Virology Hantaviruses
Hantaviruses belong to the bunyavirus family of viruses. There are 5 genera within the
family: bunyavirus, phlebovirus, nairovirus, tospovirus, and hantavirus. Each is made up
of negative-sensed, single-stranded RNA viruses. All these genera include arthropod-borne
viruses, with the exception of hantavirus, which is rodent-borne.
Like other members of the bunyavirus family, hantaviruses are enveloped viruses with a
genome that consists of three single-stranded RNA segments designated S (small), M
(medium), and L (large). All hantaviral genes are encoded in the negative (genome
complementary) sense. The S RNA encodes the nucleocapsid (N) protein. The M RNA encodes a
polyprotein that is cotranslationally cleaved to yield the envelope glycoproteins G1 and
G2. The L RNA encodes the L protein, which functions as the viral transcriptase/replicase.
Within virions, the genomic RNAs of hantaviruses are thought to complex with the N protein
to form helical nucleocapsids, which circularize due to sequence complementarity between
the 5' and 3' terminal sequences of each genomic segment.
Hantaviruses replicate exclusively in the host cell cytoplasm. Entry into host cells is
thought to occur by attachment of virions to cellular receptors and subsequent
endocytosis. Nucleocapsids are introduced into the cytoplasm by pH-dependent fusion of the
virion with the endosomal membrane. Transcription of viral genes is initiated by
association of the L protein with the three nucleocapsid species. In addition to
transcriptase and replicase functions, the viral L protein is also thought to have an
endonuclease activity that cleaves cellular messenger RNAs (mRNAs) for the production of
capped primers used to initiate transcription of viral mRNAs. As a result of this
"cap snatching," the mRNAs of hantaviruses are believed to be capped and contain
nontemplated 5' terminal extensions. The viral N and L mRNAs are thought to undergo
translation at free ribosomes, whereas the M mRNA is translated in the endoplasmic
reticulum. G1 and G2 glycoproteins form heterodimers and are then transported from the
endoplasmic reticulum to the Golgi complex, where glycosylation is completed. The L
protein produces nascent genomes by replication via a positive-sense RNA intermediate.
Hantavirions are believed to form by association of nucleocapsids with glycoproteins
embedded in the membranes of the Golgi, followed by budding into the Golgi cisternae.
Nascent virions are then transported in secretory vesicles to the plasma membrane and
released by exocytosis.
Hantaviruses Causing HPS
Sin Nombre virus (SNV) was first isolated from rodents collected on the premises of one
of the initial HPS patients in the Four Corners region. Isolation was achieved through
blind passage in Peromyscus maniculatus and subsequent adaptation to growth in Vero
E6 cells. Additional viral strains have also been isolated from P. maniculatus
associated with a fatal case in California and P. leucopus from the vicinity of
probable infection of a New York case. Black Creek Canal virus was isolated from S.
hispidus collected near the residence of a human case in Dade County, Florida.
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Thin-section electron micrograph of Sin Nombre virus isolate, a
causative agent of hantavirus pulmonary syndrome (HPS). From the 1993 outbreak of HPS in
the southwestern United States.
Image courtesy Cynthia Goldsmith, Sherif Zaki, and Luanne Elliott.High-resolution view. |
Other Hantaviruses
Several members of the hantavirus genus cause different forms of hemorrhagic fever with
renal syndrome (HFRS), an ancient disease first described in Russia in 1913. The four
viruses that are associated with HFRS, each named for the region from where they were
first isolated, have different primary rodent hosts: Apodemus agrarius (the striped
field mouse) for Hantaan virus, Rattus norvegicus (the Norway rat) and Rattus
rattus (the black rat) for Seoul virus, Clethrionomys glareolus (the bank vole)
for Puumala virus, and Apodemus flavicollis (the yellow-necked field mouse) for
Dobrava virus. Hantaan virus from Korea and Dobrava virus from Slovenia are associated
with a severe form of HFRS characterized by renal failure that can precede pulmonary edema
and disseminated intravascular coagulation (DIC), with estimated mortality rates of 5% to
15%. A moderate form of HFRS caused by Seoul virus (which, along with its host, is
distributed worldwide) is responsible for thousands of Eurasian cases annually. Serologic
evidence for infection with Seoul-like hantaviruses has been found in rodents in major
cities of the United States, and this virus was recently implicated in human cases of HFRS
in Baltimore. One report has also associated Seoul virus with chronic renal disease. A
mild form of HFRS, caused by Puumala virus, is responsible for nephropathia epidemica in
Scandinavia, with an estimated mortality rate of 1% to 3%.
Characteristics of Some Known Hantaviruses
|
Hantaan |
Seoul |
Puumala |
Prospect Hill |
Sin Nombre |
Geographic Region |
Asia |
Worldwide |
Northern Europe |
U.S. |
North America |
Reservoir |
Field Mouse |
Domestic Rat |
Bank Vole |
Meadow Vole |
Deer Mouse |
Pathology |
Renal |
Renal |
Renal |
No known human disease |
Pulmonary |
Mortality |
5 - 15% |
1% |
1% |
N/A |
50% |
Another hantavirus, Prospect Hill, has been previously isolated in the United States,
but has not been implicated as a cause of human disease. Prospect Hill virus was
originally isolated from Microtus pennsylvanicus (meadow vole) in Frederick, MD.
