The Relationships between West Nile and Kunjin Viruses.

Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.

Kunjin (KUN) and West Nile (WN) viruses belong to the Japanese encephalitis (JE) antigenic complex of the Flavivirus genus in the family Flaviviridae (1). The Flavivirus genus comprises [is greater than] 70 antigenically related, positive-stranded RNA viruses (2,3). KUN and WN viruses are maintained in a natural transmission cycle involving mosquito vectors and bird reservoir hosts, with humans and horses believed to be incidental hosts (4,5). Clinical symptoms most commonly associated with infection with KUN and WN viruses include febrile illness or mild encephalitis. WN virus has been associated with fatal cases of acute meningoencephalitis and fulminant hepatitis (6).

Early cross-neutralization studies with polyclonal antisera raised to single strains of WN and KUN viruses revealed that these viruses shared a close relationship but were antigenically distinct (7-9). This close relationship was also shown genetically by Coia et al. (10), who compared the sequence of the MRM61C KUN isolate with that of a Ugandan strain of WN (WNFCG) (11-13) and showed that the nucleotide and amino acid sequence identity between the two viruses was 82% and 93%, respectively, in the coding region of the genome. Although genetic studies have shown that KUN virus exists in Australia as a single topotype with [is less than] 2% nucleotide divergence (14,15), Berthet et al. (16) demonstrated that WN viruses were divided into two lineages. Although these comparisons demonstrated a close relationship between the two viruses, further sequence information is needed from additional isolates of both viruses to fully establish their phylogenetic association within the genus. This report describes the results of sequence analyses of 31 Australian KUN isolates; a KUN isolate from Sarawak, Malaysia; and 28 WN isolates from Africa, India, Europe, and New York (Tables 1 and 2). These virus isolates had all been identified as WN or KUN virus by traditional antigenic means. The Koutango (KOU) isolate was also included in this study, as it belongs to the JE serogroup and is closely related to the KUN/ WN group of viruses (9,17).

Materials and Methods

Virus and Cell Culture

Virus strains sequenced in this study are listed with their sources of isolation in Table 1. African green monkey (Vero) cells were grown at 37 [degrees] C in M199 (Gibco, New York) with 20 mM HEPES (Gibco) and supplemented with 2% L-glutamine and either 10% fetal bovine serum (FBS) for growth or 2% FBS for maintenance. Viruses were cultured in Vero cells by inoculating cell monolayers with virus at a multiplicity of infection of 1. Infected cell culture supernatants were harvested when [is greater than or equal to] 70% of the cells exhibited cytopathic effect. Infected supernatant was clarified by centrifugation at 2000 x g at 4 [degrees] C for 15 min, and aliquots were stored at -70 [degrees] C. A line of Aedes albopictus (C36/36) cells was cultured in M199 without HEPES and supplemented with FBS for growth or maintenance, as described. The cells were incubated at 28 [degrees] C in a humidified atmosphere with 5% [CO.sub.2].

Table 1. West Nile and Kunjin virus isolates
and nucleotide sequences used in this study

Virus ID          Year of isolation   Source of isolation

KUN35911               1984           Horse brain
KUNP1553(b)            1994           Culex sp.
KUNCH16465C            1974           Cx. ann.
KUNCH16514C            1974           Cx. ann.
KUNCH16532C            1974           Cx. ann.
KUNCH16549E            1974           Cx. ann.
KUNM695                1982           Cx. ann.
KUNM1465               1983           Cx. ann.
KUNMRM5373             1966           Oriolus flavocintus
                                      (bird)
KUNMRM16               1960           Cx. ann.
KUNMRM61C              1960           Cx. ann.
KUNOR130               1973           Cx. ann.
KUNOR134               1973           Cx. ann.
KUNOR166               1973           Cx. ann.
KUNOR205               1973           Aedes tremulus
KUNOR354               1974           Cx. ann.
KUNOR393               1974           Cx. ann.
KUNOR4                 1972           Cx. ann.
KUNCX255               1982           Cx. ann.
KUNCX238               1982           Cx. ann.
KUNBoort               1984           Horse spinal cord
KUNFC15                1986           Cx. ann.
KUNHu6774              1991           Human
KUNK6547               1991           Cx. ann.
KUNK1738               1989           Cx. ann.
KUNK5374               1989           Cx. ann.
KUNK2499               1984           Cx. ann.
KUNK6590               1991           Cx. ann.
KUNSH183               1991           Chicken
KUNWK436               1979           Cx. ann.
KUNV407                1983           Cx. ann.
KUNMP502-66            1966           Cx. pseudovishnui
HB6343                 1989           Human
ArTB3573               1982           Tick
MgAn798                1978           Coracopsis vasa (bird)
63134Ent 280           <1963          Human
ArA1Dj                 1968           Mosquito
ArNa1047               unknown        Mosquito
G2266                  1955           Cx. vishnui
G22886                 1958           Cx. vishnui
804994                 1980           Human brain biopsy
Sarafend               unknown        unknown
KOU DakAad 5443        1968           Tatera kempi (rodent)

