B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus

Abstract

West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (microMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, an approximately 500-fold difference in serum viral load was detected in micro MT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected micro MT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.

FIG. 1.

(A) WNV antigen expression in the brains of wild-type (nine left panels) and μMT (three right panels) mice. Brains were harvested 8 or 9 days after infection with WNV, sectioned, and stained with rat anti-WNV polyclonal serum or a control negative polyclonal rat serum. Typical sections from the cerebellum, brain stem, and cerebral cortex are shown and are representative of more than 10 independent brains from either wild-type or μMT mice. At day 8 after infection, approximately 40% of the wild-type mice and 100% of the μMT mice had brains that stained positive for viral antigen by immunohistochemistry. After development with substrate, viral antigen stained dark brown. Arrows indicate examples of heavily infected neurons. In the brain stem and cortex of μMT mice, pyknotic nuclei of heavily infected neurons can be seen. (B) Scatter plot of the levels of infectious WNV in the brains of wild-type and μMT mice. Brains of five wild-type (solid circles) or μMT (open squares) mice at each time point were harvested, homogenized, and subjected to viral plaque assay in BHK21 cells. The limit of sensitivity of the plaque assay is indicated by the dotted line. Viral levels at day 6 (P < 0.03) and day 8 (P < 0.01) were statistically different between wild-type and μMT mice as determined by two-tailed Student's t test. The following percentages of mice had viral burdens below the level of detection (<10 PFU/g): 80% of day 4 wild type, 40% of day 6 wild type, 20% of day 8 and day 10 wild type, 40% of day 12 wild type, 40% of day 4 μMT and 0% of day 6 and day 8 μMT.

FIG. 1.

(A) WNV antigen expression in the brains of wild-type (nine left panels) and μMT (three right panels) mice. Brains were harvested 8 or 9 days after infection with WNV, sectioned, and stained with rat anti-WNV polyclonal serum or a control negative polyclonal rat serum. Typical sections from the cerebellum, brain stem, and cerebral cortex are shown and are representative of more than 10 independent brains from either wild-type or μMT mice. At day 8 after infection, approximately 40% of the wild-type mice and 100% of the μMT mice had brains that stained positive for viral antigen by immunohistochemistry. After development with substrate, viral antigen stained dark brown. Arrows indicate examples of heavily infected neurons. In the brain stem and cortex of μMT mice, pyknotic nuclei of heavily infected neurons can be seen. (B) Scatter plot of the levels of infectious WNV in the brains of wild-type and μMT mice. Brains of five wild-type (solid circles) or μMT (open squares) mice at each time point were harvested, homogenized, and subjected to viral plaque assay in BHK21 cells. The limit of sensitivity of the plaque assay is indicated by the dotted line. Viral levels at day 6 (P < 0.03) and day 8 (P < 0.01) were statistically different between wild-type and μMT mice as determined by two-tailed Student's t test. The following percentages of mice had viral burdens below the level of detection (<10 PFU/g): 80% of day 4 wild type, 40% of day 6 wild type, 20% of day 8 and day 10 wild type, 40% of day 12 wild type, 40% of day 4 μMT and 0% of day 6 and day 8 μMT.

FIG. 2.

Survival data for C57BL/6J mice inoculated with WNV. (A) Wild-type mice. Animals were inoculated via footpad with the indicated doses of WNV. The survival curves were constructed using data from three to six separate experiments. The numbers of animals receiving each viral dose ranged from n = 9 (10 PFU) to n = 29 (10⁶ PFU). All animals were inoculated with virus stocks from the same passage (P = 1). (B) RAG1 mice. RAG1 or wild-type C57BL/6J mice were inoculated via the footpad with 10² or 10⁶ PFU of WNV. The survival curves were constructed from data from at least three independent experiments. (C) RAG1 adoptive transfer studies. Splenocytes and purified B cells were harvested from naïve or immune congenic wild-type animals and injected into RAG1 mice 1 day prior to infection with 10² PFU of WNV. Survival data are indicated and reflect results from at least three independent experiments. (D) μMT mice. μMT or wild-type C57BL/6J mice were inoculated via the footpad with 10² or 10⁶ PFU of WNV. The results shown reflect data from at least five independent experiments.

FIG. 3.

WNV burden in peripheral nervous system and CNS tissues in wild-type and μMT mice. (A) Levels of infectious virus in peripheral tissues. Virus levels from serum, spleen, and liver of wild-type and μMT mice were measured using a viral plaque assay in BHK21 cells after tissues were harvested at the indicated days after inoculation. Data shown are the average PFU per gram of tissue or milliliter of serum for five wild-type or μMT mice per time point. The dotted line represents the limit of sensitivity of the assay. (B) Infectious virus levels in the CNS. Virus levels from brain and the inferior and superior spinal cord were determined as described above. (C) Levels of viral RNA in draining lymph nodes. WNV RNA was harvested from draining popliteal or inguinal lymph nodes at the indicated days after inoculation and quantitated using a real-time fluorogenic RT-PCR assay. Data are expressed as genomic equivalents of WNV RNA per microgram of rRNA after normalization for tissue content and represent results for tissues harvested from five wild-type or μMT mice per time point. The dotted line represents the limit of sensitivity of the assay. (D) Levels of viral RNA in serum. Viral RNA levels were determined from serum of wild-type or μMT mice after WNV infection at the indicated days using a real-time fluorogenic RT-PCR assay. Data are expressed as genomic equivalents of WNV RNA per milliliter of serum and represent the averages for five independent mice per time point.

FIG. 4.

Development of specific antibodies against WNV. Serum samples were collected from wild-type or μMT mice at the indicated days after infection. (A) Neutralizing antibody titers were determined by a PRNT assay. Samples were performed in duplicate, and results represent the average of three independent experiments with at least three mice per group. Data are expressed as the reciprocal PRNT₅₀, the antibody titers that reduced the number of plaques by 50%. (B) Isotype-specific ELISA. The development of the isotype (IgM or IgG) of specific antibodies was determined after incubation of serum with adsorbed control or viral antigen. Data are the averages of three separate experiments performed in duplicate. (C) IgM depletion studies. IgM was depleted from day 4 or day 10 serum samples after treatment with β-mercaptoethanol (see Materials and Methods). Serum was analyzed for remaining neutralizing antibodies by using the PRNT assay as described above. Data represent results from one experiment that is representative of three.

FIG. 5.

Passive administration of serum to μMT mice. Serum samples were collected from mice that were naïve or immune or at 4 days postinfection with WNV. After heat inactivation, 0.5 ml of serum was administered to μMT mice 1 day prior to and after infection with 10² PFU of WNV. Data represent results from at least three independent experiments with five mice per condition.