Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1682, USA1
Author for correspondence: Barry Beaty. Fax +1 970 491 8323. e-mail bbeaty{at}colostate.edu
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Abstract |
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Introduction |
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La Crosse (LAC) virus, a member of the family Bunyaviridae, genus Bunyavirus, is the causative agent of La Crosse encephalitis, the most commonly reported form of paediatric arboviral encephalitis in the United States (Rust et al., 1999 ; McJunkin et al., 2001
). LAC virus is transmitted horizontally and vertically by Aedes triseriatus mosquitoes, with infected female mosquitoes transmitting the virus transovarially to their progeny. LAC virus overwinters in the infected eggs, and infected progeny emerge in the spring to resume the transmission cycle (Grimstad, 1988
; Beaty & Calisher, 1991
; Beaty et al., 2000
).
LAC virus replication in its vector and vertebrate hosts has been the subject of extensive investigation, and thus presents a unique opportunity to determine if the quasispecies model enables effective LAC virus passage between the two disparate systems. The genomic coding strategies of LAC virus are known: the large (L) RNA segment (6980 nt) encodes the polymerase, the middle-sized (M) RNA segment (4526 nt) encodes the envelope glycoproteins (G1 and G2) and nonstructural protein NSM, and the small (S) RNA segment (984 nt) encodes the nucleocapsid (N) protein and nonstructural protein NSs in overlapping reading frames (Schmaljohn, 1996 ; Gonzalez-Scarano & Nathanson, 1996
; Elliott et al., 1991
). Gene structurefunction studies revealed that many important biological functions co-segregate with the M RNA segment. In vertebrates, G1 is a major determinant of tissue tropisms, membrane fusion, neuroinvasiveness and elicitation of neutralizing antibody (Gonzalez-Scarano, 1985
; Gonzalez-Scarano et al., 1992
). In the vector, midgut infection and transmission also co-segregate with the M RNA segment (Beaty et al., 1981
, 1982
). Importantly, it seems that G1 must be proteolytically cleaved in the lumen of the midgut for efficient vector infection to occur. This cleavage exposes either a receptor ligand or a hydrophobic region on G2 that conditions vector infection (Ludwig et al., 1989
). Proteolytic processing of G1 is apparently not necessary for virus disseminating from the midgut to infect secondary target organs (Ludwig et al., 1991
). G1 and G2 would thus seem to be good candidates to monitor for selection during passage. The S RNA segment and its gene products do not co-segregate with vector infection, and thus would presumably not be subject to selection during mosquito passage. Little is known about the potential variability in the sequence or differential function of the L protein in invertebrate compared to vertebrate hosts.
In these studies, three different strains of LAC virus with dramatically different laboratory passage histories were characterized genetically after Ae. triseriatus midgut infection, dissemination to salivary glands and ovaries, and transovarial transmission (TOT). At each stage of vector infection, portions of the G1, G2 and N open reading frames (ORFs) were sequenced to determine if selection occurred during virus passage in the vector. L segment sequences were not compared due to lack of knowledge about potential variability between hosts. Virus populations at each stage were also characterized for genetic diversity using single-strand conformation polymorphism (SSCP) analysis. The central hypothesis was that quasispecies populations of LAC viruses would respond genetically to the selective pressures posed by infection and replication in different mosquito tissues and organs. Further it was hypothesized that the most dramatic changes would occur in ORFs encoding the G1 and G2 proteins, which condition vector tropisms.
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Methods |
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Mosquito feeding and assay.
The Ae. triseriatus mosquitoes used in this study originated from field material collected near La Crosse, Wisconsin, in 1981, and have been colonized continuously at Colorado State University (Wasieloski et al., 1994 ). Three groups of 50100 female Ae. triseriatus mosquitoes were orally infected with an artificial blood meal containing either WT LAC, LAC ORI or SM1-78. Medium from BHK-21 cells infected with LAC virus (m.o.i. of 0·01 or 0·001) was removed 2448 h after infection, clarified and used in blood meal preparation. The blood meal consisted of equal volumes of infected cell culture medium, washed fresh sheep red blood cells and foetal bovine serum containing 10% sucrose. The blood meal was prepared immediately before feeding and warmed to 37 °C. Mosquitoes were fed by placing drops of the blood meal onto the netting of their cartons for 1 h. Virus titre of the blood meal at the end of feeding was determined by end-point assay in Vero cells.
Mosquitoes that fed to repletion were maintained for 2 weeks extrinsic incubation, then assayed for LAC virus infection by fluorescent antibody assay of leg tissue using a FITC-conjugated anti-LAC polyclonal mouse antibody (Beaty et al., 1981 ). Slides were examined at 200x using an Olympus BH2 epifluorescence microscope.