Virus ID          Place of isolation

KUN35911          Hunter Valley, NSW,(a) AU
KUNP1553(b)       Marble Bar, WA, AU
KUNCH16465C       CH, Qld, AU
KUNCH16514C       CH
KUNCH16532C       CH
KUNCH16549E       CH
KUNM695           Victoria, AU
KUNM1465          Victoria, AU
KUNMRM5373        MRM, Qld, AU
KUNMRM16          MRM
KUNMRM61C         MRM
KUNOR130          OR, East Kimberley, WA, AU
KUNOR134          OR
KUNOR166          OR
KUNOR205          OR
KUNOR354          OR
KUNOR393          OR
KUNOR4            OR
KUNCX255          Wyndham, East Kimberley
KUNCX238          Wyndham, East Kimberley
KUNBoort          Boort, Victoria, AU
KUNFC15           West Kimberley, WA, AU
KUNHu6774         Southern NSW, AU
KUNK6547          SE Kimberley, WA, AU
KUNK1738          OR
KUNK5374          SE Kimberley, WA, AU
KUNK2499          OR
KUNK6590          Broome, West Kimberley, WA, AU
KUNSH183          Victoria, AU
KUNWK436          Camballin, West Kimberley, WA, AU
KUNV407           Jabiru, NT, AU
KUNMP502-66       Sarawak, Borneo, Malaysia
HB6343            CAR
ArTB3573          CAR
MgAn798           Madagascar
63134Ent 280      Uganda
ArA1Dj            Algeria
ArNa1047          Kenya
G2266             Sathuperi, India
G22886            Sathuperi, India
804994            Bangalore Field Station, Karnataka, India
Sarafend          unknown
KOU DakAad 5443   Senegal, Africa

