Viral Genetics/Strain Differentiation
Bakonyi, T., E. Ivanics, K. Erdely, K. Ursu, E. Ferenczi, H. Weissenbock, and N. Nowotny (2006). Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerging Infectious Diseases 12(4): 618-623. ISSN: 1080-6040.
Abstract: Two different West Nile virus (WNV) strains caused lethal encephalitis in a flock of geese and a goshawk in southeastern Hungary in 2003 and 2004, respectively. During the outbreak in geese, 14 confirmed human cases of WNV encephalitis and meningitis were reported in the same area. Sequencing of complete genomes of both WNV strains and phylogenetic analyses showed that the goose-derived strain exhibits closest genetic relationship to strains isolated in 1998 in Israel and to the strain that emerged in 1999 in the United States. WNV derived from the goshawk showed the highest identity to WNV strains of lineage 2 isolated in central Africa. The same strain reemerged in 2005 in the same location, which suggests that the virus may have overwintered in Europe. The emergence of an exotic WNV strain in Hungary emphasizes the role of migrating birds in introducing new viruses to Europe.
Descriptors: Accipiter gentilis, Anser anser domesticus, viral diseases, West Nile virus, records and epidemiological implications, Hungary.
Beasley, D.W., M.C. Whiteman, and S. Zhang (2005). Envelope Protein Glycosylation Status Influences Mouse Neuroinvasion Phenotype of Genetic Lineage 1 West Nile Virus Strains. Journal of Virology: 8339-8347.
Descriptors: West Nile virus, strains, genetic linkeage, protein glycosylation, mouse neuroinvasion phenotype.
Benzaghou, I., I. Bougie, F. Picard Jean, and M. Bisaillon (2006). Energetics of RNA binding by the West Nile virus RNA triphosphatase. FEBS Letters 580(3): 867-877.
Abstract: The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.
Descriptors: acid anhydride hydrolases, viral RNA, West Nile virus genetics.
Davis, C.T., G.D. Ebel, R.S. Lanciotti, A.C. Brault, H. Guzman, M. Siirin, A. Lambert, R.E. Parsons, D.W. Beasley, R.J. Novak, D. Elizondo Quiroga, E.N. Green, D.S. Young, L.M. Stark, M.A. Drebot, H. Artsob, R.B. Tesh, L.D. Kramer, and A.D. Barrett (2005). Phylogenetic analysis of North American West Nile virus isolates, 2001-2004: Evidence for the emergence of a dominant genotype. Virology 342(2): 252-265.
Abstract: The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.
Descriptors: viral genome, West Nile virus, molecular sequence data, North America, species specificity, variation genetics, viral envelope proteins genetics.
Fayzulin, R., F. Scholle, O. Petrakova, I. Frolov, and P.W. Mason (2006). Evaluation of replicative capacity and genetic stability of West Nile virus replicons using highly efficient packaging cell lines. Virology 351(1): 196-209. ISSN: 0042-6822.
Abstract: A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.
Descriptors: variation genetics, virus assembly, virus replication physiology, West Nile virus, Cercopithecus aethiops, cricetinae, vero cells.
Jerzak, G., K.A. Bernard, L.D. Kramer, and G.D. Ebel (2005). Genetic variation in West Nile virus from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection. Journal of General Virology 86(8): 2175-2183. ISSN: 0022-1317.
Abstract: Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 3' 1159 nt of the WNV envelope (E) coding region and the 5' 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0.016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19.5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra- and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations.
Descriptors: Corvus brachyrhynchos, Corvus ossifragus, Culex pipiens, Culex restuans, Culex salinarius, Cyanocitta cristata, viral diseases, West Nile virus, virus genetic diversity, New York, Long Island.
Kinney, R.M., C.Y. Huang, M.C. Whiteman, R.A. Bowen, S.A. Langevin, B.R. Miller, and A.C. Brault (2006). Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology 87(Pt 12): 3611-3622.