RNA isolation and preparation.
The midguts, ovaries and salivary glands were dissected from live, anaesthetized, infected mosquitoes. Each organ was placed immediately in 200 µl of RNAgents (Promega) denaturation solution (guanidine thiocyanate and 2-mercaptoethanol). The organs were stored at -70 °C until processed. RNA was extracted with acid phenolchloroformisoamyl alcohol and finally dissolved in 17 µl RNase-free water.
LAC virus RNA sequence analysis.
Portions of the LAC M and S RNA segments were amplified using the Access RTPCR kit (Promega). Four µl of total cellular RNA solution was added to 46 µl of reaction mixture containing 1x AMV/Tfl reaction buffer, 0·2 mM dNTP mix, 1 µM forward and reverse primers, 1 mM MgSO4, 5 units AMV reverse transcriptase and 5 units Tfl DNA polymerase. Reactions were incubated at 48 °C for 45 min for reverse transcription. After 2 min at 94 °C for RT inactivation and template denaturation, the samples were thermocycled as follows: 94 °C for 30 s, 55 °C for 1 min, 68 °C for 2 min for 3540 cycles, final extension at 68 °C for 7 min. Primers were designed using OLIGO 4.0 (National Biosciences) for amplification of a conserved region of the nucleocapsid ORF, a variable region of the G1 ORF and a variable region of the G2 ORF. The sequences and positions of the primers are listed in Table 1.
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LAC virus RNA SSCP analysis.
A 126 nt region of the G1 ORF of WT LAC virus was amplified by RTPCR from midgut, ovaries and salivary glands of a single infected mosquito as described above using primers LMF2I and LMR2 (Table 1). PCR products were cloned into pCR2.1 using a TA cloning kit (Invitrogen). Individual clones were then amplified by PCR and analysed for SSCP (Farfan et al., 1997
). Nucleotide sequences of selected clones were determined as described above.
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Results |
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WT LAC virus RNA sequences
No nucleotide changes from the parental WT LAC nucleocapsid ORF sequence were detected in any organs of the two orally infected and one transovarially infected mosquitoes examined.
G2 ORF RNA from one or more organs of all three WT LAC orally infected mosquitoes had a synonymous mutation at nucleotide 743. This substitution was not present in the virus transmitted to the single progeny mosquito tested. A synonymous change also was detected at position 559 in the RNA from all three organs of one of the orally infected mosquitoes (WT 14).
WT LAC G1 ORF RNA was analysed from the organs of three orally infected mosquitoes and six transovarially infected progeny. Mutations had occurred in RNA from one or more organs of two of the WT LAC-infected mosquitoes at positions 1912 and 1922, resulting in amino acid changes of Arg618 to Trp and Ala621 to Val. The third WT LAC-infected mosquito had the same mutation at position 1912 and an A to G transition at position 1928, resulting in an amino acid change of Glu623 to Gly. One or two of the same mutations were detected in viral RNA extracted from all of the WT LAC-infected progeny tested, with two progeny showing additional synonymous mutations at nucleotides 1749 and 1974 (Table 3).
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The sequence of the G2 ORF region analysed also was identical for WT LAC and LAC ORI parental virus RNA. No nucleotide changes were detected in the G2 ORF of LAC ORI RNA from two of the three orally infected mosquitoes. A mutation occurred at nucleotide 609 in M RNA from the ovaries and salivary glands of the third mosquito. This substitution resulted in an amino acid change from Phe183 to Ser.
In contrast to the other two genome regions, in the G1 ORF region analysed, the LAC ORI parental virus RNA had mutations at nucleotide positions 1749, 1912, 1971 and 1974 as compared to WT LAC (Table 3). The mutation at nucleotide 1912 resulted in an amino acid change from Arg618 to Trp. LAC ORI G1 ORF RNA was analysed from the organs of four orally infected mosquitoes and one transovarially infected progeny. Upon replication and dissemination in the mosquito vector, LAC ORI acquired G1 mutations in RNA from most or all organs at positions 1749, 1912, 1922, 1971 and 1974 (Table 3
). The mutation at position 1912 resulted in a Trp618 to Arg substitution, and that at 1922 in an Ala621 to Val substitution. Interestingly, the mutations at positions 1749, 1912, 1971 and 1974 resulted in restoration of the sequence of the parental WT LAC virus. Although 77 progeny mosquitoes from LAC ORI-infected females were tested by fluorescent antibody and RTPCR, only one was found to be infected. The LAC ORI virus in this mosquito retained the same mutations that were detected in the parental female (Table 3
).