Virus ID          GenBank Accession Number

KUN35911          AF196511 (E gene)
KUNP1553(b)       AF297856 (NS5/3'UTR)
                  AF196495 (E gene)
KUNCH16465C       AF297841 (NS5/3'UTR)
                  AF196504 (E gene)
KUNCH16514C       AF297842 (NS5/3'UTR)
                  AF196501 (E gene)
KUNCH16532C       AF297843 (NS5/3'UTR)
                  AF196513 (E gene)
KUNCH16549E       AF297844 (NS5/3'UTR)
                  AF196520 (E gene)
KUNM695           AF297852 (NS5/3'UTR)
                  AF196496 (E gene)
KUNM1465          AF297851 (NS5/3'UTR)
                  AF196522 (E gene)
KUNMRM5373        AF297859 (NS5/3'UTR)
                  AF196509 (E gene)
KUNMRM16          AF196505 (E gene)
KUNMRM61C         AF196516 (E gene)
KUNOR130          AF297857 (NS5/3'UTR)
                  AF196492 (E gene)
KUNOR134          AF196506 (E gene)
KUNOR166          AF196499 (E gene)
KUNOR205          AF297858 (NS5/3'UTR)
                  AF196515 (E gene)
KUNOR354          AF297855 (NS5/3'UTR)
                  AF196518 (E gene)
KUNOR393          AF196503 (E gene)
KUNOR4            AF196523 (E gene)
KUNCX255          AF297845 (NS5/3'UTR)
                  AF196514 (E gene)
KUNCX238          AF196502 (E gene)
KUNBoort          AF297840 (NS5/3'UTR)
                  AF196519 (E gene)
KUNFC15           AF297846 (NS5/3'UTR)
                  AF196510 (E gene)
KUNHu6774         AF297847 (NS5/3'UTR)
                  AF196493 (E gene)
KUNK6547          AF196521 (E gene)
KUNK1738          AF297848 (NS5/3'UTR)
                  AF196494 (E gene)
KUNK5374          AF297849 (NS5/3'UTR)
                  AF196517 (E gene)
KUNK2499          AF196498 (E gene)
KUNK6590          AF297850 (NS5/3'UTR)
                  AF196500 (E gene)
KUNSH183          AF297853 (NS5/3'UTR)
                  AF196491 (E gene)
KUNWK436          AF297854 (NS5/3'UTR)
                  AF196507 (E gene)
KUNV407           AF196508 (E gene)
KUNMP502-66       AF196534 (E gene)
HB6343            AF196542 (NS5/3'UTR)
                  AF196528 (E gene)
ArTB3573          AF196541 (NS5/3'UTR)
                  AF196527 (E gene)
MgAn798           AF196543 (NS5/3'UTR)
63134Ent 280      AF196539 (NS5/3'UTR)
                  AF196530 (E gene)
ArA1Dj            AF196536 (NS5/3'UTR)
                  AF196529 (E gene)
ArNa1047          AF196535 (NS5/3'UTR)
G2266             AF196537 (NS5/3'UTR)
                  AF196525 (E gene)
G22886            AF196538 (NS5/3'UTR)
                  AF196524 (E gene)
804994            AF196540 (NS5/3'UTR)
                  AF196526 (E gene)
Sarafend          AF196533 (E gene)
KOU DakAad 5443   AF196532 (E gene)

(a) NSW = New South Wales; AU = Australia; WA = Western Australia;
Cx. ann. = Culex annulirostris: CH = Charleville; Qld = Queensland;
MRM = Mitchell River Mission; OR = Ord River; NT = Northern Territory;
CAR = Central African Republic; UTR = untranslated region.

(b) P1553 was isolated from a culture of C6/36 cells inoculated with
culture fluid derived from a mosquito pool from which Edge Hill (EH)
virus had also been isolated (Annette Broom, pers. comm.).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sequencing

A single-step RT-PCR procedure (22) was performed on each virus isolate. The region amplified within the envelope (E) gene used the primers KUN5276 (GCG TGT GGT TCT TCA AAC TCC A) and WN4752 (TGC GTG TCC AAC CAT GGG TGA AGC) with the isolates Sarafend, MP502-66, and a strain of KOU virus, DakAad 5443. Primer KUN5276 was used with primer KUN4778 (ATA ATG ACA AGC GGG CTG ACC C) for the remaining isolates. The region of the virus genome encompassing the terminus of the nonstructural protein, NS5 and the 5' end of the 3' untranslated region (3'UTR), was amplified by using the previously published universal flavivirus PCR primers EMF1 and VD8 (23).

Both strands of the PCR product were then sequenced on a 377 automated sequencer (Applied Biosystems International [ABI], Foster City, CA, USA) by using the same primer pair. The two sequences derived from each PCR product were initially aligned by using the program SeqEd (ABI) and a consensus sequence determined. The consensus sequences were then aligned by using the program Clustal W (24), and results were further analyzed by using phylogenetic programs in Bionavigator (http://www.bionavigator.com). Percentage nucleotide similarity was calculated by the Old Distance (GCG) program, and bootstrap confidence levels were calculated with 1,000 replicates by using the Consense program (25). Sequences determined in this study have been deposited in GenBank (National Institutes of Health, Bethesda, MD, USA) (Table 1). Additional sequences included in this analysis are listed in Table 2.

