How does wnv reproduce

For biological transmission to evolve, the factors that favor appropriate encounters among virus, competent vector, and susceptible vertebrate are of fundamental importance[ 13 ]. But, no matter how perfectly other basic requirements for the establishment of biological transmission are met, many viruses do not replicate sufficiently in certain hosts because of genetic constraints[ 13 , 23 , 24 ]. However, these mutations result in attenuation and do not completely abolish replication.

Until now, temperature has not been identified as a factor that can restrict flavivirus host range, although previous studies have suggested that alphavirus host range can be restricted, at least in part, by temperature. A second, but more complex, explanation involves the response of mammalian cells to sub-physiological temperatures. Low temperature cultivation of mammalian cells also results in prolonged generation time and maintenance of cell viability for longer periods, reduced glucose and glutamine consumption, suppressed release of waste products, delayed apoptosis, reduced protease activity, and improved tolerance to shear stress[ 33 — 36 ].

From this, we postulate that changes associated with the response to sub-physiological temperatures in mammalian cells make them susceptible to RABV infection. This is consistent with the fact that RABV could infect mosquito cell culture regardless of temperature, and it stands to reason that poikilotherm cells are more resistant to temperature-induced change because, in nature, they constantly have to deal with shifts in temperature.

Regardless of the mechanism, understanding the factors involved in the maintenance and spread of an infectious organism are crucial for timely recognition of emerging infections.

This becomes especially relevant when one considers that a large proportion of emerging viral infections are caused by multi-host zoonotic RNA viruses, because these viruses have a higher propensity to switch hosts[ 37 — 40 ]. Clearly, RABV is capable of replicating in vertebrate cells if conditions are appropriate, and it has not yet been excluded that RABV might circulate and be amplified in certain vertebrates, e. Therefore, RABV provides a powerful tool to study the evolution and molecular determinants of flavivirus host range, as well as genetic changes in the pathogen that facilitate emergence.

Identifying the mechanism s involved in mediating RABV temperature sensitivity will be the subject of future investigations. As such, reverse genetic tools currently are being developed for RABV and will be especially valuable in efforts to elucidate genetic changes that facilitate host switching, which will provide a comprehensive molecular portrait of flavivirus-host cell interactions.

Kuno G: Host range specificity of flaviviruses: correlation with in vitro replication. J Med Entomol. Syst Biol. J Gen Virol. Arch Virol. Parasit Vectors. Article PubMed Google Scholar. Cook S, Holmes EC: A multigene analysis of the phylogenetic relationships among the flaviviruses Family: Flaviviridae and the evolution of vector transmission.

J Mol Evol. Kuno G, Chang GJ: Biological transmission of arboviruses: reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends.

Clin Microbiol Rev. PLoS One. Emerg Infect Dis. Viral Immunol. Cell Host Microbe. At late times after infection, differential activation of the UPR pathways was observed in Kunjin infected mouse cells with the ATF6 and IRE1 arms but not the PERK arm being activated and this was postulated to contribute to maintaining cell survival [ , ].

In contrast, in WNV infected human neuroblastoma cells and primary rat hippocampal neurons activation of all three of the UPR pathways as well as of apoptosis pathways was observed [ ]. A number of cell proteins that bind to regions of the UTRs of flavivirus RNAs have been reported [ 58 , , , , , , , , , , ].

Three cellular proteins with molecular masses of 52, 84 and kDa were reported to bind specifically to probes consisting of the two 3' terminal SLs of the genomes of WNV and 3 other flaviviruses [ ]. The binding activity of eEF1A for the 3' terminal RNAs of four divergent flaviviruses was similar and strongly suggested that this interaction is a common characteristic of the genomes of the members of the genus Flavivirus [ 47 ].

Three of the four nts of the major binding site are typically basepaired in the 3' terminal SL. Mutation of these nts decreased in vitro eEF1A binding while disruption of the base pairs by substitution of the pairing partners increased in vitro eEF1A binding [ ]. Interestingly, increased synthesis of minus strand viral RNA reduced the amount of progeny genome produced, indicating that the correct ratio of minus to plus strand RNA is important for the overall virus life cycle.

