ISRAEL JOURNAL OF
VETERINARY MEDICINE
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VOLUME 54 ( 3) 1999
ANTEMORTEM DETECTION AND VIRUS CHARACTERIZATION
OF
THREE HUMAN RABIES FATALITIES IN ISRAEL
BETWEEN 1996-1997
D. David1, C. E. Rupprecht3 , J. S. Smith3, and Y. Stram2
1. Rabies Laboratory, Pathology Division
2. Virology Division, Kimron Veterinary Institute, Bet Dagan 50250 Israel.
3. Centers for Disease Control and Prevention, Division of Viral and Rickettsial Diseases,
Viral and Rickettsial Zoonoses Branch, 1600 Clifton Road, MS G33, Atlanta, Georgia 30333
USA.
Introduction
Materials and Methods
Results
Discussion
Summary
Twenty five years after the last reported case of human rabies in Israel, the
disease was diagnosed in 3 humans within a thirteen-month period
(November 1996- December 1997). By using a heminested RT-PCR and
direct sequencing of the RT-PCR product, rabies viral RNA was identified in
the saliva of the three individuals presenting with clinical signs. For virus
isolation, suckling mice were injected intracerebrally with human saliva or
brain extract and an epidemiological investigation based on molecular and
antigenic characterization of the viral isolates was performed. The 328 bp
sequence from the 3’ terminus of the N gene of the three isolates was
compared. The three human isolates and those isolated from infected wild
animals caught in the same vicinity revealed 100% homology with a virus
recovered from a fox. Further antigenic characterization of the three rabies
viruses based on a panel of monoclonal antibodies to the N protein revealed
that two of the human isolates were similar to phenotype 1 variant while the
third was closely related to phenotype 2 variant.
Introduction
Rabies is a major zoonotic disease in the Middle East affecting all mammals
except bats. There are two main epidemiological forms of rabies: urban, in
which stray dogs are responsible for the maintenance of the disease and its
transmission to man. The second type is wildlife (sylvatic) rabies, in which the
disease is maintained by wildlife vectors.
In most areas of Israel rabies is enzootic and its distribution includes both
epidemiological forms. Urban rabies is known since 1930 when domestic
dogs and jackals (Canis aureus) were identified as the main reservoir and
transmitting vectors (1). Mass vaccination of dogs, which became compulsory
in 1956 has decreased the number of urban cases drastically (2). In 1979
rabies became sylvatic and involved mainly foxes (Vulpes vulpes) which are
both reservoirs and transmitters of the virus.
The last case of human rabies was diagnosed in a Druse village on the
Golan Heights in 1971 (2). Twenty five years later, rabies occurred in a 20year-old (Israeli) soldier in 1996, and then in 1997 two further cases of lethal
human rabies affecting a 7 year old child and a 58 year old male (3).
Rabies virus belong to the Lyssavirus genus of the Rhabdoviridae family
and consists of an unsegmented negative – stranded RNA genome encoding
the N, M1, M2,G and L genes. Nucleoprotein gene reverse transcription and
polymerase chain reaction (RT-PCR) amplification of viral RNA was recently
introduced in diagnosis, epidemiological investigations ( 4,5,6), virus
characterization (7), and the importance of RT-PCR in ante- mortem
diagnosis of humans has been recognized (8,9). Recently a hemi-nested
RT-PCR (hnRT-PCR) test was developed to recognize rabies and related
lyssaviruses (9). hnRT-PCR has been shown to be a simple, sensitive and
rapid test for rabies(9). Early diagnosis of rabies is essential for proper patient
treatment, infection control, post-exposure vaccination of in-contact
individuals and for determining the source of the infection.
In this report, we demonstrate rapid antemortem detection of rabies virus in
infected human saliva using the hnRT-PCR. A molecular and monoclonal
antibodies (MAbs) profiling study to trace the virus origin revealed that a fox
was the most likely vector.