Table 2. Additional West Nile and Kunjin virus sequences
included in this study

                                                             GenBank
                  Year of       Source of      Place of      Accession
Virus ID         isolation      isolation      isolation     Number

KUNMP502-66      1966        Culex pseudo-
                               vishnui         Sarawak       L49311
NY99             1999        Phoenicopterus
                               chilensis
                               (Chilean
                               flamingo)       NYC(a)        AF196835
NY99             1999        Human             NYC           AF202541
ISR98            1998        Goose             Israel        AF205882
Rom96            1996        Human             Romania       AF130363
Rom97-50         1997        Unknown           Romania       AF130362
ARB310           1967        Culex sp.         CAR           AF001566
Mor96            1996        Unknown           Morocco       AF205884
Italy98          1998        Unknown           Italy         AF205883
ArD93548         1993        Cx. neavei        Senegal       AF001570
AND27875         1979        Galago
                               senegalensis    Senegal       AF001569
PaH651           1965        Human             France        AF001560
AnMg798          1978        Coracopsis
                               vasa (bird)     Madagascar    AF001559
ArMg978          1988        Cx. univittatus   Madagascar    AF001574
MP22             unknown     unknown           Uganda        AF001562
UGA-B956         unknown     unknown           Uganda        AF208017
ArD78016         1990        Aedes vexans      Senegal       AF001556
HB83P55          1983        Human             CAR           AF001557
Eg101            1951        Human             Egypt         AF001568
Eg101            1951        Human             Egypt         AF260968
ARA3212          1981        Cx. guiarti       Ivory Coast   AF001561
KUNMRM16         1960        Cx. ann.          MRM           L48979
KUNMRM61C        1960        Cx. ann.          MRM           L48978
Sarafend         unknown     unknown           unknown       L48977
KOUDakAad 5443   1968        Tatera kempi
                               (rodent)        Senegal       L48980
WNFCG            1937        Human             Uganda        M12294

                 Region of
Virus ID          genome             Reference

KUNMP502-66      NS5/3'UTR              17

NY99             E                      18
NY99             NS5/3'UTR              21
ISR98            E           V. Deubel,unpub. data
Rom96            E                      19
Rom97-50         E                      20
ARB310           E                      16
Mor96            E           V. Deubel,unpub. data
Italy98          E           V. Deubel,unpub. data
ArD93548         E                      16
AND27875         E                      16
PaH651           E                      16
AnMg798          E                      16
ArMg978          E                      16
MP22             E                      16
UGA-B956         NS5                    21
ArD78016         E                      16
HB83P55          E                      16
Eg101            E                      16
Eg101            NS5         Bowen et al., unpub. data
ARA3212          E                      16
KUNMRM16         NS5/3'UTR              17
KUNMRM61C        NS5/3'UTR              17
Sarafend         NS5/3'UTR              17
KOUDakAad 5443   NS5/3'UTR              17
WNFCG            E and NS5/3'           11
                   UTR

(a) NYC = New York City; Cx. ann. = Culex annulirostris;
CAR = Central African Republic; MRM = Mitchell River Mission;
UTR = untranslated region.

Enzyme-Linked Immunosorbent Assay (ELISA)

Antigenic profiles of each isolate were compared by using a panel of anti-KUN monoclonal antibodies (MAbs) (26,27) and anti-WN MAbs (28,29) in ELISA as described (26). All MAbs were produced to the E protein except for 3.1112G, which was specific for the NS 1 protein.

Results

Genetic Analysis

In accordance with previous reports (16,18,21), the phylogenetic trees generated from both E gene and NS5/ 3'UTR sequences grouped most of the isolates into two major lineages (Figures 1 and 2). Australian KUN isolates and WN isolates from North, West, and central Africa; southern and eastern Europe; India; the Middle East; and New York constituted lineage I. Lineage II comprised WN isolates from West, central, and East Africa and Madagascar. Genetic lineage was not significantly associated with date or source of isolation, with most isolates of both lineages coming from human, mosquito, and avian sources between 1950 and 1990. However, as noted, all viruses isolated during outbreaks of human or avian disease in the last decade belonged to lineage I. Lineage I viruses grouped together with an average sequence identity of 80% (E gene) and 77% (NS5/3'UTR), while the viruses of lineage II contained a single cluster with an average identity of 82% and 83%, respectively. The lineage I viruses were further separated into three clusters: the Australian KUN isolates; the Indian WN viruses; and WN isolates from Africa, the Middle East, Europe, and North America. The divergence observed between lineage I and lineage II viruses was in the range of 16.5% to 30.8% and 19% to 36.5% for sequences of the E gene and NS5/3'UTR, respectively. High bootstrap confidence levels (100%) for the sequences of the NS5/3'UTR also support the separation of the two lineages and the branching of the NY99 cluster of WN viruses with the Australian KUN viruses in lineage I, rather than with the WN group of viruses in lineage II. The clustering of the Indian WN group in lineage I based on sequences in the E gene, however, was at a lower bootstrap confidence level (63%).

