Emerging Infectious Diseases Volume 2 Issue 3

89 Pages·1996·6.2 MB·English

by  CDC

Emerging Vol. 2, No. 3, July-September 1996
Infectious Diseases
Tracking trends and analyzing new and reemerging infectious disease issues around the world
Molecular Identification of S-J. Gao
Unculturable Infectious Agents
DNA Vaccines R.G. Whalen
Conjugate Vaccines and
Haemophilus influenzae Type b M.L. Barbour
Molecular Techniques and D.P. Fedorko
Microsporidia
Coccidioidomycosis T.N. Kirkland
Antibody-Based Therapies A. Casadevall
HIV-1 Group O Virus in M. Rayfield
the United States
Morbilliviruses in Dolphins J.K. Taubenberger
Spotted Fever Rickettsiosis B.L. Smoak
Heterogeneous HIV-1 in the M. Massanga
Central African Republic
Legionella-Like Amebal Pathogens A. Adeleke
Enterovirus 71 and E.E. da Silva
Acute Flaccid Paralysis
Bancroftian Filariasis D.F. Thompson
Yellow Fever in Kenya E.J. Sanders
Paramyxovirus in Pteropus Bats P.L. Young
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service
Emerging Infectious Diseases
Emerging Infectious Diseases is indexed in Current Contents and in several electronic databases.
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Chief Medical Adviser Liaison to Infectious Diseases Society Joseph E. McDade, Ph.D.
Commonwealth Department of of America National Center for Infectious Diseases
Centers for Disease Control
Human Services and Health University of Virginia Medical Center
and Prevention (CDC)
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Perspectives Editor
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Pan American Health Organization Central Public Health Laboratory
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World Health Organization London, United Kingdom New York, New York, USA
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Robert Shope, M.D.
Synopses Editor
Gail Cassell, Ph.D. Professor of Research
Phillip J. Baker, Ph.D.
Liaison to American Society for Microbiology University of Texas Medical Branch Division of Microbiology and Infectious Diseases
University of Alabama at Birmingham Galveston, Texas, USA National Institute of Allergy and Infectious Diseases
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and Prevention (CDC) National Center for Infectious Diseases
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Infectious Diseases.
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Emerging Infectious Diseases Beatrice T. Divine M.B.A.
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Contents
Emerging Infectious Diseases
Volume 2 • Number 3 July–September 1996
Perspectives
Molecular Approaches to the Identification of Unculturable Infectious Agents 159 S-J. Gao and P.S. Moore
DNA Vaccines for Emerging Infectious Diseases: What If? 168 R.G. Whalen
Synopses
Conjugate Vaccines and the Carriage of Haemophilus influenzae Type b 176 M.L. Barbour
Application of Molecular Techniques to the Diagnosis of 183 D.P. Fedorko and Y.M. Hijazi
Microsporidial Infection
Coccidioidomycosis: A Reemerging Infectious Disease 192 T.N. Kirkland and J. Fierer
Antibody-Based Therapies for Emerging Infectious Diseases 200 A. Casadevall
Dispatches
HIV-1 Group O Virus Identified for the First Time in the United States 209 M.A. Rayfield, P. Sullivan, C. I.
Bandea, R. A. Otten, C.P. Pau,
D. Pieniazek, S. Subbarao,
P. Simon, C. A. Schable, A. Wright,
J. Ward, and G. Schochetman
Two Morbilliviruses Implicated in Bottlenose Dolphin Epizootics 213 J.K. Taubenberger, M. Tsai,
A.E. Krafft, J.H. Lichy, A.H.
Reid, F.Y. Schulman, and
T.P. Lipscomb
An Outbreak of Spotted Fever Rickettsiosis in U.S. Army Troops 217 B.L. Smoak, J.B. McClain,
Deployed to Botswana J.F. Brundage, L. Broadhurst,
D.J. Kelly, G.A. Dasch, and
R.N. Miller
A Highly Heterogeneous HIV-1 Epidemic in the Central African Republic 222 M. Massanga, J. Ndoyo, D.J.
