Hilda Guzman

Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage

Background Zika virus (ZIKV) is a mosquito-borne flavivirus distributed throughout much of Africa and Asia. Infection with the virus may cause acute febrile illness that clinically resembles dengue fever. A recent study indicated the existence of three geographically distinct viral lineages; however this analysis utilized only a single viral gene. Although ZIKV has been known to circulate in both Africa and Asia since at least the 1950s, little is known about the genetic relationships between geographically distinct virus strains. Moreover, the geographic origin of the strains responsible for the epidemic that occurred on Yap Island, Federated States of Micronesia in 2007, and a 2010 pediatric case in Cambodia, has not been determined. Methodology/Principal Findings To elucidate the genetic relationships of geographically distinct ZIKV strains and the origin of the strains responsible for the 2007 outbreak on Yap Island and a 2010 Cambodian pediatric case of ZIKV infection, the nucleotide sequences of the open reading frame of five isolates from Cambodia, Malaysia, Nigeria, Uganda, and Senegal collected between 1947 and 2010 were determined. Phylogenetic analyses of these and previously published ZIKV sequences revealed the existence of two main virus lineages (African and Asian) and that the strain responsible for the Yap epidemic and the Cambodian case most likely originated in Southeast Asia. Examination of the nucleotide and amino acid sequence alignments revealed the loss of a potential glycosylation site in some of the virus strains, which may correlate with the passage history of the virus. Conclusions/Significance The basal position of the ZIKV strain isolated in Malaysia in 1966 suggests that the recent outbreak in Micronesia was initiated by a strain from Southeast Asia. Because ZIKV infection in humans produces an illness clinically similar to dengue fever and many other tropical infectious diseases, it is likely greatly misdiagnosed and underreported.

Immunization with heterologous flaviviruses protective against fatal west Nile encephalitis

Prior immunization of hamsters with three heterologous flaviviruses (Japanese encephalitis virus [JEV] SA14-2-8 vaccine, wild-type St. Louis encephalitis virus [SLEV], and Yellow fever virus [YFV] 17D vaccine) reduces the severity of subsequent West Nile virus (WNV) infection. Groups of adult hamsters were immunized with each of the heterologous flaviviruses; approximately 30 days later, the animals were injected intraperitoneally with a virulent New York strain of WNV. Subsequent levels of viremia, antibody response, and deaths were compared with those in nonimmune (control) hamsters. Immunity to JEV and SLEV was protective against clinical encephalitis and death after challenge with WNV. The antibody response in the sequentially infected hamsters also illustrates the difficulty in making a serologic diagnosis of WNV infection in animals (or humans) with preexisting Flavivirus immunity.

Efficacy of Killed Virus Vaccine, Live Attenuated Chimeric Virus Vaccine, and Passive Immunization for Prevention of West Nile virus Encephalitis in Hamster Model

Results of experiments evaluating the efficacy of three immunization strategies for the prevention of West Nile virus (WNV) encephalitis are reported. Immunization strategies evaluated included a killed virus veterinary vaccine, a live attenuated chimeric virus vaccine candidate, and passive immunization with WNV-immune serum; all were tested by using a hamster model of the disease. Each product protected the animals from clinical illness and death when challenged with a hamster-virulent wild-type WNV strain 1 month after initial immunization. The live attenuated chimeric virus vaccine candidate induced the highest humoral antibody responses, as measured by hemagglutination inhibition, complement fixation, and plaque reduction neutralization tests. Although the duration of protective immunity was not determined in this study, our preliminary results and the cumulative experience of other virus vaccines suggest that the live attenuated chimeric virus provides the longest lasting immunity.

Persistent West Nile Virus Infection in the Golden Hamster: Studies on Its Mechanism and Possible Implications for Other Flavivirus Infections

Golden hamsters (Mesocricetus auratus) experimentally infected with West Nile virus (WNV) developed chronic renal infection and persistent shedding of virus in urine for up to 8 months, despite initial rapid clearance of virus from blood and the timely appearance of high levels of specific neutralizing antibodies. Infectious WNV could be recovered by direct culture of their urine and by cocultivation of kidney tissue for up to 247 days after initial infection. Only moderate histopathologic changes were observed in the kidneys or brain of the chronically infected hamsters, although WNV antigen was readily detected by immunohistochemistry within epithelium, interstitial cells, and macrophages in the distal renal tubules. Comparison of WNV isolates from serial urine samples from individual hamsters over several months indicated that the virus underwent both genetic and phenotypic changes during persistent infection. These findings are similar to previous reports of persistent infection with tickborne encephalitis and Modoc viruses.

Experimental Yellow Fever Virus Infection in the Golden Hamster (Mesocricetus auratus). II. Pathology

Subadult and adult hamsters were inoculated intraperitoneally with 10(6) TCID(50) of yellow fever (YF) virus (Jimenez strain). Four animals from each group were subjected daily to histologic examination for 9 days. The liver showed spotty necrosis on day 3 after infection, which was followed by steatosis and focally confluent necrosis. In surviving hamsters, hepatocyte regeneration began on day 8, which was accompanied by decreasing steatosis. The spleen initially exhibited lymphoid hyperplasia, which was followed by lymphoid depletion and increased phagocytosis by splenic macrophages. Focal pancreatic acinar necrosis and spotty adrenal cortical necrosis were seen transiently between days 5 and 7. Viral antigen was detected immunohistochemically in the liver and the spleen. TUNEL analysis showed a dynamic change of hepatocyte necrapoptosis, with activity corresponding to the severity of disease. The histopathologic changes were more severe in younger (subadult) animals. The YF-hamster model appears to be an accurate and inexpensive experimental system for studying the pathophysiology and treatment of YF.

Experimental Yellow Fever Virus Infection in the Golden Hamster (Mesocricetus auratus). I. Virologic, Biochemical, and Immunologic Studies

This report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (YF) virus. An accompanying paper describes the pathologic findings. Following intraperitoneal inoculation of a virulent strain of YF virus, hamsters developed a high-titered viremia (up to 109/mL) lasting 5--6 days and abnormal liver function tests. YF hemagglutination-inhibiting antibodies appeared 4 or 5 days after infection, often while viremia was still present. The mortality rate in YF-infected hamsters was variable, depending on the virus strain and the age of the animals. Clinical and pathologic changes in the infected hamsters were very similar to those described in experimentally infected macaques and in fatal human cases of YF, which indicates that the golden hamster may be an excellent alternative animal model, in place of nonhuman primates, for research on the pathogenesis and treatment of YF and other viscerotropic flavivirus diseases.

Limited evolution of West Nile virus has occurred during its southwesterly spread in the United States

Analysis of partial nucleotide sequences of nine West Nile virus strains isolated in southeast Texas during June-August 2002 revealed a maximum of 0.35% nucleotide variation from a New York 1999 strain. Two sequence subtypes were identified that differed from each other by approximately 0.5%, suggesting multiple introductions of virus to this area. Analysis of sequences from cloned PCR products for one strain revealed up to 0.6% divergence from the consensus sequence at the subpopulation level. The presence of unique patterns of small numbers of mutations in North American West Nile strains studied to date may suggest the absence of a strong selective pressure to drive the emergence of dominant variants.

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.

Negevirus: a Proposed New Taxon of Insect-Specific Viruses with Wide Geographic Distribution

ABSTRACT Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ∼45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 1010 PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.