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The sequence of the virus from Malaysia, KUN MP502-66, grouped outside the two lineages described. Similarly, the KOU virus, which was 72%-73% identical to KUN MP502-66, did not group with either lineage. The range of percentage divergence between KUN MP502-66 and KOU viruses with the lineage I and lineage II viruses (Table 3) shows that these two isolates display similar divergence from all other isolates in this study, supporting their grouping outside the two main lineages.

Table 3. Range of percentage divergence between the Malaysian and
Koutango isolates with lineage I and lineage II viruses

                  E gene                  NS5/3'UTR

           Lineage I   Lineage II   Lineage I   Lineage II

MP502-66   20%-30%     20%-30%      21%-35%     21%-25%
KOU        25%-30%     29%-32%      26%-39%     22%-25%

UTR = untranslated region.

The viruses of lineage I group together in three tight clusters. The first of these includes the Australian KUN viruses, which were 94% identical when sequences of the E gene were compared and 90% when the sequences of the NS5/ 3'UTR were compared. High bootstrap confidence levels (100% for sequences from the E gene and 99% for sequences from the NS5/3'UTR) separated the Australian KUN viruses from the other isolates. However, extremely low bootstrap confidence levels were observed for most of the branches between the Australian KUN viruses in both dendrograms, which also suggests that these viruses are closely related and cannot be definitively separated from each other. The Indian viruses also cluster together, with a sequence identity of 97% and 98% for sequences of the E gene and NS5/3'UTR, respectively. The WN isolates in the remaining cluster of lineage I are 90% and 97% identical, respectively, for the regions sequenced. When compared with the Australian KUN isolates, this cluster, which includes the 1999 New York isolate, shared a sequence identity of 89% for the E gene and 88% for the NS5/3'UTR. Similarly, when the sequences of the Australian KUN isolates were compared with those of the WN Indian viruses, they were 80% identical for the E gene and 77% identical for the NS5/3'UTR. In comparison, the two clusters of WN viruses in lineage I and the WN isolates in lineage II shared an average sequence identity of only 78% and 71% for the E gene and NS5/3'UTR, respectively. These results demonstrate that the sequences of some WN isolates are more closely related to the Australian KUN viruses than to other WN isolates.

The high degree of nucleotide sequence homology within clusters is consistent with the observed similarity of the amino acid sequences. The most notable variation in amino acid sequence in this study appears around the potential glycosylation site at amino acid 154 of the E protein (Figure 3). The Australian KUN viruses generally contain either the glycosylation motif NYS at this position or the sequence NYF, which abolishes glycosylation of the E protein. In contrast, the KUN virus SH183 has a 154N [right arrow] K substitution, which also ablates the potential for glycosylation at this site. In comparison with the KUN prototype, the amino acids 159 (T [right arrow] I, T [right arrow] V, or T [right arrow] Q) and 162 (A [right arrow] T) of all the WN isolates in this study contain an amino acid substitution. The KUN isolate P1553 also differs from the KUN prototype at amino acid 159 (T [right arrow] I). Two aberrant isolates, 63134Ent280 and WNFCG, incur a deletion of four amino acids (154 through 157), which also abolishes the glycosylation site.

