transmission of WN virus has been confirmed in four recipients of solid organs from a single organ donor. (N7.51.w2, N7.51.w3, W380.Dec02.wnv1, J222.348.w1, P39.4.w2)
    • Blood transfusion was considered to be the probable source of infection for an organ donor from whom infection was subsequently transmitted to four other individuals. One blood donor seroconverted and retrieved stored plasma was found to be WN virus-positive by PCR. (J222.348.w1, P39.4.w2)
    • Testing of blood donations in the USA in 2003 detected 1,285 donations initially reactive for WN virus by nucleic acid-amplification (NAT) tests on pooled samples, with further testing indicating 601 presumptive viral donations (0.02% of total donations); results on a further 209 initially reactive donations were still pending. (N7.52.w1, N7.52.w4)

Laboratory-acquired percutaneous transmission:

  • Infections from laboratory accidents (laceration from a contaminated scalpel and puncture by a contaminated needle) have been documented. Infection occurred despite cleansing of the wounds, with onset at four and three days after exposure respectively. (N7.51.w7)
  • In 2001 a suspect case of laboratory-acquired infection was reported in New York. In 2002 two cases of occupationally-acquired infection occurred in laboratory personnel, both by percutaneous injury and both developing WN fever. (P39.4.w2)
  • Transmission can occur from an infected animal to a person through punctures and cuts. (W181.28Jan04.WNV6)
  • More than 20 cases of laboratory-acquired infection were reported up to 2002. (W181.28Jan04.WNV6)
    • Potential sources of infection in the laboratory include serum, CSF, other tissues and infected arthropods. (W181.28Jan04.WNV6)
    • Primary hazards are from procedures producing aerosols and from percutaneous inoculation; there are special hazards from animals (including mosquitoes) which are either naturally or experimentally infected. (W181.28Jan04.WNV6)

Transplacental transmission:

  • Transplacental transmission was recorded for the first time in 2002 in humans. Intrauterine infection was confirmed by the presence of WNV-specific IgM antibodies in serum and cerebrospinal fluid of the infant, which was born with severe cerebral abnormalities following maternal WNV infection in about the 27th week of pregnancy. (W27.19Dec02.wnv1, N7.51.w8)
    • Infection occurred late in the second trimester. Cord and heel-stick blood from the infant were WN virus IgM-positive and the placenta/umbilical cord was WN virus positive or equivocal in two laboratories. The mother had had a prolonged clinical illness. The infant presented with chorioretinitis, bilateral temporal/occipital loss of white matter and a temporal lobe cyst. (P39.4.w2)
    • In three other women with WN viral illness during pregnancy there was no infection in the infants. (P39.4.w2)

Maternal breast milk-associated transmission:

  • Transmission via breast milk was recorded for the first time in 2002 in humans. USA, 2002. WN virus was detected in human breast milk, by TaqMan PCR, from a woman who developed febrile illness with headache progressing to meningoencephalitis, onset eight days following receipt of WN virus-contaminated blood after delivery of her baby. The milk was positive also for WN virus-specific IgM and IgG. The woman's infant, who was breast fed, tested positive for WN virus IgM antibodies at 25 days old (the youngest person to be confirmed IgM positive in the USA to that date). The infant showed no clinical signs of illness. It was considered that the IgM antibodies in the infant indicated WNV infection in the infant, since transmission of IgM antibodies through milk is inefficient, and that the most likely source of the virus in the infant was from the mother's breast milk. (W27.29Sept02.wnv1, W27.04Oct02.wnv1, N7.51.w4)
    • WN virus genetic material was transiently present in milk from the mother. (P39.4.w2)
  • In hamsters (Mesocricetus auratus - Golden Hamster), transmission of WN virus in the milk to suckling hamsters has been described. (J64.19.w1)

Aerosol transmission:

  • Flavivirus general information: "Aerosols present a hazard of laboratory infection."  (B243.31.w1)
  • There is a single recorded instance of aerosol transmission to a human being (laboratory infection), and experimentally mice (Mus domesticus - Laboratory mouse) exposed to an aerosol of WN virus developed infection. Early finding of virus in the olfactory bulbs of the brain suggested an intranasal/olfactory route of CNS invasion by the virus, although lung infection also occurred. (J127.46.w1)

Other routes:

  • Failure of transmission has been demonstrated following application of large quantities of WN virus to the skin or eyes of laboratory mice (a susceptible species). (J71.54.w1, J127.46.w1)
  • In outbreaks of disease in farmed alligators (Florida Alligators (Alligator mississippiensis - American alligator (Alligatoridae - Alligators & Caimans (Family)))) it has been suggested that infection may be transmitted between alligators via the water in the holding tanks. (W27.20Nov03.wnv1)