SM1-78 virus RNA sequences
The parental SM1-78 N ORF sequence had a C to T transition at position 179 by comparison to WT LAC RNA, resulting in a predicted amino change of Ser33 to Leu. No nucleotide changes from the parental RNA sequence were detected in the two orally infected and one transovarially infected mosquitoes examined.
By comparison to WT LAC parental RNA, SM1-78 G2 ORF RNA had a single nucleotide transition of C to T at position 786, resulting in an amino acid change of Thr242 to Ile. No nucleotide changes from the parental RNA sequence were detected in this region of the G2 ORF from any of the SM1-78 orally infected mosquitoes or in the RNA of the single SM1-78-infected progeny mosquito tested.
In the G1 ORF region analysed, SM1-78 parental RNA differed from WT LAC at nucleotide positions 1749, 1912 and 1974, resulting in the amino acid change Arg618 to Trp (Table 3). LAC ORI and SM1-78 parental RNA differed from each other only at nucleotide 1971 and had identical amino acid sequences in this region. In the organs of the three SM1-78-infected mosquitoes analysed, only a single mutation in one midgut was detected in the LAC G1 ORF. The substitution occurred at nucleotide 1915, resulting in an amino acid change from Asp619 to Asn. None of the six SM1-78-infected progeny tested contained viruses with mutations in this region of the G1 ORF (data not shown).
In one organ of two mosquitoes tested (WT 5 and ORI 4), there appeared to be equal amounts of viral RNA with two different nucleotides present at the same position (Table 3). This was detected by the presence of two overlapping electropherogram peaks, which were present in both strands of DNA, at the same position. SSCP analysis confirmed the presence of more than one genotype in a single organ.
SSCP analysis of quasispecies
To characterize genetic diversity in a population of LAC viruses during replication and dissemination in a single vector, a sequence of 126 nt within the G1 ORF corresponding to the coding sequence for an antigenic determinant in the G1 glycoprotein (Grady et al., 1983 , 1987
) was amplified from the organs of a WT LAC orally infected mosquito by RTPCR and cloned. Thirty clones each from the midgut, ovaries and salivary glands were then amplified by PCR and analysed for SSCP (Fig. 1
). To compare diversity in the more homogeneous environment of cultured mosquito cells, another 30 clones were prepared from WT LAC persistently infected C6/36 cells and analysed. Each group of DNA samples with an identical migration pattern was designated a genotype. The 30 midgut samples gave rise to 13 different genotypes, the ovary samples gave rise to 11 genotypes, salivary gland samples to nine genotypes, and the C6/36 cell culture samples to four genotypes. The predominant genotype for each organ was designated the consensus genotype. When samples with the consensus genotypes from each organ were analysed by SSCP together, they were shown to have the same migration pattern (Fig. 1
).
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Nucleotide sequences were determined for eight ovary samples, four of which had consensus and four had different genotypes, along with a consensus midgut sample, a consensus salivary gland sample and a consensus C6/36 sample. Sequences were aligned using the CLUSTAL W program and analysed for identity. All the consensus sequences had the same 126 bp sequence, which was identical to that determined for the G1 ORF from WT LAC parental RNA used for oral infection of mosquitoes (Table 3). The four ovary samples that had genotypes different from the consensus and from each other had from one to four nucleotide differences when compared to the consensus sequence and from four to eight nucleotide differences when compared to each other (Table 4
).
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Discussion |
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Surprisingly, the analysed regions of the glycoprotein G2 ORF as well as the nucleocapsid ORF were relatively stable; however, changes regularly occurred at six positions within a 225 nt sequence of the 410 nt amplicon from the G1 ORF of WT LAC and LAC ORI (Table 3). Seventy-five percent of these substitutions occurred initially in the midgut (18/24 substitutions) and 21% (5/24) occurred initially in the ovaries and salivary glands. One synonymous substitution occurred only in two transovarially infected progeny. All of the substitutions detected in virus genomes from the midgut also were present in virus RNA from the salivary glands and ovaries of the same mosquito. Therefore, it appears that a majority of the genetic changes resulted from selection in the midgut, and these mutated viruses disseminated to the ovaries and the salivary glands. Of the mutations encoding amino acid substitutions that were detected in adult female mosquitoes, 77% were present in the genomes of viruses transmitted to progeny.