Interestingly, substitution of this nt with a C had a greater negative effect on in vitro eEF1A binding activity than deletion of the entire sHP. Consistent with the different eEF1A binding activities, the G87C mutation was lethal in an infectious clone but viral RNAs with either an A or a U at this position were still able to carry out a sufficient amount of viral RNA synthesis to generate a revertant with the parental G. Even though the nts involved in flavivirus genome 3'—5' basepairing have been identified, little is known about the mechanisms regulating the formation and disruption of this long distance RNA-RNA interaction.

A cis -acting metastable structural feature located in the middle of the terminal 3' SL that consists of small, symmetrical bulges flanking two base pairs was identified [ 47 ]. It is expected that initiation of plus strand synthesis would occur from the minus strand product.

However, how the 3' end of a nascent minus strand is released from the duplex formed with the genome template so that it can initiate plus strand synthesis is not known Figure 2 C. The copying of a nascent genome from the new minus strand would release the original genome template.

During the early stage of the virus replication cycle, the reinitiation of genome synthesis is not efficient and similar low levels of plus and minus RNA are made Figure 2 D. The binding of the NS5 MTase to the 5' terminal nts of a nascent plus strand product and then to sites on the 5' SLA as it forms in the nascent plus stand as part of the cap addition process may facilitate release of the 3' end of the minus strand and formation of the terminal 3'SL on the minus strand template needed for reinitiation of plus stand synthesis.

These multifunctional proteins are members of the RRM family of RNA-binding proteins, bind to AU rich RNA sequences, are expressed in most tissues and shuttle between the nucleus and cytoplasm of infected cells [ , , , ]. Deletion or C substitution of the AU-rich sequence in either loop in a WNV infectious clone was lethal and partial deletion or substitution of these sequences reduced the efficiency of virus replication [ ].

These mutations did not affect the translation efficiency of the genome RNA. The observation that the replication of WNV, but not that of viruses from other families, was less efficient in TIAR-knockout cell lines than in wild type cells also suggested a functional role for these proteins during WNV replication [ ].

In a recent study, individual WNV nonstructural proteins were expressed in Vero cells in different combinations and their interactions were analyzed by various imaging techniques, including confocal microscopy, fluorescence resonance energy transfer, and biologic fluorescence complementation [ ].

NS5 was found to only interact with NS3. Previous studies done with dengue NS3 and NS5 proteins also demonstrated interaction between these two proteins and suggested that the phosphorylation state of NS5 affected the interaction [ ]. The interacting regions of the dengue proteins were mapped to the C -terminus of NS3 residues to and the N -terminus of NS5 residues to with Lys identified as the NS3 helicase domain interacting residue [ , ].

Although similar in vitro studies have not yet been done with WNV NS3 and NS5 proteins, it is speculated that the results would be similar to those found with the dengue proteins. In the same recent study, consistent with previous data, NS3 was found to also interact with its membrane associated co-factor NS2B [ ].

No interactions between NS1 and other nonstructural proteins were detected in this study. Dengue NS1 was previously found not to trans-complement a yellow fever virus genome with an NS1 deletion but genome variants were selected that could utilize the dengue NS1 and sequencing detected a single point mutation in the NS4A gene suggesting that an interaction between NS1 and NS4A is required for early viral RNA synthesis [ ].

A compensatory mutation in the viral NS4B gene rescued the impaired replication phenotype of the NS1 mutant but had no effect on the replication of the wildtype genome.

At early times of infection when the number of genomes present is low, genomes are expected to switch back and forth between the cyclized replication form and the non-cyclized translation form. How this switching is regulated is not known.

Natural lineage 1 and lineage 2 WNV strains produce low levels of viral RNA at early times after infection but produce a sufficient amount of viral protein to block the Type I IFN response [ , ]. These findings suggest that translation is favored over minus strand synthesis at early times of infection. Lower early viral protein levels and less efficient counteraction of the Type 1 IFN response were also observed in WIC infected cells suggesting that cyclized genomes predominated at early times [ ].

The phenotype of the WIC chimera suggested that interactions between specific residues of the viral nonstructural proteins may be involved in down-regulating the efficiency of early viral RNA synthesis. The low early viral RNA level phenotype of the natural virus strains was rescued by replacement of the combination of the C -terminal 23 aa of E plus NS1 plus the C -terminal aa of NS3 plus the N -terminal aa of NS4A with Eg sequence in a clone with the chimeric NS5 but not by replacement of any of these individual regions.

Interestingly, viral replication was less efficient for chimeras with a hybrid NS5 than for ones with full length Eg NS5. The majority of the residues that differ between the DB3 and Eg NS5 sequences are predicted to be on the protein surface and none appear to be directly involved in RdRp function supporting the hypothesis that some of these residues may be protein interaction sites.