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Routes of Infection - entry of the virus into a new host body

Source Information
  • Flaviviridae (Virus Family): "natural infections result from relatively low-dose peripheral inoculation by arthropod bite." (B240.14.w14).
  • Flaviviridae (Virus Family): "After inoculation into the skin, the virus replicates in local tissues and regional lymph nodes. Virus is then carried via lymphatics to the thoracic duct and into the bloodstream. This primary viraemia seeds extraneural tissues, which in turn support further viral replication and serve as a source for release of virus into the circulation. Major extraneural sites of flavivirus replication include connective tissue, skeletal muscle and myocardium, smooth muscle, vascular endothelium, lymphoreticular tissues, and endocrine and exocrine glands." (B240.14.w14)
    • Following initial inoculation mainly in extravascular tissues, the virus replicates locally then spreads via lymphatics to regional lymph nodes. (B324.33.w33)
  • Flaviviridae (Virus Family): The route of neuroinvasion "remains controvertible", and may be haematogenous or by infection of olfactory neurons (unprotected by the blood-brain barrier) with axonal transport to the olfactory lobe of the brain and subsequent rapid spread from this site. (B240.14.w14)

SPREAD WITHIN THE VERTEBRATE HOST:

  • Initial replication is assumed to occur within dendritic cells at the site of inoculation. The virus moves to local lymph nodes, probably in Langerhans' dendritic cells migrating from the skin, enters the lymph system and then the vascular system. By the vascular route it spreads to peripheral organs such as spleen, liver, kidneys, heart and lungs. If it crosses the blood-brain barrier virus also replicates in neurons. (J214.267.w9, P48.1.w8)
  • Replication in peripheral tissues appears to be important in WNV infection. (J214.267.w9)
  • Replication in epithelial cells appears to be important for the WN virus to cross the blood-brain barrier.(J214.267.w9)
  • Although perivascular cuffing with inflammatory cells occurs the cells do not appear to transport viral antigen across the blood-brain barrier. (J214.267.w9)
  • An alternative route for entry into the CNS may be via the olfactory mucosa where the blood-brain barrier is absent. (J214.267.w9)
  • Entry into cells occurs via attachment to a specific receptor on the cell surface; the virus enters the cell, its RNA is translated, and replication of virus occurs. (P48.1.w8)

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Cell Infection and Virus Replication

Source Information Entry into the cell and replication: (J285.3.w1, J286.44.w1, B324.32.w32, B324.33.w33,)
  • The virus attaches to the host cell surface via an as-yet unidentified receptor and the virion is taken up into the cell probably by receptor-mediated endocytosis, into a vesicle. (J285.3.w1, B324.32.w32)
    • The E protein is thought to be involved in the binding of the virion to cell receptors. (B324.32.w32, B324.33.w33, J286.44.w1)
  • Single virions and virion aggregates are found in clathrin-coated pits in the cell surface; attachment is rapidly followed by uptake into coated vesicles. Later the virions are found in uncoated prelysosomal vesicles. The nucleocapsid is released from the vesicle by acid-catalysed membrane fusion. Low pH induces a conformational change in the viral E peptide to expose the fusogenic domain. (J285.3.w1, B324.32.w32, J286.44.w1)
  • After entry and fusion presumably nucleocapsids are disassembled, after which genomic RNA is translated and RNA is initiated. (B324.32.w32)
  • The flavivirus genome is translated as a large polypeptide and this is processed co-translationally and post-translationally by cellular proteases and a virally encoded serine protease into ten distinct products (three structural and seven non-structural). Translation is associated with the rough endoplasmic reticulum, with some peptides being translocated into the lumen of the endoplasmic reticulum while other remain in the cytoplasm. (J285.3.w1, B324.32.w32)
  • Translation can begin with the first AUG within the long open reading frame (ORF); there is a second in-frame AUG 12 to 14 codons downstream from this, and this may also act as a translation initiations site. (J286.44.w1.)
  • Translation termination for WN virus is at the termination codon UAA. (J286.44.w1)
  • The individual viral proteins are produced by proteolysis. (J286.44.w1)
  • The polypeptide is cleaved by cellular signal peptidase (signalase) in the lumen of the ER to generate the N-termini of prM, E, NS1 and NS4b. The peptide prM is cleaved, during the egress of the virion, by host enzyme furin to form the structural protein M and the N-terminal pr segment. The other polypeptide cleavages are mediated by NS2b-3 protease to generate the N-termini of NS2b, NS3, NS4a and NS5. (J285.3.w1)
  • The C-prM cleavage by signalase and core glycosylation of prM occur in the lumen of the ER. (J286.44.w1)
  • The roles of the non-structural proteins are mainly in the replication of viral RNA. 
    • RNA replication requires NS1 and its interaction with NS4a. 
    • NS2a peptide, which is hydrophobic, functions in the assembly and/or release of infectious flavivirus particles. 
    • NS2b forma a complex with NS3; it is a required co-factor for the serine protease function of NS3. 
    • NS3 peptide has multiple functions, acting as a serine protease, nucleoside triphosphatase and helicase. 
    • The function of NS5 is as the viral RNA-dependant RNA polymerase as well as the methylltransferase involved in methylation of the 5 RNA cap structure. 
  • "It seems likely that translation in associating with the rough ER (RER), translocation, cleavage, acquisition of the correct protein topology, assembly of the replication complexes, and virion morphogenesis are all closely related phenomena." (J286.44.w1)
  • The plus-strand genomic RNA is also transcribed into a complementary minus-strand RNA which then serves as a template for synthesis of further plus-strand RNA; about 10-100 times more plus-strand than minus-strand RNA is synthesised. (J285.3.w1, B324.32.w32, J286.44.w1)
    • One synthesised the plus-strand RNA may be used for translation of viral polypeptides, synthesis of minus strand RNA or be encapsulated into virions. (J286.44.w1)