Case reports
Case 1
On October 6, 1996, a soldier on the Golan Heights was bitten on his lip by
an unidentified animal while sleeping. The wound was cleansed and sutured
but the patient did not receive specific anti-rabies treatment. Clinical signs
started 39 days later on November 14 with a high fever and headache. On
November 16, he was admitted to the Hillel Yaffe Hospital emergency room
suffering from hallucinations, difficulty in swallowing and generalized
weakness. Rabies was considered in the differential diagnosis. After receiving
passive and active immunization against rabies his neurological condition had
worsened and he was transferred to Chaim Sheba Medical Center. Three
days later he became comatose. On November 21, samples of saliva ,serum,
CSF, skin biopsy and corneal impressions were sent to the Pasteur Institute,
Paris, France. On November 22, samples of saliva and CSF were submitted
to the Kimron Veterinary Institute and hnRT- PCR was performed. The
soldier was diagnosed as rabid on November 24, eight days after the
appearance of clinical signs. Four days later, the Pasteur Institute confirmed
the PCR results. On December 15, 35 days after the appearance of clinical
symptoms the patient died despite supportive therapy.
Case 2
A 7 years old child from Kalansawa village was admitted to hospital on
November 20, 1997 presenting diarrhea, fever and vomiting that appeared 3
days earlier and was treated for dehydration. The only potential exposure to
rabies identified in her case history was a wound inflicted two months earlier
by an unidentified animal that attacked her during her sleep. A day later on
November 21, generalized convulsions began and deterioration in her
consciousness appeared with gasping. She was treated with phenobarbital
and idantoin, ventilated and transferred to the Pediatric Intensive Care Unit
(PICU) at the Schneider Children’s Medical Center, Petach Tikvah. On
admission she was unconscious with no verbal response, there was some
undirected movement of her hand in response to painful stimuli and much less
movement of the legs. The pupils responded to light, the doll’s eye sign was
normal, There was no cough reflex, a normal gag reflex, gasping with
ineffective breathing. There were very weak tendon reflexes in the legs with a
positive Babinski sign bilaterally. The tendon reflexes were very active in the
arms. During the following days, progressive deterioration in her brain stem
function was noted. All her brain reflexes disappeared but there was still
gasping and convulsions. Her evaluation included negative blood and CSF
bacterial cultures. Viral cultures for herpes and entroviruses were negative,
but had a positive mycoplasma serum antibody titer (IgG and IgM). CT scan
of brain was done 3 times and was normal as well as two MRIs of brain and
spinal cord. Her treatment included cetriaxon and tetracycline, and acyclovir
until the herpes virus results were completed. Saliva was positive for rabies in
a hnRT-PCR on December 1, whereas the CSF was negative. On December
3, samples of saliva, serum, CSF, skin and brain biopsies were sent to the
CDC to confirm the diagnosis, and on December 5, the diagnosis was
confirmed based on the RFFIT (Rapid Fluorescent Focus Inhibition Test) on
the serum and CSF and dIFA (Direct Immunofluorecence Assay) on the brain
tissue. The child died on December 7 despite supportive care.
Case 3
On December 11, 1997 a 58-year-old male from Judeida near Naharia was
admitted to the emergency room of Western Galilee Hospital with a fever,
headache and sore throat. He was diagnosed as having pharyngitis, and
received an oral antibiotic. Three days later he was admitted to ICU (Intensive
Care Unit) of the same hospital with fever and confusion. It transpired that he
had been bitten by unidentified animal on his left hand and face 3 months
earlier while sleeping. On admission to the hospital, the Lumbar Puncture, CT
scan and EEG were all normal. There were no significant abnormalities on
routine testing. A chest X-ray was normal. Because of significant
abdodistension and tenderness, abdominal ultrasound and CT scans were
performed and were normal. He received ceftriaxone IV, acyclovir and
citroflaxon empirically. On the third day he had respiratory arrest, and during
intubation, an acute laryngospasm with copious amounts of salivation
occurred. On December 13, rabies was suspected and viral RNA in the saliva
was detected by hnRT-PCR on December 15, four days after the patient was
hospitalized. The diagnosis was confirmed at the CDC. The patient died on
December 16, 1997.