Hu, C-P. Pau, S. Lee-Thomas,
R. Hawkins, D. Senekian, M.A.
Rayfield, J.R. George, A. Zengais,
N.N. Yatere, V. Yossangang,
A Samori, G. Schochetman, and
T. J. Dondero
Legionella-Like Amebal Pathogens—Phylogenetic Status and Possible 225 A. Adeleke, J. Pruckler,
Role in Respiratory Disease R. Benson, T. Rowbotham,
M. Halablab, and B. Fields
Role of Enterovirus 71 in Acute Flaccid Paralysis After the Eradication 231 E.E. da Silva, M.T. Winkler,
of Poliovirus in Brazil F. Ferreira, and M. Pallansch
Bancroftian Filariasis Distribution and Diurnal Temperature 234 D.F. Thompson, J.B. Malone,
Differences in the Southern Nile Delta M. Harb, R. Faris, O.K. Huh,
A.A. Buck, and B.L. Cline
Sentinel Surveillance for Yellow Fever in Kenya, 1993 to 1995 236 E.J. Sanders, P. Borus,
G. Ademba, G. Kuria, P.M.
Tukei, and J.W. LeDuc
Serologic Evidence for the Presence in Pteropus Bats of a Paramyxovirus 239 P.L. Young, K. Halpin,
Related to Equine Morbillivirus P.W. Selleck, H. Field, J.L.
Gravel, M.A. Kelly, and
J.S. Mackenzie
Letters
Acute Cervical Lymphadenopathy 241 M.B. Pasticci, F. Baldelli,
F. Bistoni, C. Piersimoni,
G. Sbaraglia, G. Stagni, and
S. Pauluzzi
AIDS: Déjà Vu in Ancient Egypt? 242 R.J. Ablin
News and Notes
International Meeting on Research Advances and 243 C.E. Rupprecht
Rabies Control in the Americas
Symposium Notice 243 Infect. Dis. Assoc. of California
Perspectives
Molecular Approaches to the Identification
of Unculturable Infectious Agents
Shou-Jiang Gao, Ph.D., and
Patrick S. Moore, M.D., M.P.H., M.Phil.
School of Public Health, Columbia University,
New York, New York, USA
New molecular biologic techniques, particularly representational difference analysis,
consensus sequence–based polymerase chain reaction, and complementary DNA library
screening, have led to the identification of several previously unculturable infectious agents.
New agents have been found in tissues from patients with Kaposi’s sarcoma, non-A, non-
B hepatitis, hantavirus pulmonary syndrome, bacillary angiomatosis, and Whipple’s disease
by using these techniques without direct culture. The new methods rely on identifying
subgenomic fragments from the suspected agent. After a unique nucleic acid fragment
belonging to an agent is isolated from diseased tissues, the fragment can be sequenced and
used as a probe to identify additional infected tissues or obtain extended portions of the
agent’s genome. For agents that cannot be cultured by standard techniques, these
approaches have proved invaluable for identification and characterization studies. Applying
these techniques to other human diseases of suspected infectious etiology may rapidly
elucidate novel candidate pathogens.
Identifying the causative agent of an infectious Although they have revolutionized our ability to
disease is the cornerstone for its eventual control. identify new pathogens, innovative molecular bio-
In recent years, a great deal of progress has been logic techniques must be applied in conjunction with
made in identifying new agents associated with both traditional epidemiologic procedures. Beral, Jaffe and
well-known and newly emerging infectious diseases. colleagues, for example, showed that AIDS-related
A number of syndromes exist, however, in which Kaposi’s sarcoma (KS) is likely to be caused by a
infectious etiology is likely, but the pathogen resists transmissible agent other than human immunodefi-
cultivation with standard microbiologic techniques. ciency virus (HIV), before any likely causative agent
For emerging diseases, rapid identification and char- was isolated (3, 4). These findings focused the atten-
acterization of the responsible agent are crucial first tion of investigators on AIDS-KS, which resulted in
steps for epidemic control. the isolation of viral DNA from AIDS-KS lesions and
The rapid identification of a hantavirus respon- the description of a new human herpesvirus (5, 6).