All of the G1 ORF mutations resulting in amino acid substitutions occurred in a 17 nt region of the 410 nt sequence analysed. The most frequent amino acid substitution occurred at position 618 of the G1 glycoprotein and involved either Arg replacing Trp (WT LAC) or Trp replacing Arg (LAC ORI). SM1-78 RNA had a Trp codon at this position that did not change upon mosquito passage. These data suggest that the presence of Arg at position 618 may be a result of passage in cell culture (Table 3). It is possible that mutations consistently occur at this position in response to changes in a region of the G1 ORF outside the sequence analysed in order to preserve structural properties of the protein, since amino acids 607 to 638 are proposed to occur on the surface of the G1 protein and form an antigenic determinant (Grady et al., 1983
, 1987
). Other mutations resulting in amino acid substitutions occurred less frequently: Asp619 to Asn in the midgut of a single SM1-78-infected mosquito; Ala621 to Val in 29% (6/21) of midguts, ovaries and salivary glands of mosquitoes infected with either WT LAC or LAC ORI; and Glu623 to Gly in a single WT LAC-infected mosquito, also transmitted to progeny.
It has been proposed that in the mature virion, the G1 glycoprotein masks G2, making it unavailable for binding to mosquito midgut cells (Ludwig et al., 1989 ), and that in the midgut G1 is cleaved by host proteases. The exposed G2 glycoprotein then mediates binding of the virus to midgut cells. However, following release of progeny virus into the haemocoel, G1 is no longer exposed to proteolytic conditions present in the midgut and may mediate virus attachment to cells in the ovaries and salivary glands (Ludwig et al., 1991
). Therefore, at least two modes of selection may act on the G1 glycoprotein after virus infection of a mosquito. Viruses with G1 glycoproteins that are most readily cleaved by midgut proteases may be most fit for infection of midgut cells upon ingestion in a blood meal. When the virus progeny disseminate, selection for viruses with G1 glycoproteins that bind efficiently to the receptors on secondary target organs may occur. The cellular receptor(s) for G1 has not been identified; it is possible that G1 binds to a receptor present on both vertebrate and invertebrate cells. In that case, viruses that have adapted to passage in cell culture may not be at a disadvantage when infecting the secondary organs of the mosquito vector. It is worth noting the correlation between the lower oral infection rate for mosquitoes by LAC ORI (Table 2
) and the observation that 66% of the G1 ORF mutations detected were in LAC ORI RNA vs 30·5% of mutations in WT LAC and 6·6% in SM1-78.
Mutations resulting in a conservative change from Ala621 to Val in the G1 glycoprotein of WT LAC and LAC ORI and synonymous substitutions at nucleotide positions 1749, 1971 and 1974 in the M RNA of WT LAC and LAC ORI (Table 3) may reflect differences in codon usage between cultured vertebrate cells and mosquito cells.
The SSCP analysis of genetic diversity within the virus population in a single mosquito suggested that a greater variety of quasispecies were fit to replicate in mosquito organs than in cell culture. In persistently infected C6/36 cells only four genotypes were detected among 30 samples. The greatest variety of genotypes was detected in the mosquito midgut, and as the infection disseminated from the midgut to other organs, the number of genotypes was reduced. The consensus genotype was identical in midguts, salivary glands and ovaries as revealed by both SSCP and sequence analyses.
The results of this study demonstrate that strains of LAC virus that have been serially passaged in cell culture may undergo balancing selection when reintroduced into the mosquito host due to the presence of different selective constraints present in the two disparate amplification systems. Repeated passage of WT LAC and LAC ORI in cell culture may have selected for viruses that were less fit for replication in mosquitoes. When reintroduced into mosquitoes, these viruses underwent genetic changes necessary to readapt to efficient replication in the mosquito host. SM1-78 virus showed the least amount of change during oral and transovarial passage in mosquitoes. This may be attributable to the fact that SM1-78 virus had not been repeatedly passaged in vertebrate cells. Most of the genetic changes were detected in the G1 ORF, whereas the sequences of the G2 and N ORFs were relatively stable. If genetic changes are indeed a function of selection based on tissue tropisms, it is surprising that few changes occurred in the G2 sequence analysed. However, since it is postulated that glycoprotein G2 is involved only in mosquito midgut infection and not in binding to vertebrate cells, cell culture passage would not be expected to select for different genotypes.
It is important to note that virus mutations acquired during replication in parental mosquitoes were detected in their progeny. Thus, TOT can serve to amplify and to maintain new virus genotypes resulting from vector passage in nature.
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Acknowledgments |
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Footnotes |
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b Present address: USDA, Agricultural Research Service, Animal Disease Research Unit, 3003 ADBF, Washington State University, Pullman, WA 99164-6630, USA.
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References |
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Received 4 June 2001;
accepted 30 August 2001.