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Discussion
Introduction
Materials and Methods
Results
Materials and Methods
Saliva
Saliva was collected 6 days, 14 days and 14 days respectivly after the
appearance of clinical symptoms from each of the three patients, and was
transferred on ice to the rabies laboratory in the Kimron Veterinary Institute.
Brain tissue
Samples of brain tissue were taken from patients 1and 3 postmortem and
from patient 2 antemortem. Samples of brain tissue were collected from
animals that died of rabies in the vicinity of the human cases were found to be
positive for rabies virus by direct immunofluorescence antibody (dIFA) test
(Centocor, Malvern, PA, USA) and PCR examination.
RT-PCR and nested PCR on saliva
Total RNA was extracted from infected and uninfected saliva for PCR and
nested PCR assay. The RNA (200-300 ng total RNA) was extracted using TRI
reagent (Molecular Research Center, Cincinnati, OH, USA) according to the
manufacturer’s instructions, was heated to 950C for 1 min, cooled on ice, and
added to 20 µl mixture of reverse transcription reaction containing AMV RT
reaction buffer (25 mM Tris-HCl, Ph 8.3 at 420C, 25 mM KCl, 5 mM MgCl2, 5
mM DTT, 0.25 mM spermidine), 250 µM of each of four deoxynucleotides,
100 pmol of hexamer random primer or specific primer 113 (5’GTAGGATGATATATGGG-‘3), 25 U of RNAsin (Promega, Madison), and 10
U of AMV reverse transcriptase (Promega, Madison). After incubation at 42 0C
for 90 min, 1 µl cDNA product was added to PCR solution in a 25 µl total
volume reaction mixture (60 mM Tris HCl, 15 mM (NH4)2SO4, 1.5 mM MgCl2,
Ph 8.5) containing 100 µM of each of the four nucleotides, 5 U of Taq
polymerase (Amplitaq, Perkin Elmer),100 ng of the primers 509 (5’GAGAAAGAACTTCAAGA-‘3) and 304 (5’-GAGTCACTCGAATATGTC-‘3) for
the PCR reaction and primers 509 and 105 (5’TTCTTATGAGTCACTCGAATATGTCTTGTTTAG-‘3) for the nested PCR
reaction. The reaction mixture was overlaid with mineral oil. Forty cycles of 45
sec. at 940C, 45 sec. at 370C and 90 seconds at 720C were applied. For the
nested PCR reaction 1 µl of PCR product was transferred to the reaction
mixture, which was the same as that used for the PCR reaction except that it
contained 100 ng of the primers 105 and 509. After 20 more cycles, as
described above, the nested PCR product was analyzed on 1.5% agarose
gels containing ethidium bromide
Virus isolation
To isolate the virus, antemortem saliva and postmortem brain tissue
suspensions from the infected patients, was inoculated intracerebrally in
suckling mice as previously described (13).
Molecular study
Genetic analysis was performed on rabies virus PCR products from the
dead patients and from brain samples; RNA was obtained directly from
infected brain tissue by TRI reagent. Cycling sequencing using an automatic
sequencer (Applied Biosystems) was employed on purified PCR fragments.(
Wizard PCR prep DNA purification system ; Promega). The fragment used for
the sequencing of the N gene was 328 bp in length (nt 1156 to 1484). The
fragment contained 264 bp from the 3’ of the N gene and 64 bp of the 3’
untranslated sequences of the N-NS non-coding region. Sequence analysis
was done using the pileup program, a part of Genetics Computer Group
(GCG, Wisconsin Sequence Analysis Package) (14).
Immunofluorescent staining for nucleocapsid antigen
The indirect immunofluorescent staining was used for detection of
nucleocapsid antigen on acetone- fixed brain impressions. The brain material
from the first and third cases were tested with a panel of 19 anti - N protein
monoclonal antibodies (Mabs) (CDC, Atlanta, GA, USA) for 30 min. at 370C,
washed to remove unbound antibody, and restained with fluoresceinconjugated goat anti mouse IgG ( Jackson ImmunoResearch Laboratories,
Inc.). Slides were examined x 200 in a fluorescent microscope BX-40
(Olympus Optical Co., Ltd. ).
