Journal of General Virology

The effect of mosquito passage on the La Crosse virus genotype

Monica K. Borucki,† Brian J. Kempf, Carol D. Blair and Barry J. Beaty

Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1682, USA

The genetic consequences of passing three different strains of La Crosse (LAC) virus orally and transovarially in Aedes triseriatus mosquitoes were examined. Two of the LAC strains (WT LAC and LAC ORI) had been passaged numerous times in cell culture; the third strain (SM1-78) had been passaged only once in suckling mice. Genetic changes were monitored in three regions of the LAC genome after oral infection and dissemination in the mosquito, and transovarial transmission (TOT) of the virus to progeny. Sequence analyses were used to characterize the genetic changes occurring in regions of G1, G2 and N open reading frames (ORFs) during passage. Only one mutation was detected in the G1 ORF of SM1-78 virus after mosquito passage; however, numerous nucleotide and amino acid substitutions were detected in the G1 ORF of WT LAC and LAC ORI (cell culture-adapted viruses). In contrast to G1, the N and G2 ORF sequences examined were stable. Mutations introduced into viral genomes during replication in parental mosquitoes were expressed in progeny mosquitoes following TOT. Genetic diversity of virus populations from a single mosquito was examined by single-strand conformation polymorphisms analysis of the variable region of glycoprotein G1. LAC virus RNA genotype diversity was greatest in virus that infected and replicated in the midgut, and declined as virus disseminated from the midgut and infected ovaries and salivary glands.

Arthropod-borne viruses are maintained in nature principally through biological transmission between susceptible vertebrate hosts and haematophagous arthropod vectors (Beaty et al., 1997). Therefore, these viruses must be able to infect and replicate in cells of both vertebrates and invertebrates. The ability of arboviruses to bridge the gap between vertebrate and invertebrate hosts is likely to be a function of their RNA genomes. RNA viruses have extremely high mutation rates (because of the poor fidelity of RNA-dependent RNA polymerases and lack of proofreading enzymes) and are proposed to exist as quasispecies populations (Holland et al., 1982). The quasispecies represents a balance between the expansive influence of mutation and the conservative forces of selection and is envisaged to contain a cloud of genotypes that provide the genetic variability that enables RNA viruses rapidly to exploit new niches (Holland et al., 1982; Eigen & Biebricher, 1988). The quasispecies is an excellent model to explain the remarkable ability of arboviruses to pass rapidly and continuously between vectors and vertebrate hosts. Certainly, passage of arboviruses in laboratory systems rapidly selects for specific virus subpopulations that are more fit in the respective system (de la Torre et al., 1988; Barrett et al., 1990; Novella et al., 1995).

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 NS_M, 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 structure–function 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.

Viruses. Three strains of LAC virus were used, two of which had undergone moderate to extensive passage exclusively in vertebrate cells, and one with minimal vertebrate passage. Prototype LAC virus (WT LAC) originally came from the Yale Arbovirus Research Unit, New Haven, CT, USA. The virus had been passaged three times in suckling mice and six times in BHK-21 cells. LAC ORI was derived from WT LAC virus. It was plaque-purified and used to generate reassortant viruses for gene structure–biological function studies in vertebrates (Gonzalez-Scarano et al., 1992). The virus was kindly provided by Dr Neal Nathanson (U. Pennsylvania) in 1985. It had been passaged many times in BHK-21 cells after plaque purification. LAC ORI is phenotypically distinguishable from WT LAC virus due to its low infection rate in Ae. triseriatus mosquitoes (unpublished data; see Table 2). The SM1-78 strain of LAC virus was isolated directly from the brain of a patient who died of LAC encephalitis in 1978 via intracranial inoculation into suckling mice of homogenized tissue from the brain cortex (Chandler et al., 1998). Virus isolated in the first suckling mouse passage (SM1-78) was used in this study; the virus was not passaged in cell culture.

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 50–100 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 24–48 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 phenol–chloroform–isoamyl 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 RT–PCR 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 35–40 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.

Table 1. RT–PCR primers used to amplify regions of the LAC virus M and S segments

Three pairs of primers were designed to amplify portions of the N, G1 and G2 ORFs from LAC virus RNA for direct sequence analysis. The fourth primer pair was designed to amplify a smaller region at the 3´ end of the G1 amplicon for SSCP and sequence analysis.