Substitutions at R, RK, R and KR in a dengue NS5 had no effect on in vitro polymerase activity but were lethal in an infectious clone [ ]. Also, dengue NS5 mutants that had wildtype in vitro polymerase activity as recombinant proteins caused impaired virus replication when they were present in an infectious clone [ ]. The finding that substitution of single residues in the WNV RdRp located near the rNTP binding pocket but not in direct contact with the incoming rNTP altered both plaque morphology and the kinetics of viral RNA replication also suggest subtle changes in the RdRp that are remote from the active site are able to affect function.

The biological fitness of these mutants was tested in a number of insect cell lines and Vero cells as well as in one day old chickens and Culex pipiens mosquitoes. While all of these mutants were significantly attenuated for virus growth in the cell lines and chickens with the NS5 AN mutant having the greatest negative effect, all of the mutants grew as efficiently as wildtype virus in Culex mosquitoes indicating that the effects on virus fitness were host dependent [ ].

The data from all of the above studies suggest that interactions between NS5 and other viral nonstructural proteins as well as cellular proteins regulate the RNA synthesis functions of NS5 and this regulation may differ at early and late times after infection and in different host species.

However, these and other modifications could also regulate interactions between these viral proteins and cell proteins. A number of cell proteins have been reported to interact with flavivirus NS5 or NS3. In another study, a high-throughput yeast two-hybrid screen identified human proteins interacting with either NS3 or NS5 or with both proteins [ ]. The functional relevance of the majority of the cellular proteins reported to interact with NS5 has not yet been tested.

However, in some cases, even though a robust in vitro interaction was demonstrated, the functional relevance of the interaction in infected cells could not be demonstrated [ ]. It is possible that the surfaces of NS5 that are exposed when it is expressed alone as a recombinant protein differ from when it is in the context of the complete replication complex in an infected cell.

During the exponential genome synthesis phase, nascent genomes exit replication complex vesicles through a pore [ ]. A recent study suggested that the nascent flaviviral RNAs may exit into a specialized compartment in the cytosol that provides a protective environment [ ]. Nascent genomes can be replicated, translated or assembled into a virion in association with ER membranes.

It is thought that the hydrophobic face of C dimers binds to the cytoplasmic side of the ER membrane while the charged face binds to genomic RNA in a sequence nonspecific manner [ 10 , ]. This idea is consistent with the observation that large deletions in the central hydrophobic region of the C protein of TBEV were tolerated [ ] and that no specific encapsidation signal sequence in flavivirus genomic RNA that was recognized by the C protein could be identified [ ].

The transmembrane domains of the E and prM proteins insert into the ER membrane with their exodomains located in the ER lumen. The assembly of virions is thought to occur due to interaction of a genomic RNA with membrane-associated capsid dimers in areas where E and preM proteins are also located leading to immature virion budding into the ER lumen.

Recent data also suggest that NS2A may play a role in virion assembly [ ]. In this conformation, the E fusion peptide is protected from triggering premature fusion with cellular membranes in the mildly acidic compartments of the cell secretory pathway during virion egress [ , ].

The immature virions are transported through the Golgi and secretory pathway where glycans on E and prM are modified. Mature virions are transported to the plasma membrane in vesicles and released by exocytosis. Typically, WN virions are released from infected cells starting at 8 to 10 h after infection and peak extracellular virus titers are usually observed by 24 h.

Smooth, noninfectious, subviral particles called slowly sedimenting hemagglutinin SHA , that are composed of a circular cellular membrane inserted with E and M proteins as well as some prM are also produced by infected cells [ , ]. The specific involvement of a number of cell proteins in the assembly, transport and release of flaviviruses has been suggested. Interaction of WNV C protein with the nucleolar helicase DDX56 was reported to enhance the assembly of infectious virions [ ] and a subsequent study showed that the helicase activity of DDX56 was required for its role in assembling infectious virions [ ].

Studies with a WNV chimera lacking the C coding region and expressing dengue 2 prM and E instead of the WNV surface proteins, showed that mutations in NS2A or NS3 independently enhanced genome packaging and also that genomes that did not express NS1' were packaged more efficiently [ ]. Src family kinase c-Yes activity was reported to be required for transit of assembled immature WN virions from the ER through the secretory pathway [ ].