sible for an outbreak of severe pulmonary distress Spurious associations between infectious agents and
syndrome in the southwestern United States (1, 2) diseases are common, however, and only through
demonstrated that applying molecular biologic ap- careful epidemiologic studies can a causal link be-
proaches can accelerate the identification of an tween an organism and a disease be established.
unknown agent. With extensive nucleic acid and Epidemiologic criteria for causality (7-9), supersed-
protein databases readily available, isolating and ing Koch’s postulates, have been used for 30 years,
sequencing genomic fragments from an unknown and a critical phase in the process of new pathogen
agent can provide important clues regarding its ori- discovery involves the unambiguous establishment
gin and biologic behavior. Once a new agent’s that an agent is central to the disease process.
phylogenetic relationship to other known organisms The various molecular biologic approaches to
is established, appropriate culture conditions, sero- agent identification differ in technical detail, but all
logic tests, and perhaps even therapeutic strategies rely on isolating nucleic acid fragments belonging to
can be rapidly developed. the agent’s genome from diseased tissue. The for-
midable tasks of identifying and separating small
Address for correspondence: Shou-Jiang Gao, Ph.D., School unique nucleic acid fragments from human genomic
of Public Health, Columbia University, P&S 14-442, 630 West-
material have been approached by various means,
168th St., New York, NY 10032; fax: [phone]; e-mail:
[email protected]. each with its own particular strengths and weak-
159
Vol. 2, No. 3—July-September 1996 Emerging Infectious Diseases
nesses. The appropriate technique depends on the
tional difference analysis is also an important tool
t y p e o f i n f e c t i o u s a g e n t i n v o l v e d ( e . g . , b a c t e r i a l o r
for identifying polymorphic DNA sequences associ-
viral), whether the disease occurs in a normally ster-
ated with noninfectious diseases (11).
ile site, and whether it can be passaged through
R e p r e s e n t a t i o n a l d i f f e r e n c e a n a l y s i s d e p e n d s
animals.
first on digesting DNA from both healthy and dis-
Once a fragment from the agent’s genome is iso-
eased tissues by using a restriction enzyme and then
l a t e d a n d s e q u e n c e d , s t a n d a r d g e n o m i c w a l k i n g
on separately “simplifying” the resulting genomes
t e c h n i q u e s a r e u s e d t o e x t e n d t h e k n o w n s e q u e n c e ,
to reduce their sequence complexity. This is done by
a n d c o m p u t e r h o m o l o g y s e a r c h e s c a n b e u s e d t o
l i g a t i n g P C R p r i m e r s t o b o t h s e t s o f D N A a n d
identify the likely phylogenetic relationship of the
nonspecifically amplifying the mixtures. Since PCR
agent to other known organisms. In this article, we
most efficiently amplifies fragments of 150 to1500
highlight recent successful situations when molecu-
bp, restriction fragments in this size range are en-
lar approaches were used to identify and characterize
r i c h e d , a n d t h e f r a g m e n t s o f t h e s e q u e n c e
unknown agents of infectious diseases.
represented outside this size range (90%) are re-
duced.