[ILLUSTRATION OMITTED]

Our results concur with those of Berthet et al. (16), who suggested the presence of signature motifs within the E gene that support the segregation of WN viruses into two lineages. These signature residues include the amino acid substitutions from lineage I [right arrow] II as follows: 172A [right arrow] S, 205T [right arrow] S, and 210T [right arrow] S. The amino acid substitution 208T [right arrow] A holds true in general; however, two of the Indian isolates (lineage I) have K at this position and WNFCG (lineage II) has E. Of particular note is the substitution at amino acid 199. The Australian KUN isolates (199S) share the same amino acid as the lineage II WN viruses, while the lineage I viruses contain an N residue at this position. We have also identified an additional three signature motifs (I [right arrow] II) at amino acids 128R [right arrow] W, 129T [right arrow] I, and 131L [right arrow] Q. When we attempted to place the Malaysian KUN isolate within either lineage by using these signature motifs, the residues at 128, 129, 131, 172, and 208 were similar to those of lineage I viruses, but the residues at 205 and 210 were consistent with those of lineage II viruses. Residue 199 (D) was unlike any of the other viruses. The KOU isolate displayed more similarities with the lineage II WN viruses (residues 131, 172, 199, and 210) when signature motifs were compared. Residues 129 and 208 differed from viruses of both lineages.

We have identified signature motifs within the NS5 protein that correlate with the separation of the two lineages. Substitutions between lineages I [right arrow] II include 860A [right arrow] T, 869Q [right arrow] H, 878I [right arrow] V (except for the isolate MgAn798, which has 878I [right arrow] L), and 899L [right arrow] V (except for the isolate ArNa1047, which has 899L [right arrow] I) (Figure 4). At amino acid 877, the lineage I WN viruses are separated again from the lineage II WN viruses with an A [right arrow] S substitution however, the KUN isolates (including MP502-66 from Malaysia) have the same motif as the lineage II WN viruses (877S). The amino acid substitution at 903 separates the Indian WN viruses (903S) from the WN and KUN viruses of both lineages (903T), instead grouping them with the Malaysian isolate and the KOU virus. Once again, the signature motifs cannot be used to classify the Malaysian isolate and KOU virus into either lineage.

[ILLUSTRATION OMITTED]

Nucleotide sequences in the 3'UTR of the viruses included in this study had a highly variable region in both length and nucleotide sequence immediately downstream of the open reading frame stop codon (Figure 5). Deletions as well as point mutations were observed in this region, which varied from 38 (MgAn798) to 129 (ArNa1047) nt in length. The Australian KUN viruses displayed only point mutations when compared with the KUN prototype, except for the isolate P1553, which contained a 7-nt insertion, consistent with the WN viruses of lineage I. The long deletion in the nucleotide sequence immediately downstream of the stop codon of the WN prototype virus, WNFCG (53 nt), has been described (31); it is also present in the sequences of another two lineage II WN viruses analyzed in this study, Sarafend (53 nt) and MgAn798 (65 nt). The rest of the 3'UTR for these viruses was found to be highly conserved.

[ILLUSTRATION OMITTED]

Antigenic Analysis

The MAb 10A1, produced to the KUN isolate OR393 (26), reacted specifically with the Australian KUN isolates in ELISA and did not react with the KUN isolate from Malaysia (MP502-66) nor with KOU virus or any of the lineage I or lineage II WN viruses (Table 4). The MAb 546 (29), produced to the WN strain Eg101, reacted with all the lineage I and lineage II WN isolates except WN-Sarafend; it did not react with the KOU, KUN, or Malaysian viruses. The MAbs 2B2, produced to the KUN isolate MRM 16 (27), and 2B4, produced to the WN isolate H442 (28), reacted with all the isolates in the study, while the MAbs 3.67G and 3.91D, again produced to the KUN isolate OR393 (26), reacted with all the isolates except WN-Sarafend. The MAb 3.1112G, produced to the NS1 protein of KUN isolate OR393 (26), reacted with all isolates except KOU. The Mab binding patterns (Table 4) clearly digress and fail to differentiate KUN and WN isolates into two distinct groups. Instead, they define five distinct antigenic groups: Australian KUN viruses, Malaysian KUN virus, lineage I and lineage II WN viruses, WN-Sarafend, and KOU virus.

