This page was
updated on July 24, 2002 to incorporate the corrections
in Vol. 8, No. 9
Dispatch
Phylogenetic Analysis
of a Human Isolate from the 2000 Israel West Nile virus Epidemic
Thomas Briese,* Andrew Rambaut,† Melissa Pathmajeyan,* Jihad
Bishara,‡ Miriam Weinberger,‡ Silvio Pitlik,‡ and W. Ian Lipkin*
*University of California at Irvine, Irvine, California, USA;
†University of Oxford, Oxford, UK; and ‡Rabin Medical Center–Beilinson
Campus, Petah Tikva, Israel
Specimens
from a patient of the 2000 Israel West Nile virus epidemic
were analyzed by reverse transcription-polymerase chain reaction.
Products corresponding to E, NS3, and NS5 sequences were amplified
from cerebellar but not from cortical samples. Phylogenetic analyses
indicated a closer relationship of this isolate to 1996 Romanian
and 1999 Russian than to 1998-99 Israeli or 1999 New York isolates.
West Nile fever is typically a mild febrile illness characterized
by headache, myalgias, lymphadenopathy, and a maculopapular rash;
West Nile fever occurs sporadically throughout endemic areas of
northeastern Africa and tropical Asia (1-5). The
causative agent, West Nile virus (WNV), is a member of the
genus Flavivirus (family Flaviviridae), which is grouped
together with Alfuy virus, Cacipacore virus, Koutango
virus, Japanese encephalitis virus, Kunjin virus, Murray
Valley encephalitis virus, St. Louis encephalitis virus,
Usutu virus, and Yaounde virus in the Japanese
encephalitis virus antigenic complex (6,7).
Two different lineages (I and II) of WNV are characterized genetically
(8). Whereas lineage II viruses are isolated in
endemic areas, lineage I viruses are isolated during epidemic outbreaks
of WNV infection and may cause severe encephalitis in the elderly
or immunosuppressed persons. Epidemics of West Nile fever were first
reported in the 1950s in Israel (2) and the 1970s
in South Africa (9). Sites of notable recent outbreaks
include Romania (1996, 1997), Italy (1998), Russia (1999), France
(2000), United States (1999, 2000) and Israel (2000) (5,10).
Sequence analysis of the WNV responsible for the United States outbreak
in 1999 (WNV-NY1999) showed a close phylogenetic relationship to
a WNV isolated from a goose in Israel in 1998 (WNV-ISR1998) (11,12).
WNV is transmitted mainly by mosquito vectors, although it has
also been isolated in several tick species (3,5,13).
Birds are an important WNV reservoir. In several avian species,
virus replication generates serum titers sufficient to sustain arthropod
transmission (4,5). Birds, during seasonal migrations,
are also believed to be instrumental in the geographic spread of
WNV (3-5,14). The virus is only
occasionally transmitted to humans or other mammals. Viremia in
mammals is low level; thus, mammals are considered to be dead-end
hosts.
The
Study
From July through November 2000, a WNV epidemic occurred in central
and northern Israel. More than 430 people were diagnosed with WNV
infection; 29 of these patients had fatal encephalitis. We report
phylogenetic analysis of WNV sequences isolated from the brain of
an encephalitis patient from the 2000 Israel epidemic.
A 72-year-old woman with a history of recurrent meningioma of the
sphenoidal ridge, dementia, and depression was hospitalized because
of fever and general deterioration of 5 days’ duration. On admission,
the patient was responsive only to painful stimuli and had generalized
muscle stiffness and limb tremors. Clinical and laboratory values
were consistent with viral encephalitis; thus, the patient was initially
treated with intravenous acyclovir for presumptive herpes simplex
encephalitis. When polymerase chain reaction (PCR) analysis of cerebrospinal
fluid (CSF) showed no evidence of herpes simplex virus infection,
and WNV antibodies were detected in serum and CSF, acyclovir was
discontinued and ribavirin was initiated at an oral dosage of 2.4
g per day. The patient’s clinical status continued to deteriorate
with aspiration pneumonia and intermittent generalized seizures.
Intravenous immunoglobulin was added (35 g/d for 2 days) without
improvement. Approximately one month after onset of illness, the
patient died of respiratory failure.
Postmortem examination of the brain showed multiple meningiomas,
generalized atrophy, and surgical resection of the right parietal
lobe. Histology was remarkable for neurofibrillary plaques consistent
with Alzheimer’s disease, and scattered microglial nodules and perivascular
lymphocytic inflammation in the medulla, pons, and midbrain were
consistent with viral encephalitis.