Unique strands of DNA from diseased tissue rep-
R e p r e s e n t a t i o n a l D i f f e r e n c e A n a l y s i s
r e s e n t i n g r e s t r i c t i o n f r a g m e n t s o f a n e x o g e n o u s
Representational difference analysis is one of the
agent are isolated in a subtractive hybridization pro-
more robust methods of identifying new agents since
c e s s c o u p l e d t o P C R a m p l i f i c a t i o n . F i r s t , t h e
i t d o e s n o t r e q u i r e p r i o r k n o w l e d g e o f t h e a g e n t ’ s
priming sequences ligated on DNA restriction frag-
class (10). The technique is based on polymerase
m e n t s o f b o t h n o r m a l a n d i n f e c t e d t i s s u e s a r e
chain reaction (PCR) enrichment of DNA fragments
r e m o v e d . N e w p r i m e r s e q u e n c e s a r e l i g a t e d o n l y
present in diseased tissue but absent from healthy
to the diseased tissue DNA fragments. These are
tissues of the same patient (Figure). Representa-
then hybridized with an excess of the healthy tis-
F i g u r e . S c h e m a t i c r e p r e s e n t a t i o n o f d i f f e r e n t m o l e c u l a r a p p r o a c h e s t o t h e i d e n t i f i c a t i o n o f u n c u l t u r a b l e
i n f e c t i o u s a g e n t s .
Perspectives
Emerging Infectious Diseases 160 Vol. 2, No. 3—July-September 1996
Perspectives
sue representation. Human DNA fragments com- AIDS or KS were examined, and none showed evi-
mon to both diseased and healthy tissues reanneal dence of KSHV infection.
to each other and, since the healthy tissue frag- Although controversy remains over the role of
ments are in excess, any given human fragment KSHV in KS (18-20), epidemiologic and biologic stud-
derived from the diseased tissue will reanneal to ies strongly suggest that the agent is a causal factor
a complementary strand from the healthy tissue in KS (21). KSHV has now been identified in 211
representation. Thus, common human sequences (94.2%) of 224 KS lesions examined by a number of
found in both representations will only have one groups around the world, and the virus is found in
PCR priming sequence or none present on two all forms of KS, AIDS-related and non-AIDS-related
complementary strands. However, DNA fragments (22-29). Two independent studies have shown that
from the infectious agent will not find complemen- KSHV is present in peripheral blood mononuclear
tary strands in the healthy tissue representation cells of AIDS-KS patients before onset of KS (30,
and will reanneal with each other. Only hybrids 31), indicating that the virus is unlikely to be a “pas-
with both strands derived from the diseased tis- senger virus” in KS lesions. Further, the virus has
sue representation will have priming sites and be been localized to KS tumor tissues by
able to undergo subsequent exponential PCR am- semiquantitative PCR (25) and by PCR in-situ hy-
plification. Several rounds of representational bridization (26). The virus has only been found in 8
difference analysis are performed, which succes- (1.8%) of 449 solid tissues from control patients with-
sively enrich the mixture for unique DNA out KS (5, 22, 25, 27, 28, 32, 38) and thus appears to
sequences present only in the diseased tissue rep- be specifically associated with KS. Although early
resentation. reports suggested that the virus is present in skin
tumors from transplant patients without KS (19),
KS-Associated Herpesvirus and KS subsequent studies have not confirmed this finding
(33, 34). One study has found evidence of viral DNA
The power of representational difference analy-
by nested PCR, but not unnested PCR, in semen
sis and the difficulties encountered in establishing
from (23%) of healthy donors; these results suggest
the etiology of disease are illustrated by its applica-
that the prevalence of infection in North America
tion to KS (5), a vascular neoplasm that frequently
may be higher than that indicated by tissue or lym-
occurrs in homosexual men with AIDS (3). Geo-
phocyte studies (35). This intriguing finding has not
graphic clustering (12, 13) and association with
been reproduced by other groups (23, 36); however,
specific sexual behavior (4, 14) suggest that the dis-
it should be explored in large rigorous studies.
ease is caused by a sexually transmitted agent.