Abstract: The NY99 genotype of West Nile virus (WNV) introduced into North America has demonstrated high virulence for American crows (AMCRs), whilst a closely related WNV strain (KEN-3829) from Kenya exhibits substantially reduced virulence in AMCRs [Brault, A. C., Langevin, S. A., Bowen, R. A., Panella, N. A., Biggerstaff, B. J., Miller, B. R. & Nicholas, K. (2004). Emerg Infect Dis 10, 2161-2168]. Viruses rescued from infectious cDNA clones of both the NY99 and KEN-3829 strains demonstrated virulence comparable to that of their parental strains in AMCRs. To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. Growth curves of the two WNV strains were performed in African green monkey kidney (Vero; 37-42 degrees C) and duck embryonic fibroblast (DEF; 37-45 degrees C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 degrees C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 degrees C relative to levels of intracellular virus-specific RNA measured at 37 degrees C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 degrees C demonstrated only a 17-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important factor leading to the increased avian virulence and emergence of this strain of WNV.
Descriptors: bird diseases, crows, virus replication, West Nile fever, West Nile virus, body temperature, cell line, Cercopithecus aethiops, ducks, plaque assay, viral RNA biosynthesis, survival analysis, temperature, vero cells.
Kononova, I., V.A. Ternovoi, M. Shchelkanov, E.V. Protopopova, S.I. Zolotykh, A.K. Iurlov, A.V. Druziaka, A.A. Slavskii, A.M. Shestopalov, D.K. L'vov, and V.B. Loktev (2006). [West Nile virus genotyping among wild birds belonging to ground and tree-brush bird populations on the territories of the Baraba forest-steppe and Kulunda steppe (2003-2004)]. Voprosy Virusologii 51(4): 19-23.
Abstract: The paper gives the results of the 2003-2004 examinations of 104 wild birds belonging to land tree-brush complexes from the Baraba forest-steppe and Kulunda steppe for the detection and genotyping West Nile virus (WNV). ELISA and RT-PCR were used to show that in the forest-steppe and steppe zones of the south of Western Siberia, WNV circulates among both migrating and settled birds. An analysis of the nucleotide sequence of a protein E gene fragment showed the circulation of WNV genotype Ia in the study birds. A number of revealed amino acid substitutions in surface glycoprotein E are unique for the 2003-2004 Western-Siberian WNV variants and absent in the 2002 Western-Siberian variants, which suggests that there are regional features of the evolution of WNV genotype Ia.
Descriptors: wild animals, birds, West Nile virus, amino acid sequence, animals, antigens, ecosystem, evolution, molecular sequence data, phylogeny, sequence alignment, Siberia, species specificity, trees, viral envelope proteins genetics.
Language of Text: Russian.
Langevin, S.A., A.C. Brault, N.A. Panella, R.A. Bowen, and N. Komar (2005). Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus). American Journal of Tropical Medicine and Hygiene 72(1): 99-102. ISSN: 0002-9637.
Abstract: The observation of avian mortality associated with West Nile virus (WNV) infection has become a hallmark epiderniologic feature in the recent emergence of this pathogen in Israel and North America. To determine if phenotypic differences exist among different WNV isolates, we exposed house sparrows (Passer doniesticits) to low passage, lineage 1 WNV strains from North America (NY99), Kenya (KEN), and Australia (KUN; also known as Kunjin virus). House sparrows inoculated with the NY99 and KEN strains experienced similar mortality rates and viremia profiles. The KUN strain elicited significantly lower-titered viremia when compared with the other strains and induced no mortality. This study suggests that natural mortality in house sparrows due to Old World strains of WNV may be occurring where the KEN strain occurs.
Descriptors: Passer domesticus, viral diseases, West Nile virus, virulence, variation between strains, mortality.
Li, J., R. Bhuvanakantham, J. Howe, and M.L. Ng (2006). The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes. Journal of General Virology 87(Pt 3): 613-622.
Abstract: The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/3' untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.