Alix, a cellular protein associated with the ESCRT machinery, was reported to interact with yellow fever virus NS3 and expression of dominant negative Alix proteins inhibited virion release [ ]. Increased accumulation of intracellular infectious virions was observed in dengue virus infected cells deficient in AP-1, a heterotetrameric adaptor protein complex involved in trafficking cargo molecules in the biosynthetic pathway back and forth between the trans-Golgi network and endosomes [ ].

A conserved capsid region sequence in the dengue genome RNA was also reported to enhance infectious virion assembly [ ] but it is not known what proteins interact with this RNA region. Although much has already been learned about the replication and molecular biology of flaviviruses, there are still many unanswered questions. Data from multiple studies confirm that flaviviruses utilize cell proteins during each step of their replication cycles but the roles of most of these host factors in virus replication are still not well understood.

WNV infection alternates between insect vectors and vertebrates in nature and some of the host factor proteins used by flaviviruses may differ in mammalian and insect hosts. Interactions between viral nonstructural proteins and also between these proteins and cellular proteins are likely to be complex and may occur only after expression of the viral proteins from the viral polyprotein and in the context of an infected cell. Also, infections initiated by genomic RNA transfection instead of virus infection would not activate the cell signaling pathways typically activated by virus attachment and entry.

Activation of cell signaling pathways at early times have been shown to have sustained effects throughout the infection cycle. Activation of cell signaling pathways may also be required for appropriate post-translational modification of viral proteins needed to regulate their functions. Although the 3' and 5' genome sequences involved in the long distance RNA-RNA interaction have been identified, the mechanisms regulating switching between the linear and cyclized forms of the genome are not known.

Although a number of interesting insights have been obtained about the early stages of the flavivirus replication cycle and about the mechanisms used by different flaviviruses to remodel infected cells, there is still much that is not known. National Center for Biotechnology Information , U. Journal List Viruses v. Published online Dec Margo A. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC.

Keywords: flavivirus replication, nonstructural proteins, nonstructural protein interactions, cis -acting RNA sequences, conserved RNA structures, host factors, RNA-protein interactions, host cell remodeling. Introduction West Nile virus WNV is maintained in nature in a mosquito-bird transmission cycle and has recently become endemic in the Western hemisphere.

Open in a separate window. Figure 1. Figure 2. Viral Polyprotein A single open reading frame ORF of 10, nt in most WNV isolates encodes a polyprotein that is co- and post-translationally processed by the viral serine protease complex NS2B-NS3 and various cellular proteases into 10 mature viral proteins [ 89 , 90 ]. Viral Nonstructural Proteins Although the functions of the WNV nonstructural proteins have not yet been completely characterized, all seven are directly or indirectly involved in viral RNA synthesis and additional functions for some of these proteins have been identified.

Nonstructural Protein 1 NS1 is a glycoprotein with three conserved N -linked glycosylation sites and multiple conserved cysteines that form disulfide bonds essential for virus viability [ , , ]. Nonstructural Protein 3 The N -terminal residues of NS3 comprise a serine protease that is active only when complexed with NS2B [ , , ]. Nonstructural Protein 5 NS5 is located at the C -terminus of the viral polyprotein Figure 1 A and is the largest and most conserved of the flavivirus proteins.

Efficient WNV replication in infected cells has been shown to require fatty acid synthesis but WNV infections do not redistribute phosphatidylinositolphosphate lipids to their replication complex vesicles [ ] At early times after infection, translation of viral RNA likely occurs by a cap-dependent mechanism.

Nonstructural Protein Interactions and Regulation of Viral RNA Replication In a recent study, individual WNV nonstructural proteins were expressed in Vero cells in different combinations and their interactions were analyzed by various imaging techniques, including confocal microscopy, fluorescence resonance energy transfer, and biologic fluorescence complementation [ ]. Progeny Virus Assembly and Release During the exponential genome synthesis phase, nascent genomes exit replication complex vesicles through a pore [ ].

Conclusions Although much has already been learned about the replication and molecular biology of flaviviruses, there are still many unanswered questions. Conflicts of Interest The author declares no conflict of interest.

References and Notes 1. Berthet F. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses.

Jia X. Genetic analysis of West Nile New York encephalitis virus. Lanciotti R. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Beasley D. Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype.