Lymphoproliferative disorders are common sec-
Several agents have been investigated, including
ondary malignancies in KS patients with and
human cytomegalovirus (CMV), human papil-
without AIDS (37). KSHV has also been found in a
lomavirus, human herpesvirus 6 (HHV-6), and HIV
rare subset of AIDS-related, body cavity-based
(for review, see [15]), but no convincing etiologic link
lymphomas, which are manifested as primary ef-
has been established.
fusions (5, 38). In these AIDS-related lymphomas,
Using representational difference analysis,
tumor cells are coinfected with EBV (38, 39) but,
Chang and colleagues isolated two unique DNA se-
unlike tumor cells in EBV-related Burkitt’s
quences (KS330Bam and KS631Bam) from a KS
lymphomas, these cells do not exhibit c-myc gene
lesion in an AIDS patient (5). These DNA sequences
rearrangement. EBV-uninfected, KSHV-related
are homologous to portions of minor capsid and tegu-
body cavity-based lymphomas have also recently
ment protein genes from Epstein-Barr virus (EBV)
been identified (40, 43). Another
and herpesvirus saimiri (HVS), a New World mon-
lymphoproliferative syndrome associated with KS
key herpesvirus. Both EBV and HVS are gamma
is Castleman’s disease in both AIDS patients and
herpesviruses associated with neoplastic disorders
HIV-seronegative patients. KSHV has been found
in humans (EBV) (16) and nonhuman primates (HVS)
in AIDS-related multicentric Castleman’s disease
(17). These results suggested that a new human her-
lesions at a high copy number and less frequently
pesvirus, now called KS-associated herpesvirus
in tissues from Castleman’s disease patients with-
(KSHV or HHV-8), could be the KS agent.
out AIDS (41).
The strong association between KS and KSHV
Identifying KSHV DNA sequences by repre-
was demonstrated by using Southern hybridization
sentational difference analysis also led to the quick
and PCR to amplify a 233-bp DNA fragment (KS330 )
233 identification of an in vitro culture system for grow-
internal to KS330Bam from all 25 intact and
ing the virus. KS330Bam and KS631Bam probes were
amplifiable DNA samples from KS lesions that were
used to identify a B-cell line derived from a body
examined (5). Ninety tissues from patients without
161
Vol. 2, No. 3—July-September 1996 Emerging Infectious Diseases
Perspectives
cavity-based lymphoma that was stably infected difference analysis sequence by PCR (44). Although
with both KSHV and EBV (42). Extended sequenc- case-patients and controls cannot be differenti-
ing and transmission studies have been performed ated by the presence or absence of these HHV-6
with body cavity-base lymphoma cell lines, which sequences, only MS tissues showed nuclear stain-
clearly define KSHV as a new human herpesvirus ing of oligodendrocytes surrounding plaques when
of the genus Rhadinovirus (6). This has recently monoclonal antibodies against HHV-6 viral pro-
been confirmed by detecting herpesvirus particles teins were used (44). There may be a subtle
in a body cavity-based lymphoma cell line by elec- difference in tissue distribution for the virus in
tron microscopy (43). MS patients not found in controls, or the HHV-6
Discovery of a continuously infected cell line variant B group 2 infecting MS plaques may be
has allowed the first generation of serologic as- particularly prone to generating the autoimmune
says for KSHV antibodies to be developed (6). response seen in this disease.
Second-generation immunoblotting assays, which
appear to be both sensitive and specific for de- GB Hepatitis Viruses
tecting KSHV antibodies, indicate that KS patients
Since representational difference analysis relies
are infected with the virus months to years be-
on PCR amplification, the technique is suited for
fore the disease develops and that few North
detecting agents with a DNA-based genome; se-
American blood donors are infected (S-J Gao, P.S.
quences can be amplified by generating a cDNA
Moore, unpublished observation). Thus, identify-
intermediate, but detecting RNA viruses is problem-
ing a virus associated with KS by using molecular
atic since mRNA expression patterns differ between
approaches has rapidly led to new assays that use
tissues and are likely to give spurious representa-
traditional serologic techniques for detecting in-
tional difference analysis bands. Simons and
fection.