Descriptors: viral genome, viral envelope proteins, West Nile virus, amino acid sequence, cell line, Cercopithecus aethiops, glycosylation, molecular sequence data, sequence alignment, species specificity, vero cells, viral envelope proteins.
Pierson, T., M. Diamond, A. Ahmed, L. Valentine, C. Davis, M. Samuel, S. Hanna, B. Puffer, and R. Doms (2005). An infectious West Nile Virus that expresses a GFP reporter gene. Virology 334(1): 28-40. ISSN: 0042-6822.
Descriptors: West Nile virus, genetic vectors, green fluorescent protein, reporter genes, gene expression regulation, neutralization, virus replication, humans, cultured cells, nucleotide sequences, viral-antibodies, molecular-sequence-data, Internet-resource.
Ramanathan, M.P., J.A. Chambers, J. Taylor, B.T. Korber, M.D. Lee, A. Nalca, K. Dang, P. Pankhong, W. Attatippaholkun, and D.B. Weiner (2005). Expression and evolutionary analysis of West Nile virus (Merion strain). Journal of Neurovirology 11(6): 544-556.
Abstract: The authors report a new strain of West Nile virus (WNV) with the expression analysis of its individual open reading frames. Since its sudden appearance in the summer of 1999 in New York City, the virus has spread rapidly across the continental United States into Canada and Mexico. Besides, its rapid transmission by various vectors, the spread of this virus through organ transplantation, blood transfusion, and mother-child transmission through breast milk is of concern. In order to understand molecular variations of WNV in North America and to generate new tools for understanding WNV biology, a complete clone of WNV has been constructed. Investigations so far have focused only on half of its genes products and a detailed molecular and cell biological aspects on all of WNV gene have yet to be clearly established. The open reading frames of WNV were recovered through an reverse transcriptase-polymerase chain reaction (RT-PCR)-PCR using brain tissue from a dead crow collected in Merion, PA, and cloned into a mammalian expression vector. The deduced amino acid sequences of individual open reading frames were analyzed to determine various structural motifs and functional domains. Expression analysis shows that in neuronal cells, C, NS1, and NS5 proteins are nuclear localized whereas the rest of the antigens are confined to the cytoplasm when they are expressed in the absence of other viral antigens. This is the first report that provides an expression analysis as well as intracellular distribution pattern for all of WNV gene products, cloned from an infected bird. Evolutionary analysis of Merion strain sequences indicates that this strain is distinct phylogenetically from the previously reported WNV strains.
Descriptors: viral gene expression regulation, West Nile fever, West Nile virus, amino acid sequence, bird diseases, birds, molecular sequence data, phylogeny, reverse transcriptase polymerase chain reaction.
Rasgon, J.L., M. Venkatesan, C.J. Westbrook, and M.C. Hauer (2006). Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis. Molecular Ecology Notes. 6(3): 680-682. ISSN: 1471-8278.
Abstract: Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42-96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA-based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito.
Descriptors: Internet-resource, West Nile virus, vectors, C. tarsalis, genomic libraries, loci, microsatellites, genetic markers.
Scholle, F. and P.W. Mason (2005). West Nile virus replication interferes with both poly(I:C)-induced interferon gene transcription and response to interferon treatment. Virology 342(1): 77-87. ISSN: 0042-6822.
Abstract: West Nile virus (WNV), the leading cause of viral encephalitis in the United States, is an arthropod-transmitted member of the family Flaviviridae. We have explored the interaction of this positive-strand RNA virus with signaling pathways involved in induction of the host's innate immune response. Phosphorylation of STAT-1 in response to interferon (IFN) treatment and the ability of IFN to establish an antiviral state were reduced in WNV replicon-bearing cell lines. Similarly, the activation of IRF3 and stimulation of IFN-beta transcription in response to the double-stranded RNA (dsRNA) mimetic poly(I:C) were inhibited in replicon-bearing and WNV-infected HeLa cells. In contrast, WNV replicons did not affect IRF3 activation by Sendai virus infection, suggesting that not all IRF3 activating pathways are inhibited by WNV. Taken together, these findings demonstrate that WNV replication in cultured cells interferes with both the response to IFN and synthesis of IFN-beta in response to dsRNA.