Virus assembly and release:

  • Flaviviridae (Virus Family)- Flaviviruses: virus particles may be found within cisternae of the endoplasmic reticulum. Virions are released from infected cells: "Mature [virus] particles within the cisternae of membranous organelles are released extracellularly either by fusion with the plasma membrane (reverse pinocytosis) or (in severely damaged cells) by lysis of the plasma membrane." (B240.14.w14)
  • Virus assembly takes place within the lumen of the endoplasmic reticulum. (J285.3.w1)
    • Morphogenesis of virions occurs in association with intracellular membranes and by electron microscopy morphologically mature virions have been demonstrated within the lumen of the endoplasmic reticulum. (B324.32.w32, J286.44.w1)
  • Interaction of the highly basic C protein with viral genomic RNA in the cytoplasm results in formation of a nucleocapsid precursor. (B324.32.w32, J286.44.w1)
  • It is thought that the hydrophobic segment of the anchored C protein may serve to localise nucleocapsid assembly to membrane sites. (J286.44.w1)
  • The nucleocapsids are thought to acquire their envelope by budding into the lumen of the endoplasmic reticulum. (B324.32.w32)
  • Fusion-incompetent immature virions are produced, in which the E-protein forms a stable heterodimer complex with prM. (J285.3.w1)
    • It is thought that the prM-E heterodimer prevents the E protein from undergoing acid-catalysed conformation change while the immature virion is transiting through the acidic intracellular compartment. (J285.3.w1, B324.32.w32)
  • The immature virions are transported through the host secretory pathway to the cell surface where exostosis occurs. (J285.3.w1)
  • Transport of nascent virions from the endoplasmic reticulum to the cell surface (where exostosis occurs) is thought to involve vesicles which may arise from either the endoplasmic reticulum or other components of the host's secretory system. (J286.44.w1)
  • Late in infection dissolution of vesicles may accompany cytolysis. (J286.44.w1)
  • The peptide prM is cleaved to pr and M by furin, a host protease, just before virion release from the cell; this destabilises the E-M heterodimer (J285.3.w1, B324.32.w32, J286.44.w1) and the E peptides apparently form trimeric complexes in WN virus (J286.44.w1) producing infectious mature virions: the specific infectivity of extracellular WN virus (about 600 particles per plaque forming unit) is about 60-fold higher than that of intracellular virus. (J286.44.w1)
  • It is thought that nascent virions are transported to the cell surface, where exostosis occurs, through the secretory pathway. (B324.32.w32)
  • Synthesis of viral RNA can be detected at three to six hours after infection and release of infectious virus at 12 hours. (J286.44.w1)
  • Infection is commonly lethal for the cell although some vertebrate cell types may become chronically infected with flaviviruses. (J286.44.w1) Infections of arthropod cells with flaviviruses are generally noncytopathic. (J286.44.w1)
  • Infection may cause dramatic cytopathic and ultrastuctural changes in cells of vertebrates, including vacuolation and proliferation of intracellular membranes, although no major inhibition of host macromolecule sythesis is observed. (J286.44.w1)

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Authors & Referees

Authors Debra Bourne MA VetMB PhD MRCVS (V.w5)
Referee Suzanne I. Boardman (V.w6); Becki Lawson (V.w26); Dr Robert G. McLean (V.w42); Charalambos Billinis DVM DrMedVet (V.w174), George Valikos DVM (V.w175)

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