colleagues overcame this problem by passing an
agent associated with non-A, non-B (NANB) hepati-
HHV-6 and Multiple Sclerosis tis through primates and using cell-free extracts of
The difficulties of using representational dif- primate plasma for representational difference analy-
ference analysis for new pathogen identification sis and discovered two new human hepatitis viruses
are also illustrated by the search for the agent (52, 53).
that causes multiple sclerosis (MS) (44). An infec- A novel form of NANB hepatitis was first identi-
tious cause for MS has been proposed (45-47), and fied in a physician who became ill with hepatitis,
geographic and household clustering of cases con- and the disease was transmitted to primates by in-
sistent with an infectious process have been shown jecting serum from patients into the animals (54).
(45, 48-50). Challoner and colleagues used DNA Extensive studies demonstrated that the virus,
from sclerotic plaques in brain tissues from MS named the GB agent, was different from all known
patients and performed representational differ- human hepatitis viruses (hepatitis A [HAV], hepati-
ence analysis against pooled DNA from peripheral tis B [HBV], hepatitis C [HCV], and hepatitis E)
blood leukocytes of healthy donors. Use of pooled (55-57). Total RNA from the samples was reverse-
lymphocyte DNA in representational difference transcribed to obtain cDNA by using cell-free
analysis opens the possibility for examining rare preinfection and acute-phase plasma from infected
banked tissues for which healthy control tissues monkeys. After cDNA synthesis, representational
are not available. A 341-bp representational dif- difference analysis was performed, and seven cDNA
ference analysis band was isolated from MS tissues clones were found to be specifically associated with
with near sequence identity to a region of the ma- this form of hepatitis. Sequence analysis and com-
jor DNA-binding protein gene of HHV-6 (44). parison with other known hepatitis viruses
Detecting exogenous DNA in diseased tissue by identified two unique flaviviruses, GBV-A and GBV-
this technique, however, does not exclude the pos- B, as the GB agents (52, 53, 58). A third virus,
sibility that a commensal agent may be identified GBV-C, was subsequently identified using the
that is not the cause of the disease being exam- known GBV-A, GBV-B, and HVC consensus se-
ined. HHV-6 is a neurotrophic virus present in quences (52b and see below).GBV-A and GBV-C
brain tissues from healthy control patients (51), are closely related phylogenetically. Cross-chal-
and more than (70%) of both MS patients and con- lenge experiments showed that GBV-C probably
trols were positive for the representational originated in human hepatitis patients, whereas
Emerging Infectious Diseases 162 Vol. 2, No. 3—July-September 1996
Perspectives
GBV-A and B may be tamarin monkey viruses that among many of the classes of viruses that eventu-
were inadvertently passaged along with the hu- ally could be used in screening panels of diseased
man virus (53, 58). tissues when a viral cause is suspected.
In addition to having difficulty in identifying
RNA viruses, representational difference analy- Bartonella: Bacillary Angiomatosis and
sis has several other limitations. Polymorphic
Cat-Scratch Disease
human DNA can be amplified through this tech-
Phylogenetic studies of bacteria have relied on
nique (5), and not all bands generated by it belong
comparisons of highly conserved rRNA gene se-
to the suspected agent. Representational differ-
quences present in prokaryotes (62). rRNA genes
ence analysis may produce DNA fragments from
are present in all living cells and contain regions
an agent whose genome has not been sequenced
of highly conserved sequences with intervening
(e.g., most bacteria and fungi), and sequence ho-
variable regions. Conserved sequences can be used
mology searches may be unable to distinguish the
to amplify and sequence the intervening variable
agent’s genomic DNA fragments from unsequenced
regions by PCR. This technique was exploited by
human DNA. Only normally sterile site tissues are
Relman and colleagues to identify the bacillus associated with
appropriate for this technique because normal flora
bacillary angiomatosis (60).