RNA was extracted from frontal cortex and cerebellum with TRI-Reagent
(Molecular Research Center, Cincinnati, OH). Four micrograms of
total RNA from each brain region were used as a template for reverse
transcription-polymerase chain reaction (RT-PCR) with primer sets
representing three regions of sequence conservation in flavivirus
genomes: NS3-1 (EDL/Fla-U5004, 5´- GGA ACD TCM GGH TCN CCH AT and
EDL/Fla-L5457, 5´- GTG AAR TGD GCY TCR TCC AT), NS5-1.1 (EDL/Fla-U9093,
5´- AGY MGR GCH ATH TGG TWY ATG TGG and EDL/Fla-L9279, 5´- TCC CAV
CCD GCK GTR TCA TC), and NS5-2 (EDL/Fla-U9954, 5´- GSS AAA KCH TAY
GCN CAV ATG TGG and EDL/Fla-L10098 5´- AGC ATR TCT TCH GTN GTC ATC
CA) (15,16). Amplification products were obtained
with RNA derived from the cerebellum in reactions with all three
primer sets; no amplification products were obtained with RNA from
the cortex. These amplification products were cloned into the pGEM-Teasy
vector (Promega, Madison, WI) and subjected to automated
dideoxy sequencing (ABI Prism Model 377, Foster City, CA).
Sequences were submitted to GenBank (NS3, GenBank accession no.
AF394218; NS5, GenBank accession nos. AF394219 and AF394220).
Signal of cerebellar amplification products in ethidium bromide-stained
gels was reduced in comparison with similar studies performed with
brain materials from four patients of the 1999 New York City outbreak
(data not shown; New York patients were 75 years to 80 years
of age, 3 male, 1 female, who died of severe WNV encephalitis during
the 1999 outbreak [16]). The relative virus load
was 140 copies/200 ng RNA in the Israeli sample, indicated by 5´-nuclease
real-time RT-PCR (17), compared with 7000 to 20
copies/200 ng RNA in specimens analyzed from the New York City outbreak
(Table). However, since the virus load
of the sample from Israel was within the range of virus loads observed
with the New York samples, this result for a single Israeli sample
may not indicate a strain difference. Quantitative analysis was
restricted to the NS5 target because no signal was obtained with
primer/probe set prNS3 (fwd, 5´- GCa CTG AGA GGA CTG CCc AT; probe,
5´-6FAM-TAc CAG ACA TCc GCA GTG cCC AGA-T-TAMRA; rev, 5´- TGg GTG
AGG GTa GCA TGa CA), because of point mutations in WNV-ISR2000 sequence
that prevented efficient hybridization with the primer and probe
oligonucleotides (fwd–2 mismatches, probe–3 mismatches, rev–3 mismatches;
given above in lower case). Sensitivity was not substantially reduced
in assays with primer/probe set prNS5, which had one mismatch in
the 3´-terminal sequence of the probe oligonucleotide (Table).
Sequence analysis of the cloned NS3 and NS5 gene fragments indicated
similarity to completely sequenced Romanian and Russian WNV isolates
WNV-RO97-50-1996 and WNV-RUS-VLG4-1999, respectively; thus, to facilitate
detailed phylogenetic analysis, E gene sequence from the Israel
human brain sample was amplified. An E gene sequence of 1509 nucleotides
(GenBank Accession Number AF394217) was amplified from total RNA
by using GeneChoice UNIPOL polymerase (PGC Scientific, Gaithersburg,
MD) and primers EDL/E-U1006 (5´- GGA GTG TCT GGA GCA ACA TGG GT)
or EDL/E-U1476 (5´- TCC TGC GGC GCC TTC AT) and EDL/E-L2244 (5´-
CCC CTC CAA CTG ATC CAA AGT CC) or EDL/E-L2538 (5´- TCC ATC CAA
GCC TCC ACA TCA), respectively. Sequence analysis of this fragment
confirmed data from NS3 and NS5 sequence analyses, indicating a
closer relationship of WNV-ISR-hISR2000 sequence to Romanian and
Russian isolates than to the 1997/98/99 Israeli and the WNV-NY1999
isolates (Figure).
The extent to which this WNV genotype contributed to human disease
in the 2000 epidemic remains to be determined. WNV-ISR-hISR2000
may have been carried into Israel by migrating birds from reservoirs
in southeastern Europe or reservoirs in northeastern Africa, where
a highly related virus was isolated in 1998 (WNV-KEN-KN3829-1998)
(18). The 2000 Israel isolates
in birds (strains ISR-00GooMaS and ISR-00PigC) were different from
the previous Israeli isolates (1997/98/99; strains ISR-97Goo1, ISR-98Goo1,
ISR-IS98St1, ISR-99Goo, and ISR-99Gull [Figure]),
but similar to the human isolate. Nonetheless, precedent exists
for implicating more than one genotypic variant in a WNV outbreak.