Heinz F. Family Flaviviridae. In: Regenmortel C. Virus Taxonomy. Murray J. Processing of the dengue virus type 2 proteins prM and C-prM. Adams S. Glycosylation and antigenic variation among Kunjin virus isolates. Zhang W. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus.

Kiermayr S. Isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particles. Solution structure of dengue virus capsid protein reveals another fold. Pierson T. In: Knipe D. Fields Virology. Mukhopadhyay S. A structural perspective of the flavivirus life cycle. Lee E. Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses.

Davis C. Rios M. West Nile virus adheres to human red blood cells in whole blood. Jemielity S. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.

PLoS pathog. Chu J. Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. Bogachek M. Characterization of glycoprotein E C-end of West Nile virus and evaluation of its interaction force with alphaVbeta3 integrin as putative cellular receptor.

Lee J. Schmidt K. Integrins modulate the infection efficiency of West Nile virus into cells. Wan S. Medigeshi G. Barrows N. Factors affecting reproducibility between genome-scale siRNA-based screens. Krishnan M. RNA interference screen for human genes associated with West Nile virus infection. Sessions O. Discovery of insect and human dengue virus host factors. Le Sommer C. G protein-coupled receptor kinase 2 promotes flaviviridae entry and replication.

PLoS Negl. Fernandez-Garcia M. Cheng G. A C-type lectin collaborates with a CD45 phosphatase homolog to facilitate West Nile virus infection of mosquitoes. Rab 5 is required for the cellular entry of dengue and West Nile viruses.

Van der Schaar H. Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog. Allison S. Oligomeric rearrangementof tick-borne encephalitis virus envelope proteins induced by an acidic pH.

Structures and mechanisms in flavivirus fusion. Virus Res. Ivanyi-Nagy R. RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.

Nucleic Acids Res. Pong W. FEBS Lett. Scherbik S. Urbanowski M. Studies showed that for the protection against lineage I, perforin played the most important role and, in contrast, lineage II strain Sarafend was controlled more efficiently by granzymes [ , ]. All the above data provide solid evidence that a combination of various aspects of both innate and adaptive immune response cooperate to control WNV infection in the periphery and CNS. Various studies especially in the last decade have recognized a variety of genetic determinants of virulence for West Nile virus strains.

Specific mutations have been found to attenuate or strengthen virus pathogenicity via various mechanisms. Those that have been found to be the most important will be reported here, focusing on the ones that seem to have major impact on the replication mechanisms of WNV. This glycosylation motif has been recognized to various flaviviruses and spatially is located in close proximity to the center of the fusion peptide of DII of E protein, and thus is considered to increase the stability of the protein to a fusion-active form even at high temperatures [ , ].

This proved to be really important for the multiplication of the virus to avian cell and animal models: results showed that E glycosylated WNV variants multiplied more efficiently to avian cell cultures and at high temperatures, causing at the same time high viremic titers and pathogenicity to chicks [ ]. Most of the Lineage I virulent strains as well as recent virulent Lineage II strains associated with the Greek outbreak carry the N-glycosylation site, suggesting it a prerequisite for the efficient circulation and amplification of the virus in a mosquito-avian transmission cycle [, Valiakos et al.

Of course it is possible that E glycosylation affects other aspects of the WNV replication cycle as well such as target cell tropism, virion assembly and release etc. Studies proved that substitution of cysteine an amicoacid which is often critical for the proper function of a protein with serine at position of NS4B, CysSer leads to sensitivity to high temperatures as well as attenuation of the neuroinvasive and neurovirulent phenotypes in mice [ ].

However this mutation implemented in North American Lineage 1 strains did not cause significant changes to phenotype indicating that in many cases the effect of mutations under study can be strain-specific. D73H and MK were mutations found to be related to poor replication and non mortality to mice [ ]. The introduction of a TP in North American Linage 1 strain was found to be sufficient to generate a phenotype virulent to American crows [ 99 ].

A HP mutation is considered to be the main cause of increased virulence of Lineage 2 strain that caused the major WNV disease outbreak in , in Greece.

Only the Greek sequences, detected in mosquito pools, corvids and chickens [5, , Valiakos et al. Hence, a proline residue in position of the NS3 position is not sufficient to enhance virulence, at least in certain cases [ , ]. Theoretically, substitutions of hydrophobic to hydrophilic amino acids and vice versa as well as substitutions of glycine, proline and cysteine residues are considered to have a potential effect on the secondary structure of proteins. West Nile virus is considered a serious public health threat, especially for high risk groups very young and elderly, imunocompromised.