that differs between tissue sites could result in
spurious amplification. Finally, for viruses with Bacillary angiomatosis is an inflammatory vascular prolif-
small genomes, multiple restriction digests may erative process that affects the skin, lymph nodes, and visceral
be required to identify a unique restriction fragment organs of AIDS patients (63). Although bacilli can be identified
of the appropriate size that can be efficiently ampli- by Warthin-Starry staining of lesions (64, 65), the suspected
fied. Although the technique has been successfully causal organism was resistant to cultivation by standard tech-
used by several groups to identify polymorphic DNA, niques. Consensus oligonucleotide primers complementary to
the procedure is complex and not uniformly repro- the 16S rRNA genes of eubacteria were used to amplify 16S
ducible. rRNA gene fragments directly from tissue samples (60). Phy-
logenetic analysis of the amplified DNA sequence showed that
the organism belonged to the genus Rochalimaea (now re-
Consensus Sequence–Based PCR
named Bartonella) (60). Bartonella organisms
Consensus sequence PCR relies on the use of were also cultured from bacillary angiomatosis le-
highly conserved DNA sequences, such as riboso- sions (66) and blood from bacteremic patients (67,
mal RNA (rRNA) gene sequences from known organisms, to 68), and serologic analyses have associated the or-
amplify DNA from related organisms not yet discovered (59). ganisms with cat-scratch disease (69). Current
This technique is simple and extremely successful in identifying evidence suggests that bacillary angiomatosis in
new human pathogens. Subunit rRNA genes evolve in a rela- HIV-seropositive patients results from infection
tively slow and uniform manner, which makes these sequences with either B. quitana or a newly described
extremely useful for establishing phylogenetic relationships (59). Bartonella species, B. henselae (68, 70), whereas
By using conserved DNA sequences from bacterial 16S rRNA cat-scratch disease is primarily caused by infec-
genes, at least two new bacteria associated with human dis- tion with B. henselae (71) (for
eases have been identified (60, 61), and PCR amplification of review, see [72]).
conserved hantaviral capsid DNA sequences resulted in the
rapid identification of a new hantavirus associated with an out-
Whipple’s Disease
break of severe pulmonary disease (1).
Consensus sequence PCR was also used to iden-
Unlike using representational difference analysis, using
tify an organism associated with Whipple’s disease,
consensus sequences to amplify DNA requires knowledge of
one of the most persistent mysteries in microbiol-
the suspected agent’s phylogenetic relationship to other or-
ogy (61). Whipple’s disease is a systemic illness, first
ganisms. The technique generally should be used on normally
described in 1907, characterized by arthralgias, di-
sterile site tissues if sequences from normal flora are also likely
arrhea, abdominal pain, and weight loss (73).
to be amplified. Although the lack of broadly amplifiable con-
Rod-shaped bacilli were identified histologically in
sensus primers limits use of this technique to prokaryotic and
Whipple’s disease lesions in the early 1960s (74),
eukaryotic pathogens, consensus sequences are to likely exist
but the suspected bacteria were not culturable by
standard techniques (73). The agent was found to
be a gram-positive actinomycete (Tropheryma
whippelii gen.nov.sp.nov.), unrelated to any char-
163
Vol. 2, No. 3—July-September 1996 Emerging Infectious Diseases
Perspectives
acterized species when a bacterial 165 rRNA se- were not caused by either virus (81, 82). Conven-
quence was amplified and sequenced directly from tional techniques failed to identify the agent responsible for most
infected tissue (61, 75). cases of NANB hepatitis (83), despite evidence that the dis-
ease was caused by a bloodborne, small, enveloped virus readily
transmissible to chimpanzees (84, 85).
Hantavirus Pulmonary Syndrome
Since the virus was presumed to be an RNA virus, Choo
Consensus PCR primers have been successfully
and colleagues made a cDNA expression library from RNA
used to identify and classify bacteria, and similar
isolated from an infected chimpanzee’s plasma (86). While the
techniques can be used to diagnose new viral agents.