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Discussion
Introduction
Materials and Methods
Results
Antemortem detection of rabies nucleic acid in saliva
Results
All three patients were diagnosed antemortem. By using the hnRT-PCR,
rabies RNA were detected in the saliva of all three patients whereas the CSF
were negative. In all three cases RNA detected by hnRT-PCR at eight, 14 and
14 days respectively after the appearance of clinical symptoms. The first case
was confirmed by Pasteur Institute, and the other two by CDC.
Figure 1 show the detection of rabies nucleic acid from the saliva of the
second case using RT-PCR and hnRT-PCR. RNA was extracted from
undiluted saliva and diluted 1:3. PCR was performed with primers 509-304
and gave a product of 377 bp (Fig.1A). hnRT-PCR was performed with
primers 509-105 and a 270 bp fragment was detected in the undiluted saliva
and diluted 1:3 (Fig. 1A and 1B lane 1,2). Both dilutions of the CSF were
negative in PCR and hnRT-PCR (Fig. 1A and 1B lane 3,4). In parallel, the
same procedures were performed on saliva from an uninfected person (Fig.
1A and 1B lane 5,6) and on brain tissue from an uninfected cow (Fig.1A and
1B lane 7); all these controls were negative in the PCR and hnRT-PCR
reaction.
Fig. 1
Agarose gel detection of rabies virus N gene from saliva of human case 2 using hnPCR method. (A)
··³ bp PCR product (B) 270 bp hnPCR product. A & B) lane 1 and lane 2: infected saliva - undiluted,
diluted 1:3 respectively; lane 3 and lane 4: CSF respectively; lane 5 and lane 6: control salivaundiluted, diluted 1:3; lane 7: normal cattle brain tissue; lane 9 and lane 10: rabies-infected cow brain
tissue. M: 1kb DNA marker.
Virus Isolation
Rabies virus was isolated from the soldier and the male but not from the
saliva of the child. In the first case, two of 15 suckling mice died 14 days postinoculation. The presence of rabies virus antigen in the brain tissue was
confirmed by dIFA, and viral RNA was detected by RT-PCR. Mice inoculated
with brain tissue of the first case did not show clinical signs until 30 days postinoculation. All the mice injected with the brain and saliva samples from the
third case died 14 days later.
Genetic analysis
Brain tissues were taken postmortem from the two patients and a brain
biopsy was collected antemortem from the child. Samples of brain tissue were
collected from dIFA and RT-PCR positive animals that died of rabies in the
region of the human cases. Phylogenetic trees, based on nucleotide
alignment of the 328 bp revealed three groups of sequences of which Golan
Heights and Galilee differed from each other in one nucleotide (Fig. 2) and
Shomron - Sharon differed from the others with two nucleotides. The Golan
Heights isolate had at nt 1201 a T to C substitution. The Galilee isolate
characterized at nt 1337 a A to T substitution and Shomron - Sharon isolate
has characterized at nt 1452 C to T and 1444 C to T in the non - coding
region of the N-M1 genes.
Fig. 2
Dendrogram showing the percentages of nucleotide changes of rabies virus isolates from human and
animals in the three regions as determined by analysis of 328 bp of the N gene.
Antigenic analysis
The reaction pattern obtained was compared with those of rabies variants
found in rabid animals in the geographical vicinity of the human cases. Two
antigenic variants 1 and 2 were found (Fig. 3). A single pattern of reactivity,
designated antigenic variant 1 (MAb C18 negative) characterized the two
isolates from human cases 1 and 3. This pattern was found in rabies virus
isolates from 10 foxes, one jackal and four cattle in the same regions (Fig. 3).
Antigenic variant 2 (MAbs C2, C7, C12, C13, C18 negative) characterized a
rabies virus isolate from 4 foxes, one dog and one cow in the vicinity of the
second human case (Fig. 3).