Currently there has not been established any antiviral treatment to WNV infections; only supportive care may be administered. Vaccine development is still at an early stage for humans.

Hence, preventive measures rely still on reduction of mosquito populations and minimizing vector-host contact. Various diagnostic techniques have been developed the last decades, both molecular and serological, trying to minimize the difficulties arisen from other cross-reactive closely related flaviviruses.

Data presented here prove the complexity of the host-virus interaction: Specific host-pathogen-vector interface, cellular tropism, viral structure diversity regarding maturation, immune system recognition and response, genetic diversity are all factors characterized by great variation rendering WNV control extremely difficult.

Continuous studies are being demanded to understand the extent of this complexity to further elucidate biological relationships among host, vector and virus that will lead to improved disease control.

As more is learned about the biological characteristics of WNV infection, one continuing objective will be to relate this knowledge to the clinical features of disease. An important viral-host determinant is virus attachment, mediated by cellular receptor and allowing subsequent infection. Host defensive behaviors that could affect virus acquisition and transmission should be also further studied.

This may help in the design and implementation of more efficient and cost-effective control strategies since introduction of WN virus is an ongoing risk and reality. The ultimate challenge will be to apply the knowledge gained in understanding viral replication and unraveling the complexity leading to pathogenesis in order to prevent and control West Nile virus and its severe manifestations.

The research leading to these results received partial funding from the European Union Seventh Framework Programme under grant agreement no. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.

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Downloaded: Introduction West Nile virus WNV was first isolated in Uganda West Nile district in from the blood of a native Ugandan woman [ 1 ] and until the end of the 20 th century was considered a cause of viral encephalitis limited only in Africa and Asia. Table 1. Functions of West Nile virus nonstructural proteins.

Acknowledgement The research leading to these results received partial funding from the European Union Seventh Framework Programme under grant agreement no. More Print chapter. How to cite and reference Link to this chapter Copy to clipboard. Available from:. Over 21, IntechOpen readers like this topic Help us write another book on this subject and reach those readers Suggest a book topic Books open for submissions. More statistics for editors and authors Login to your personal dashboard for more detailed statistics on your publications.

Our replicon particles system is associated with several advantages: 1 the virus readily forms plaques in BWNV-CME cells and can be used to investigate the neutralization of WNV; 2 the standard PRNT approach involves the use of live infectious virus, which must be handled by a skilled investigator in an appropriate biocontainment facility, whereas our replicon particles system does not require BSL3 containment.

Moreover, the inhibition of viral entry can also be tested using a variety of cell types, including those that do not support the formation of plaques. DQ lacking the C-prM-E gene was constructed by chemical synthesis. This was performed to ensure the production of flavivirus RNA with an authentic start and terminus following transcription by a cellular RNA polymerase in the nucleus of the transfected cells. In addition, the 2A autoprotease of foot and mouth disease virus FMDV was cloned downstream of the insertion, which can liberate the GFP gene from the flavivirus polyprotein during translation Fig.

Two days later, the transfected cells were digested and cloned by limited dilution in well plates and growth in medium containing G The amount of E antigen produced by the cloned cells was examined and compared by Western blotting. One clone designated BWNV-CME that exhibited more efficient expression of the E antigen was selected and maintained in Gsupplemented medium for further characterisation and antigen production.

GFP fluorescence was observed at various time points post-infection. The viral titres were determined using a fluorescence unit counting method following infection of BHK or other permissive cells.

After a six-day incubation, the plate was stained in a 0. Then the stain solution was discarded, the cells were rinsed with distilled water and the plaques were counted. The efficiency of infection in vitro with the same preparation of WNV differs among different cell lines. To precisely estimate the number of reporter replicon particles produced by an infection and analyse the replicon secretion kinetics over time, we collected the culture fluid from infected cells at daily intervals and analysed it using BWNV-CME cells by performing plaque assays.

The presence of WNV neutralizing antibodies were tested using a plaque reduction assay. The cells were air-dried and the plaques were counted. Viral titre reduction assays were performed to examine the antiviral activity of ribavirin and 6-Azauridine. The cells were infected with individual virus MOI of 0. Brinton, M. The molecular biology of West Nile Virus: a new invader of the western hemisphere. Annu Rev Microbiol 56 , — Hayes, E. Epidemiology and transmission dynamics of West Nile virus disease.

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