Variation in Western Equine Encephalomyelitis Viral Strain Growth in Mammalian, Avian, and Mosquito Cells Fails to Explain Temporal Changes in Enzootic and Epidemic Activity in California
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Abstract:
The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20–30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.Keywords:
Enzootic
Togaviridae
Vero cell
Sindbis virus
Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells
ABSTRACT Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells—presumably a prerequisite for alphavirus maintenance in nature.
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Alphaviruses are members of the Togaviridae family of icosahedral, enveloped, single-strand, message-sense RNA viruses. The mosquito-borne alpha-viruses are important causes of encephalomyelitis in the Americas and are on the category B list of agents of biodefense concern. Eastern equine encephalitis (EEE), western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE) viruses are the neurotropic alphaviruses of greatest importance as causes of human encephalomyelitis and were initially recognized for their ability to cause disease in horses. Semliki Forest virus (SFV) and Sindbis virus (SINV) do not usually cause encephalitis in humans, but are studied frequently in mice as model systems for alphavirus encephalomyelitis.
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Sindbis virus
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Venezuelan equine encephalitis (VEE) is caused by a virus in the family Togaviridae genus Alphavirus. It is an enveloped virus with an icosahedral capsid 60 to 70 nm in diameter with a linear, single-stranded positive-sense RNA nonsegmented genome of approximately 11.4 kilobases (4). VEE periodically occurs in South and Central America and occurred in Texas in 1971 (6). VEE viruses exist in two settings: (i) a continuous cycle maintained between Culex mosquito vectors and rodents (enzootic), and (ii) epidemics that involve several mosquito species that feed on mammals (epizootic). Epizootic VEE virus varients occur in irregular epizootic cycles and cause clinical disease and deaths in equines only during those cycles. Sylvatic or enzootic VEE viruses may be found at any time in enzootic cycles involving rodents; equine disease is rarely associated with infection by sylvatic VEE viruses. Aedes taeniorhynchus is thought to be the main mosquito vector (1); however, other mosquitoes are thought to play a role, and during an epidemic in horses in 1969 to 1971 Aedes aegypti, Culex tarsalis, Deinocerites pseudes, and Psorophora confinnis mosquitoes were shown to be infected. This epidemic began in 1969 in northern South America and by the time it ended in 1971, it had resulted in the deaths of hundreds of thousands of horses throughout Central America, Mexico, and Texas (3). Female mosquitoes ingest the virus when bloodfeeding on infected rodents or horses to obtain protein for egg production and after a 7 to 20 day extrinsic incubation period can transmit the virus when feeding on a new host. The mosquitoes remain infected for life. The principal mosquito vector for human infections is thought to be Culex pipiens although more than 30 other species have been implicated (5)
Usually 0.5 to 5 days after being bitten by an infected mosquito, horses begin to show signs. Infections range from asymptomatic, to mild (anorexia, fever), or severe either with fatality or without. Horses with a severe response show a distinctive lack of coordination that leads to a leaning stance and circling due to the swelling of the brain (Fig. 1–3). Other signs include fever, lack of appetite with rapid weight loss, and depression, and may include seizures. The sylvatic virus is endemic in northern South America, Trinidad, Central America, Mexico, and Florida. Epizootic virus appears sporadically in epizootics mostly in Mexico, Central and South America. The photos shown here were taken in Gualaca, Panama. Prognosis is poor for horses infected with epizootic viruses (50 to 90% mortality). Horses often die from trauma induced during seizures. Figure 4 shows a horse that has died, and shows lesions on the eyes and face incurred during seizures. Also note the lack of vegetation around the head, which is caused as the horse's head swings back and forth during the seizures. Similar defoliation is also often noted near the legs of horses that die of VEE as their legs will swing in a paddling motion. Upon necropsy the brain shows signs of encephalitis (swelling of the brain) and hemorrhaging that is actually caused by head trauma during the seizures rather than viral damage (Fig. 5). Horses are often euthanatized before they reach this point, as recovery in cases this severe is rare (3, 6). There is a vaccine to control this disease that should be administered yearly and also contains western and eastern encephalitis viruses along with VEE viruses.
Similar to western and eastern encephalitis, humans can become infected with VEE. In humans, signs of VEE infection include fever, exhaustion, back pain, nausea, vomiting, and headache; children are at greatest risk for developing central nervous system infections. The overall mortality rate in epidemics is 0.5 to 1%. In patients who develop encephalitis, the mortality rate is about 20%, in the absence of adequate medical care this can approach 25 to 30%. Encephalitis is clinically diagnosed in only 1 to 4% of adults and 3 to 5% of children. There is no vaccination for humans (3).