Table 4. Binding patterns of anti-KUN and anti-WN monoclonal
antibodies to virus isolates in enzyme-linked immunosorbent
assay (ELISA)(a)

                            Monoclonal antibodies (MAb)

Virus          10A1   546   2B2   2B4   3.91D   3.67G   3.1112G

KUN(b)         +(c)    -     +     +      +       +        +
KUN MP502-66   -       -     +     +      +       +        +
WN(d)          -       +     +     +      +       +        +
WN Sarafend    -       -     +     +      -       -        +
KOU            -       -     +     +      +       +        -

(a) Infected C6/36 cell monolayers in 96-well plates were fixed
with acetone and used as the antigen in the ELISA.

(b) All Australian KUN isolates exhibited identical MAb
binding patterns.

(c) A result was considered positive if consecutive twofold
dilutions of MAb produced an OD > 0.25 and at least twice
that shown on uninfected cells.

(d) All West Nile isolates except Sarafend produced identical
MAb binding patterns.

Conclusion

The results of the phylogenic analysis in this report clearly illustrate that the KUN, WN, and KOU viruses make up a closely related group of viruses, which can be further subdivided into several subgroups on the basis of genetic and antigenic data. Previous phylogenic studies have also shown that KUN and WN viruses share a close relationship (16-18,21). This report however, further defines this relationship by using a comprehensive panel of both viruses. Also included in this study were several anomalous isolates, including an isolate from Southeast Asia (MP502-66), a laboratory-adapted WN strain of uncertain passage history and origin (Sarafend), and a flavivirus from West Africa (KOU), which has been shown to be closely related to the KUN/WN group of viruses.

The region sequenced in the E gene spans a glycosylation site that, although highly conserved among viruses of the JE antigenic subgroup, is absent from many KUN and WN isolates (16,26; Scherret JH, Khromykh AA, Mackenzie JS, Hall RA, unpub, data). While glycosylation at this site has been associated with neuroinvasiveness of WN isolates in mice (32,33), the biological significance of E protein glycosylation is still unclear. Indeed, sequence analysis of the E gene of WN viruses responsible for fatal outbreaks of encephalitis in Romania (Rom 96) and New York (NY99) showed that only the latter contained a potential glycosylation site, casting doubt on the importance of E protein glycosylation in viral pathogenesis. However, our studies and those of others have shown that limited passage of WN and KUN viruses in some cell types can alter the glycosylation status of the E protein and that analysis of passaged viral isolates should be interpreted with caution (33; Scherret JH, Khromykh AA, Mackenzie JS, Hall RA, unpub. data).

The 3'UTR of flaviviruses ranges in length from 400 nt to 600 nt and is thought to play a crucial role in the initiation and regulation of viral translation, replication, and assembly. It includes a potential stable secondary RNA structure at its terminus (2,34-38), and upstream it contains several domains that appear to be conserved among mosquito-borne flaviviruses (2,39, 40). Men et al. (41) have suggested that deletions in the distal 80 nt to 90 nt would most likely lead to disruption of the stem-loop and loss of viability. In contrast, the region sequenced in this study contains highly variable regions suitable for genetic classification and analysis of the relationships among viruses, which had been subjected to deletions or insertions or both during evolution (17).

Phylogenetic trees constructed from sequence data from both regions identified two major lineages, consistent with previous reports (16,18,21). These two lineages did not separate the KUN isolates from the WN isolates; rather, they emphasized the close link between KUN and WN viruses of lineage I. Nevertheless, within lineage I, the Australian KUN isolates formed a tight cluster with an average nucleotide divergence of 6% for the E gene and 10% for the NS5/3'UTR. In contrast, the WN isolates were spread between the two lineages in three clusters, with a divergence of up to 30.6% for sequences of the E gene and 28.3% for sequences of the NS5/3'UTR. Signature motifs in the deduced amino acid sequences of the E and NS5 proteins also support the separation of the viruses into two lineages.

The virus from Malaysia, KUN MP502-66, and the African virus, KOU, pose a conundrum as to their relationship with the WN and KUN group of viruses. Statistical support for clustering with either of the WN lineages was poor, suggesting that they represent two single-isolate lineages. Although our previous findings suggested that the Malaysian KUN isolate may represent an evolutionary link between the KUN and WN viruses (17), the lack of sequence identity between KUN MP502-66 and the KUN/WN group of viruses in our study suggests that these viruses have evolved separately from a common ancestor.