GBV-A, B, C were identified by direct detection of viral cDNA
In May 1993, an outbreak of unexplained acute res-
through representational difference analysis, HCV cDNA was
piratory illness with a high death rate occurred in
identified by immunologic detection of cDNA that was encod-
the southwestern United States (76). In the initial
ing viral protein. Viral antigens expressed from
phases of the investigation, the cause of the syn-
the cDNA library were identified by
drome was not clearly known to be infectious. Serologic tests
immunoscreening with convalescent-phase human
quickly detected cross-reactive antibodies to
sera. A cDNA clone was isolated encoding an anti-
known hantaviral antigens in the serum of pa-
gen that could be used to screen
tients; these results suggested that a previously
convalescent-phase sera from patients with NANB
unrecognized hantavirus was the cause of the dis-
hepatitis. Identification of the clone rapidly led to
ease (77). PCR primers, based on consensus
the production of a recombinant antigen used for
sequences within the G2 protein coding region of
serologic screening to detect specific antibodies
the M segment of the genomes of known
in infected chimpanzees and patients with hepati-
hantaviruses, were designed and used in nested
tis after transfusion. Extended sequence analysis
reverse-transcription PCR to amplify a short seg-
demonstrated that the agent, now known as HCV,
ment of the viral genome from diseased tissues
is closely related to the family Flaviviridae and is
(1).
the major cause of NANB hepatitis throughout
Sequence analysis of the PCR products showed
the world (86, 87). Recently, the same approach
that the amplicon differed from the other known
was successfully used to identify HGV, which has
hantaviral sequences by least 30%, and phyloge-
been found to be almost identical to GBV-C, the
netic analysis demonstrated that the new virus is
human hepatitis virus identified by representa-
most closely related to Prospect Hill hantavirus
tional difference analysis (88). The potential role
(1), a zoonotic hantavirus endemic in North
of HGV and GBV-C in human disease still remains
America (78). Hantavirus antigens have been de-
uncertain (88b).
tected in endothelial cells from patients (1, 79),
Using convalescent-phase sera to screen cDNA
and virus particles have been identified in infected
libraries from diseased tissues is a novel method
pulmonary endothelial cells and macrophages (80).
for pathogen identification. It is a potentially use-
Deer mice (Peromyscus maniculatus) are the pri-
ful technique for diseases in which well-defined
mary host for the virus (1), and the virus has been
convalescent-phase sera are available, and it re-
passaged through laboratory-bred deer mice and
quires tissues with a high titers of the agent. On
cultured in Vero E6 cells (2). This newly recog-
the other hand, constructing and screening DNA
nized virus was originally named Muerto Canyon
libraries are laborious, and cross-reactive antigens
virus (2) but has since been renamed Sin Nombre
are likely to be detected, especially for diseases in
virus in light of nomenclatural disputes regarding
which autoantibodies are common.
the appropriateness of a descriptive name.
Future Directions
Complementary cDNA Library Screening
Nucleic acid database information is rapidly ex-
Identification of HCV and HGV
panding for all classes of organisms, and a significant fraction of
A third major approach to new organism identi-
the human genome has already been sequenced. Even small
fication relies on screening cDNA libraries made from
laboratories can exploit new and relatively inexpensive mo-
diseased tissues by using hyperimmune serum from
lecular biologic technologies to search for new pathogens. Once
specimens. This method was successfully used to
a small, unique nucleic acid fragment from a pathogen has been
identify the cause of most cases of NANB hepatitis,
identified, nucleic acid detection and serologic assays can often
HCV. When serologic tests for HAV and HBV be-
be rapidly developed to establish an etiologic link with disease.
came available, it became clear that most cases of
New pathogens are likely to be identified by some of these
transfusion-associated hepatitis in the United States
molecular biologic approaches. A number of diseases have long
Emerging Infectious Diseases 164 Vol. 2, No. 3—July-September 1996