The virus is cultivated typically in
cell culture and quantified using plaque assays; virus is serially diluted and plated on the African green monkey kidney cell line Vero V76 cells and cytopathic effect is quantified. Diagnosis is usually attempted using paired serums (acute and convalescent; 2 weeks apart) assaying for a four-fold increase in serum neutralizing antibody titers. This assay detects antibodies in the serum, if animals have had an immune response. However, this is problematic as many animals die before the second, 2 week, convalescent sample can be obtained (6).
See also:
Venezuelan Equine Encephalitis Virus
References.
1. Brault, A. C., A. M. Powers, and S. C. Weaver. 2002. Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein. J. Virol. 76
:6387–6392.
2. Committee on Foreign Animal Diseases of the United States Animal Health Association. 1998, revision date. The Gray book of foreign animal diseases, 6th ed. United States Animal Health Association, Richmond, Va. [Online.] http://www.vet.uga.edu/VPP/gray_book/FAD/index.htm.
3. Fenner, F., P. A. Bachmann, E. P. J. Gibbs, F. A. Murphy, M. J. Studdert, and D. O. White. 1987. Veterinary virology, p. 460–462. Academic Press, Inc., Orlando, Fla.
4. Griffin, D. E. 2001. Alphaviruses, p. 917–962. In D. M. Knipe and P. M. Howley (ed.), Fields virology. Lippincott Williams and Wilkins, Philadelphia, Pa.
5. Nasci, R. S., and B. R. Miller. Culicine mosquitoes and the agents they transmit, p. 85–97. In B. J. Beaty and W. C. Marquardt (ed.), The biology of disease vectors. University Press of Colorado, Niwot, Colo.
6. Roberts, W. A., and G. A. Carter. 1976. Essentials of veterinary virology, p. 107. Michigan State University Press, East Lansing, Mich.
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Historically, western equine encephalomyelitis virus (WEEV) caused large equine and human epidemics in the Americas from Canada into Argentina. Despite recent enhanced surveillance for West Nile virus, there have been few reports of equine or human cases and little documented enzootic activity of WEEV. During the past three years, WEEV has been active again in California, but without human or equine cases. In the current study, we compared host and vector competence of representative WEEV isolates made during each decade over the past 60 years using white-crowned sparrows, house sparrows, and Culex tarsalis Coquillett as representative hosts. Results indicated limited time-related change in virulence among WEEV strains in birds and little difference in vector competence in Cx. tarsalis. Although temporal and spatial genetic changes have been documented, these seem to present limited phenotypic change in host competence and cannot explain the absence of equine and human cases.
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Field studies of the ecology of eastern equine encephalitis virus (EEEV; family Togaviridae , genus Alphavirus ) in the southeastern United States have demonstrated that Culex erraticus (Dyar and Knab) is the most common mosquito at many enzootic sites and is often infected with the virus. However, the competence of Cx. erraticus for EEEV has not been explored in detail. Culex erraticus females were collected from the field and fed upon EEEV-infected chicks. The infected mosquitoes were provided honey for nutrition and to monitor for time to infectiveness. Of the mosquitoes that survived the 14-d postfeeding period, 89% were infected and 84% had evidence of a disseminated infection, though titers were generally low. EEEV was first detected in honey 6 d postinfection and was detected in samples collected from 94% of the mosquitoes with a disseminated infection overall. These data and others were then employed to estimate the relative vectorial capacity of Cx. erraticus at an EEEV enzootic site in Alabama. The vectorial capacity of Cx. erraticus at this site was 44% of Culiseta melanura (Coquillett), the accepted enzootic vector, suggesting Cx. erraticus may play a role in transmitting EEEV in areas where it is abundant and Cs. melanura rare.
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Western equine encephalitis (WEE) was once prevalent and routinely isolated from mosquitoes in Colorado; however, isolations of Western equine encephalitis virus (WEEV) have not been reported from mosquito pools since the early 1990s. The objective of the present study was to test pools of Culex tarsalis (Coquillett) mosquitoes sampled from Weld County, CO, in 2016 for evidence of WEEV infection. Over 7,000 mosquitoes were tested, but none were positive for WEEV RNA. These data indicate that WEEV either was not circulating enzootically in Northern Colorado, was very rare, and would require much more extensive mosquito sampling to detect, or was heterogeneously distributed spatially and temporally and happened to not be present in the area sampled during 2016. Even though the reported incidence of WEE remains null, screening for WEEV viral RNA in mosquito vectors offers forewarning toward the detection and prevention of future outbreaks.
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Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.
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The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20–30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.
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The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.
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