Fig. 3
Reactivity patterns of Mabs with rabies virus isolates from animals and two human patients. The
numbers of isolates tested are in boxes. The open box represents a positive reaction and solid boxes
negative staining. The BBL product is Anti-Rabies Globulin, Fluorescein Labeled, a polyclonal
antibody which used as a positive control (Becton - Dickinson, MD, USA).
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Discussion
Introduction
Materials and Methods
Results
Conclusions
Early antemortem diagnosis of rabies virus in an infected human is of the
utmost importance as it can significantly reduce the number of potential
exposures to the virus during contact with the patient and permit the
identification of persons who are candidates for prophylaxis. The system
described in this communication could therefore be applied in the future.
Checking the virus in the saliva can overcome the issue of sampling from
suspect humans and the sensitivity of hnRT- PCR makes it the technique of
choice for detecting limited amounts of virus in the saliva that is loaded with
proteases and nucleases. From the results presented in this communication
the hnRT- PCR appears to be a feasible technique for detecting rabies RNA
sequences in saliva. A recent report (9) regarding intravitam diagnosis
confirmed that RT-PCR of saliva and DFA assay of skin sections combined
together yielded a positive diagnosis in nine laboratory-confirmed cases of
human rabies. By using RT-PCR alone rabies virus was identified in the
saliva of 5 out of 9 patients (9). By using hnRT- PCR a 10- fold increase in
sensitivity compared with that of the external RT-PCR was achieved (10). The
mean time of diagnosis after clinical manifestation was 12 days, 3 days longer
than reported in the U.S which was 9.2 days (11). The difference was due to
the time the saliva sample were send to the laboratory and not lack the
sensitivity, since viral RNA could be detected immediately upon arrival.
In the first case we were unable to isolate the virus from brain tissue
despite positive RT-PCR and dIFA results. It is our believe that the prolonged
period of coma of the soldier resulted in loss of viral infectivity while some viral
RNA and protein persisted in the brain.
Antigenic characterization by MAbs of human rabies virus isolates in this
study revealed two phenotypic variants, 1 and 2. The variant phenotype 1 is
distributed in northern and southern Israel. The phenotypic variant 2 is found
in the central part of Israel.
Molecular analysis of the rabies virus isolates from animals and humans
showed that there are three genetic variants associated with the three
different geographic regions (Fig. 2). The three variants differed from each
other by one or two nucleotides. Previous work (5) showed that rabies viruses
isolated from an outbreak area tended to be very similar in nucleotide
sequence, regardless of the year of collection. Four field rabies samples
collected in 1988 from dogs in Mexico gave results similar to ours; analysis of
a 200-bp region of the N gene showed only one nucleotide difference
between them (5). Moreover, two samples from a region in western Mexico,
isolated 30 years apart, were identical in sequence (5). Incorporation of the
reference strains Pasteur and SAD B19 (12,15) into our phylogenetic tree
indicated that the viruses isolated from the three human cases belong to
lyssavirus genotype 1. Based on the homology of the nt sequences between
the three patients and foxes caught in the same regions, we conclude that the
reservoir for rabies in foxes is responsible for infection in all three humans.
These data clearly demonstrate that even age-old zoonoses such as rabies
are still important within the context of emerging infectious diseases, that
novel molecular tools can readily provide answer to key epidemiological
facets of such maladies, and that costs associated with biomedical
complacence may have disastrous public health consequences.
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Discussion
Introduction
Materials and Methods
Results
Acknowledgment
We express our gratitude to Dr. H. Bourhy at the Rabies Unit, Pasteur
Institute, Paris France for the confirmation of the rabies diagnosis. We
indebted to those who sent us the clinical cases; Dr. J. Ben Ari, PICU,
Schneider children’s Medical Center, Petach Tikvah, Israel; Dr. Mahoul, ICU
Western Galillee Hospital, Naharia, Israel ; Dr. A. Greenfeld, RICU, Tel
Hashomer Medical Center, Ramat Gan Israel; Prof. R. Caraso, Neurology
Department, Hillel Yaffe Hospital, Hadera, Israel.
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Discussion
Introduction
Materials and Methods
Results