RT–PCR products were purified using a Wizard PCR Prep kit (Promega) and sequenced directly by Macromolecular Resources (Colorado State University) using an ABI Prism automated sequencing apparatus. The primers used for RT–PCR amplification were also used as sequencing primers. Nucleotide sequences were aligned using SEQMAN II (DNASTAR) and CLUSTAL W version 1.6 (Thompson et al., 1994).

LAC virus RNA SSCP analysis. A 126 nt region of the G1 ORF of WT LAC virus was amplified by RT–PCR 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.

Oral infection rates

The oral and filial infection rates (FIR) for the three virus strains are presented in Table 2. Oral infection rates ranged from approximately 50 % for WT LAC and SM1-78 to 28 % for LAC ORI. Infected mosquitoes were then provided with an uninfected blood meal to stimulate a second gonadotropic cycle and FIRs were determined for these progeny. The mean FIRs ranged from 68 % for SM1-78 to only 1 % for LAC ORI (Table 2).

Table 2. Aedes triseriatus oral and filial infection rates (FIRs) with three strains of LAC virus

* Log₁₀ TCID₅₀ determined by end-point assay in Vero cells.

† Per cent adult female mosquitoes orally infected as determined by immunofluorescent assay of leg tissue (no. infected/no. fed).

‡ Per cent of total progeny mosquitoes transovarially infected as determined by immunofluorescent assay of head tissue (no. infected/total progeny)

§ Range of FIRs among the progeny of three, two and four female mosquitoes assayed after infection with WT LAC, LAC ORI and SM1-78, respectively.

Sequence comparison of LAC RNA from three virus strains after mosquito passage

For each of the three LAC virus strains, the genomic RNA sequences of virus used to prepare the blood meal (designated parental); viral RNA amplified from the midgut, ovaries and salivary glands of two to four orally infected mosquitoes; and viral RNA amplified from one to six transovarially infected progeny mosquitoes (designated virus strain adult number-progeny number, e.g. WT 9-5) were determined. A 266 nt region of the S segment nucleocapsid ORF, a 410 nt region of the M segment G1 ORF, and a 599 nt region of the M segment G2 ORF of each sample were sequenced. All sequences were compared to those of the parental WT LAC RNA. Overall, the N and G2 ORFs were found to be less variable than the G1 ORF.

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).

Table 3. Amino acid and nucleotide substitutions in the G1 ORF of WT LAC, LAC ORI and SM1-78

Six positions in a 410 nt region of the G1 ORF of WT LAC (WT) and LAC ORI (ORI) consistently acquired either synonymous (shown as nucleotide bases) or nonsynonymous (shown as amino acids) mutations as compared to parental (Par) sequence when virus infected the midgut (MG) and disseminated to the ovaries (OV) and salivary glands (SG). Six WT LAC-infected progeny (WT 9-5, 9-6, 9-9; WT 14-1, 14-2, 14-3) and one LAC ORI-infected progeny (ORI 82-1) were analysed also for mutations in transovarially transmitted virus. SM1-78 RNA acquired only a single mutation in one midgut sample after replication in mosquitoes (not shown).

Of the total of 12 mutations in non-consensus ovary sequences examined, only four resulted in amino acid changes. In ovary 28, a T to C transition at position 1919 resulted in a Phe₆₂₀ to Leu change and an A to G transition at nucleotide 1958 resulted in a Thr₆₃₃ to Ala change. In ovary 43, an A to G transition at position 1935 resulted in a Lys₆₂₅ to Arg change and an A to T transversion at nucleotide 1968 resulted in an Asp ₆₃₆ to Gly change.

These studies tested the hypothesis that passage in vectors results in purifying selection for certain LAC virus genotypes in the quasispecies that are more fit for replication in the respective organ systems. Viruses with dramatically different laboratory passage histories (and thus different genotypes) were used to orally infect Ae. triseriatus and the resulting progeny viruses were compared to parental virus for genetic changes during vector passage. Specifically, genetic changes in the ORFs for three structural proteins of LAC virus were monitored during replication in mosquitoes and after TOT of the virus to mosquito progeny. Genetic and phenotypic changes are epidemiologically significant only if mutant viruses are transmitted to vertebrate hosts orally and/or to progeny transovarially. Therefore, virus populations were sampled from the midgut, ovaries, salivary glands and progeny of infected mosquitoes.

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.

This research was supported by grant AI 32543 from the National Institute of Allergy and Infectious Diseases. We thank Cindy Meredith for expert assistance in the insectary.

† Present address: USDA, Agricultural Research Service, Animal Disease Research Unit, 3003 ADBF, Washington State University, Pullman, WA 99164-6630, USA.

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