During the 1999 outbreak in Volgograd, Russia, two different genotypes
were isolated: WNV-RUS-ASTR986-1999 (similar to 1997/98/99 Israeli
and the WNV-NY1999 isolates, genotype lineage I subtype 2a) and
WNV-VLG22889/WNV-RUS-VLG4-1999 (similar to WNV-ISR-hISR2000, subtype
2b [Figure]) (19). Indeed,
even more divergent genotypes were identified during the 1996-97
WNV outbreak in Romania (WNV-RO97-50-1996 similar to WNV-ISR-hISR2000,
genotype lineage I subtype 2b; WNV-RO96-1030-1996 and WNV-ROM96(0334)-1996,
belonging to a different subtype, subtype 1 [Figure])
(20). The fact that no such divergence of genotypes
of WNV isolates was observed during the 1999 New York epidemic (Figure)
was interpreted as being compatible with a single, new introduction
of this virus to the Western Hemisphere. While this manuscript was
under review, another group reported WNV sequences from four patients
of the 2000 Israel outbreak: two isolates most closely related to
WNV-R097-50-1996 and two identical to the WNV-NY1999 isolates (21).
Analysis of additional isolates from the Israel 2000 and other outbreaks,
including isolates obtained in 2000, 2001, and subsequent years
in the USA, will be required to establish the extent to which avian
migration and viral mutation contribute to the epidemiology of WNV-related
disease.
This study was supported by the National Institutes of Health (NS-29425).
Dr. Briese is associate director of the Emerging Diseases Laboratory
and assistant professor at the University of California, Irvine.
In summer 2002, he will join the faculty in the Mailman School of
Public Health at Columbia University as an associate professor of
Epidemiology.
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Table.
Real Time reverse transcription-polymerase chain reaction
(RT-PCR) analysis of RNA extracts from 2000 Israel West Nile
patient specimens using primer set NY1999-NS5 |
|
NS5 standard NY1999
|
Armored RNA
|
Armored RNA
extract
|
NY1999 specimens
|
NS5 standard ISR2000
|
ISR2000 specimens
|
|
|
|
|
|
|
Amounta
|
CTb
|
Dil.c
|
Amountd
|
Dil.e
|
Amountd
|
Patient no.
|
CT
|
Amountd
|
Amountf
|
CT
|
Sample
|
CT
|
Amountg
|
|
2.5x106
|
16.4
|
1:101
|
4.5x106
|
1:101
|
4.4x105
|
1
|
29.3
|
6.9x102
|
2.5x106
|
16.5
|
cereb.
|
31.1
|
1.4x102
|
2.5x105
|
20.0
|
1:102
|
3.4x105
|
1:102
|
4.0x104
|
2
|
25.6
|
7.3x103
|
2.5x105
|
20.0
|
cortex
|
36.4h
|
1.9x100
|
2.5x104
|
23.6
|
1:103
|
3.1x104
|
1:103
|
4.3x103
|
3
|
30.0
|
4.6x102
|
2.5x104
|
23.1
|
|
|
|
2.5x103
|
27.2
|
1:104
|
3.1x103
|
1:104
|
6.0x102
|
5
|
34.8
|
2.3 x101
|
2.5x103
|
26.7
|
|
|
|
2.5x102
|
31.1
|
1:105
|
3.4x102
|
1:105
|
9.8x101
|
|
|
|
2.5x102
|
30.3
|
|
|
|
2.5x101
|
34.9
|
1:106
|
3.1x101
|
1:10
|
2.1x100
|
|
|
|
2.5x101
|
34.1
|
|
|
|
2.5x100
|
36.8h
|
1:107
|
3.0x100 h
|
1:107
|
n.di
|
|
|
|
2.5x100
|
37.3h
|
|
|
|
|
>45
|
0
|
0
|
0
|
0
|
|
|
|
0
|
>45
|
|
|
|
|
aPlasmid DNA p88-D-21 was quantitated
spectrophotometrically, and dilutions containing the indicated
copy number of target sequence were added to each polymerase
chain reaction (PCR) assay.
bCT , Cycle number at which signal crosses
threshold.
cArmored RNA West Nile virus (HNY1999) standard
(Ambion, Austin, TX) was diluted 1:10, boiled, reverse
transcribed, and then diluted to result in amounts per PCR
assay equivalent to the indicated dilution of the stock (5
µL).
dAmount calculated based on calibration curve obtained
with NS5 Standard NY1999 (column 1).
eDilutions of Armored RNA West Nile virus
(HNY1999) standard (Ambion) were extracted with TRI-Reagent
(Molecular Research Center, Cincinnati, OH) and then subjected
to RT-PCR to result in amounts per assay equivalent to the
indicated dilution of the stock (5 µL).
fPlasmid DNA pISR-Dfrag-D6 was quantitated spectrophotometrically,
and dilutions containing the indicated copy number of target
sequence were added to each PCR assay.
gAmount calculated based
on calibration curve obtained with NS5 Standard ISR2000 (column
5).
hPoisson effects take place
at low template concentration; duplicate assay deviations:
36.2 / 37.4, NY1999; 6.0 x100 / 0, armored RNA;
37.4 / 37.1, ISR2000; and 36.4 / >45, cortex.
in.d. = not determined.
|
|