The binding patterns of MAbs to KUN and WN isolates did not differentiate these viruses into the same phylogenetic lineages observed in the dendrograms, although they did support the sequencing results by identifying the Australian KUN viruses, the Malaysian KUN virus, and KOU virus as distinct antigenic groups. The WN-specific MAb used in this study, 546, could not distinguish subgroups within the WN group of viruses; however, Besselaar and Blackburn (28) and Damle et al. (42) have differentiated Indian WN isolates from lineage I South African strains by using MAbs, consistent with the earlier studies of Hammam et al. (43,44). These findings support our sequence data, which show tight clustering of the Indian isolates on a separate branch from other WN isolates in the phylogenetic trees (Figures 1 and 2). Additional MAbs to the E protein of WN viruses may be required to differentiate between lineage I and lineage II viruses.

The unique binding pattern of anti-E MAbs to the Sarafend WN isolate is difficult to explain in light of the E gene sequencing results and amino acid alignments, which show that this virus is similar to other lineage II viruses. However, Sarafend also differs from other WN viruses in the way that it buds from the cell membrane of infected cells (45). Sequencing of the entire prM and E genes of this virus may identify the basis for structural differences in the envelope heterodimer that account for the loss of MAb binding sites and unusual virion maturation.

Phylogenetic analyses enable more precise determination of the relationships among similar viruses and consequently aid in identifying the origin of unknown viruses in subsequent outbreaks. The importance of defining the relationship between the KUN and WN viruses was emphasized during the 1999 outbreak of viral encephalitis in New York City (46,47). Until recently, WN and KUN had been classified as distinct virus types in the Flavivirus genus. However, the latest report by the International Committee on Taxonomy of Viruses (25) recognized that KUN and WN should not be classified as two separate species and designated KUN as a subtype of WN. Our results suggest that this definition requires further consideration. The species should perhaps be further subdivided into at least six subtypes on the basis of the clusters of viruses displayed in the phylogenetic trees. Subtypes would then include lineage II WN group, Indian WN group, Australian KUN group, lineage I WN group, Malaysian group, and KOU group.

Indeed, the assessment of viruses from each subgroup for transmissibility by the major mosquito vectors of each geographic region and relative virulence and amplification in primate, equine, and avian species will provide valuable information on the likelihood and possible consequences of the spread of these viruses to new geographic regions. Additional studies of cross-protection between subgroups by natural infection or immunization with vaccines derived from these viruses and the specificity and sensitivity of serologic and molecular assays for each subgroup in monitoring and diagnostic applications will be useful in defining control strategies.

Acknowledgments

We thank Robert Lanciotti for valuable scientific discussions regarding the New York outbreak, Terry Besselaar for supplying monoclonal antibodies, and J.P. Thakare and S.S. Gogate for providing the isolates from India. We also appreciate comments on the manuscript by Helle Bielefeldt-Ohmann and technical assistance from Petra Sedlak.

This work was supported by a research grant from the National Health and Medical Research Council of Australia.

Dr. Scherret is a postdoctoral fellow with the World Health Organization Collaborating Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas. Her dissertation concerned the molecular epidemiology and biology of Kunjin and West Nile viruses. Her research interests focus on viral hemorrhagic diseases, including dengue and Oropouche fever.

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Jacqueline H. Scherret,(*) Michael Poidinger,([dagger]) John S. Mackenzie,(*) Annette K. Broom,([double dagger]) Vincent Deubel,([sections]) W. Ian Lipkin,([paragraph]) Thomas Briese,([paragraph]) Ernest A. Gould,(#) and Roy A. Hall(*)

(*) University of Queensland, Brisbane, Queensland, Australia; ([dagger]) Bioinformatics Inc., Eveleigh, New South Wales, Australia; ([double dagger]) University of Western Australia, Nedlands, Western Australia, Australia; ([sections]) Institut Pasteur, Paris, France; ([paragraph]) University of California, Irvine, California, USA; and (#) Institute of Virology and Environmental Microbiology, Oxford, England

Address for correspondence: Roy A. Hall, Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia; fax: [phone]; e-mail: [e-mail]