Descriptors: interferon beta genetics, West Nile virus, cell nucleus active transport, dimerization, hela cells, natural immunity, interferon regulatory factor 3, viral genetics, signal transduction, transcription, genetic drug effects, virus replication.
Seligman, S.J. (2006). Single nucleotide polymorphisms in human genes and increased susceptibility to West Nile Virus disease. Journal of Infectious Diseases 193(8): 1187-1188; Author Reply 1188.
Descriptors: single nucleotide polymorphism genetics, West Nile fever, genetic predisposition to disease, natural immunity genetics, mice, West Nile virus (WNV).
Notes: Comment On: J Infect Dis. 2005 Nov 15;192(10):1741-8.
Sheets, R.L., J. Stein, T.S. Manetz, C. Andrews, R. Bailer, J. Rathmann, and P.L. Gomez (2006). Toxicological safety evaluation of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar despite differing plasmid backbones or gene-inserts. Toxicological Sciences 91(2): 620-630.
Abstract: The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies were performed to screen for potential toxicities (intrinsic and immunotoxicities). All treatment-related toxicities identified in these repeated-dose toxicology studies have been confined primarily to the sites of injection and seem to be the result of both the delivery method (as they are seen in both control and treated animals) and the intended immune response to the vaccine (as they occur with greater frequency and severity in treated animals). Reactogenicity at the site of injection is generally seen to be reversible as the frequency and severity diminished between doses and between the immediate and recovery termination time points. This observation also correlated with the biodistribution data reported in the companion article (Sheets et al., 2006), in which DNA plasmid vaccine was shown to remain at the site of injection, rather than biodistributing widely, and to clear over time. The results of these safety studies have been submitted to the Food and Drug Administration to support the safety of initiating clinical studies with these and related DNA plasmid vaccines. Thus far, standard repeated-dose toxicology studies have not identified any target organs for toxicity (other than the injection site) for our DNA plasmid vaccines at doses up to 8 mg per immunization, regardless of disease indication (i.e., expressed gene-insert) and despite differences (strengths) in the promoters used to drive this expression. As clinical data accumulate with these products, it will be possible to retrospectively compare the safety profiles of the products in the clinic to the results of the repeated-dose toxicology studies, in order to determine the utility of such toxicology studies for signaling potential immunotoxicities or intrinsic toxicities from DNA vaccines. These data build on the biodistribution studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
Descriptors: DNA vaccines, viral vaccines, acquired immunodeficiency syndrome, ebola like viruses, HIV-1 genetics, hemorrhagic fever, rabbits, severe acute respiratory syndrome, tissue distribution, West Nile fever genetics, West Nile virus.
Shirato, K., H. Miyoshi, H. Kariwa, and I. Takashima (2006). The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus. Virus Research 121(1): 11-16. ISSN: 0168-1702.
Abstract: The envelope (E) protein glycosylation status of the New York strain of West Nile (WN) virus is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and non-glycosylated WN virus infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta were up-regulated with replication of the E protein-glycosylated virus. Interferon (IFN) beta and IL-6 were up-regulated with the clearance of both viruses. These results suggest that TNFalpha and IL-1beta expression are related to the virulence of E protein-glycosylated WN virus.
Descriptors: interleukin 1 metabolism, peritoneal macrophages, tumor necrosis factor, alpha metabolism, viral envelope proteins, West Nile fever, cultured cells, glycosylation, inbred Balb C mice, up-regulation.
Sitati, E.M. and M.S. Diamond (2006). CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system. Journal of Virology 80(24): 12060-12069. ISSN: 0022-538X.
Abstract: Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.
Descriptors: West Nile virus, CNS, clearance, CD4+T cell responses, immune responses, spleen, serum, viral clearance.