Genome sequence analysis of La Crosse virus and in vitro and in vivo phenotypes

Abstract

Background: La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas.

Results: In the present study, sequence analysis of the complete LACV genomes of low-passage LACV/human/1960, LACV/mosquito/1978, and LACV/human/1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6-100%, 98.9-100%, 97.8-99.6%, and 99.2-99.7% amino acid identity for N, NsS, M polyprotein, and L proteins respectively. We identified 5 amino acid differences in the RNA polymerase and 4 nucleotide differences in the non-coding region of the L segment specific to the human virus isolates, which may result in altered disease outcomes.

Conclusion: All three wild type viruses had similar in vitro growth kinetics and phenotypes in mosquito C6/36 and Vero cells, and similar levels of neurovirulence and neuroinvasiveness in Swiss Webster mice. The biologically cloned derivative of LACV/human/1960 was significantly less neuroinvasive than its uncloned parent and differed in sequence at one amino acid position in the GN glycoprotein, identifying this residue as an attenuating mutation.

Figure 1

Alignment of 3' non-coding region of S, M, and L genome segments (cDNA presented). S segment 3' NCR shows highly conserved sequence with no nucleotide changes from the consensus. For each segment the consensus sequence consists of three or more sequences sharing the same nucleotide at a given position and areas with no clear consensus are indicated with an "N". A single nucleotide change was reported in the LACV/mosquito/1977 published sequence at position 9 of the M segment. For the 3' NCR of the L segment, 2 changes from the consensus were observed in LACV/mosquito/1978 with position 31 having no clear consensus. Underlined sequence indicates region conserved among all three segments. Putative host-specific nucleotide sequences are indicated with an arrow (↓).

Figure 2

Alignment of 5' non-coding region of S, M, and L genome segments (cDNA presented). Among the two human isolates only one nucleotide difference was observed in the NCR of the L segment at position 6888. For each segment, the consensus sequence consists of three or more sequences sharing the same nucleotide at a given position and areas with no clear consensus are indicated with and "N". Underlined sequence indicates region conserved among all three segments. Putative host-specific nucleotide sequences are indicated with an arrow (↓).

Figure 3

Growth kinetics and CPE of LACV strains. A. Growth kinetics of LACV/human/1960, LACV/human/1978, and LACV/mosquito/1978 in Vero cells or C6/36 cells infected at an MOI of 0.01. B. Photographs of mock or LACV/human/1960 infected Vero cell monolayers from panel "A". Cell rounding and detachment from the flask can be seen on days 2–4 post-infection in infected monolayers.