C-C motif chemokine receptor 2 and 7 synergistically control inflammatory monocyte recruitment but the infecting virus dictates monocyte function in the brain.

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function.

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis.

One of the key events in viral encephalitis is the ability of virus to enter the central nervous system (CNS). Several encephalitic viruses, including La Crosse Virus (LACV), primarily induce encephalitis in children, but not adults. This phenomenon is also observed in LACV mouse models, where the virus gains access to the CNS of weanling animals through vascular leakage of brain microvessels, likely through brain capillary endothelial cells (BCECs). To examine age and region-specific regulatory factors of vascular leakage, we used genome-wide transcriptomics and targeted siRNA screening to identify genes whose suppression affected viral pathogenesis in BCECs. Further analysis of two of these gene products, Connexin43 (Cx43/Gja1) and EphrinA2 (Efna2), showed a substantial effect on LACV pathogenesis. Induction of Cx43 by 4-phenylbutyric acid (4-PBA) inhibited neurological disease in weanling mice, while Efna2 deficiency increased disease in adult mice. Thus, we show that Efna2 and Cx43 expressed by BCECs are key mediators of LACV-induced neuroinvasion and neurological disease.

Passenger Mutations Confound Phenotypes of SARM1-Deficient Mice.

The Toll/IL-1R-domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Roles for SARM1 in TLR signaling, viral infection, inflammasome activation, and chemokine and Xaf1 expression have also been described. Much of the evidence for SARM1 function relies on SARM1-deficient mice generated in 129 ESCs and backcrossed to B6. The Sarm1 gene lies in a gene-rich region encompassing Xaf1 and chemokine loci, which remain 129 in sequence. We therefore generated additional knockout strains on the B6 background, confirming the role of SARM1 in axonal degeneration and WNV infection, but not in VSV or LACV infection, or in chemokine or Xaf1 expression. Sequence variation in proapoptotic Xaf1 between B6 and 129 results in coding changes and distinct splice variants, which may account for phenotypes previously attributed to SARM1. Reevaluation of phenotypes in these strains will be critical for understanding the function of SARM1.

[Molecular genetic analysis of Tahyna virus strains].

The partial nucleotide sequence of S and M genome segments was identified in 13 little studied Tahyna virus (Bunyaviriridae, Bunyavirus, California encephalitis serogroup) strains isolated in Czechoslovakia, Finland, Armenia, Azerbaijan, Kazakhstan, and Tajikistan. A phylogenetic analysis indicated that the examined strains form two groups with a geographical connection: European and Asian genetic groups.

Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes.

La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes.

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Comparison of the M RNA genome segments of two human isolates of La Crosse virus.

The M genomic RNA segments of La Crosse (LAC) virus isolates from the brains of two children autopsied 18 years apart in Wisconsin were molecularly cloned using a reverse transcriptase-PCR assay and the nucleotide sequences of the cDNAs determined. The M RNA of each virus contains 4526 nucleotides, similar to that reported previously for a New York mosquito isolate of LAC. There were 20 nucleotide differences between the two human isolates, which results in the prediction of 7 amino acid changes in the proteins encoded in the single, long open reading frame of the M segment. One of these predicted differences occurs in the G2 glycoprotein and six in the G1 glycoprotein. The two viruses were identical in terms of predicted amino acid sequence in the region believed to represent a nonstructural protein. These data have been further compared to those available for two other California serogroup isolates.