Comparison of HFRS and HPS
|
HFRS |
HPS |
Major Target Organ |
kidney |
lung |
First Phase |
febrile |
febrile "prodrome" |
Second Phase |
shock |
shock, pulmonary edema |
Evolution |
oliguria, diureses, convalescence |
diureses, convalescence |
Mortality |
1 - 15% |
50% |
Epidemiology in the Virology Laboratory
During the outbreak in 1993, definitive proof that the agent causing HPS was a novel
hantavirus was obtained using a genetic detection assay. Oligonucleotide primers were
designed on the basis of regions of the M segment (G2 coding region) conserved among
hantaviruses and were used in a nested RT-PCR assay to amplify hantavirus-specific DNA
fragments from RNA extracted from the tissues of patients. The amplified DNA fragments
were then sequenced. Comparative and phylogenetic analyses of derived sequence data
demonstrated that the hantavirus associated with the HPS outbreak (SNV) was a novel virus
most closely related to Prospect Hill virus (PHV). In addition, a direct genetic link was
made between the human HPS cases and the virus harbored by peridomestic P.
maniculatus rodents. Characterization of hantaviral genetic sequences recovered from
human tissues demonstrated that these sequences were identical to those from rodents
captured at the site of the patient's presumed infection. This characterization has
continued to facilitate identification of the site of infection when more than one such
site exists and therefore focus the public health response. These techniques also allow
implication of a specific rodent host in areas of overlapping hosts.
Sin Nombre Virus Sequencing
The entire genomic sequence of SNV has subsequently been determined by using RNA
extracted from autopsy material as well as RNA extracted from cell culture-adapted virus.
The L RNA is 6562 nucleotides (nt) in length; the M RNA is 3696 nt long; and the S RNA is
2059 to 2060 nt long. Interestingly, when the prototype sequence (NMH10) of SNV detected
in tissues from an HPS case was compared with the sequence of the SNV isolate (NMR11;
isolated in Vero E6 cells from P. maniculatus trapped in the residence of the same
case), only 16 nucleotide changes were found, and none of these changes resulted in
alterations in amino acid sequences of viral proteins. It had been assumed that in the
process of adaptation to cell culture, selection of SNV variants which grow optimally in
cell culture would occur, and selected variants would differ genetically from the parental
virus. Though NMH10 and NMR11 are identical in protein sequence, nucleotide substitutions
in nontranslated regions of the genome could be responsible for altered viral phenotypes,
as could changes in protein glycosylation or virus membrane components.
The nested RT-PCR assay developed during the initial HPS outbreak provided a rapid
method for the genetic characterization of novel hantaviruses that did not require a virus
isolate. Numerous new hantaviruses have been detected by RT-PCR in rodent tissues but have
yet to be associated with human disease. These include El Moro Canyon virus associated
with the western harvest mouse (Reithrodontomys megalotis), Tula virus with Microtus
arvalis and M. rossiaemeridionalis, Rio Segundo virus with the Mexican
harvest mouse (R. mexicanus), Isla Vista virus with the California vole (M.
californicus), and Prospect Hill-like viruses in Microtus species.
Phylogenetic Analysis
Phylogenetic analysis of Old World and American hantaviruses indicates that the
relationship among hantaviruses corresponds with the phylogeny of their rodent hosts.
Viruses of rodents belonging to the subfamily Murinae are monophyletic as are hantaviruses
of arvicoline and sigmodontine rodents, suggesting that long-term virus-rodent coevolution
is taking place. Hantavirus evolution is best understood as co-evolution within specific
lineages in the rodent family Muridae. The apparent coupling between hantaviruses and
their rodent hosts suggests that viruses of sigmodontine rodents share a common ancestor,
as do viruses of the subfamily Arvicolinae and Murinae. This coupling also has a
geographic and clinical correlate: viruses found in Old World murine rodents, including
Hantaan virus (HTNV), Seoul virus (SEOV) and Dobrava virus, are associated with HFRS in
Eurasia. By contrast, viruses carried by New World sigmodontine rodents, including
SNV Black Creek Canal virus (BCCV) and Bayou virus (BAYV), are associated with HPS in the
Americas. This distinction can narrow the search for a rodent host for newly discovered
HPS-like diseases and suggest disease implications for the various new viruses being
genetically amplified from rodents.
Sequence Divergence
Detection and characterization of Sin Nombre-like viruses in P. maniculatus and P.
leucopus populations have shown that multiple phylogenetic lineages of SNV exist in
North America, and in some cases similar viruses are detected in both Peromyscus
species. Sequence divergence among SNV genes has been shown to be as high as 23%
nucleotide dissimilarity and 7% amino acid dissimilarity. Comparison of SNV sequences with
those of other hantaviruses provides no obvious explanation as to why SNV and related
viruses cause HPS while other hantaviruses are associated with HFRS. The development of a
reverse genetics system for manipulation of virus genomes and an animal model for studying
pathogenesis will be necessary to define the molecular mechanism(s) of SNV pathogenicity.
Genomic reassortment by RNA viruses with segmented genomes is well documented and has
the potential to produce viruses with altered biological activity, host range, and disease
potential. For example, segment reassortment among influenza virus strains (antigenic
shift) is thought to be responsible for influenza pandemics. Genomic reassortment among
SNV variants is known to occur in nature, but the precise role of genomic reassortment in
the epidemiology of HPS and HFRS is unknown.
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