Protection of mice deficient in mature B cells from West Nile virus infection by passive and active immunization.

B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike µMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient µMT mice, but unlike µMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in µMT mice. Thus, the immature B cells present in BAFFR-/- and not µMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.

Last 20 aa of the West Nile virus NS1' protein are responsible for its retention in cells and the formation of unique heat-stable dimers.

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice.

NS1' colocalizes with NS1 and can substitute for NS1 in West Nile virus replication.

NS1' is a C-terminally extended form of the NS1 protein produced only by encephalitic flaviviruses from the Japanese encephalitis virus serogroup. Here we show that West Nile virus (WNV) NS1' and NS1 localize to the same cellular compartments when expressed from plasmid DNAs and also colocalize to viral RNA replication sites in infected cells. Using complementation analysis with NS1-deleted WNV cDNA, we demonstrated that NS1' is able to substitute for the crucial function of NS1 in virus replication.

Dendritic cell-associated MAVS is required to control West Nile virus replication and ensuing humoral immune responses.

Mitochondrial antiviral signaling protein (MAVS) is a critical innate immune signaling protein that directs the actions of the RIG-I-like receptor (RLR) signaling pathway of RNA virus recognition and initiation of anti-viral immunity against West Nile virus (WNV). In the absence of MAVS, mice die more rapidly after infection with the pathogenic WNV-Texas (TX) strain, but also produce elevated WNV-specific IgG concomitant with increased viral burden. Here we investigated whether there was a B cell intrinsic role for MAVS during the development of protective humoral immunity following WNV infection. MAVS-/- mice survived infection from the non-pathogenic WNV-Madagascar (MAD) strain, with limited signs of disease. Compared to wildtype (WT) controls, WNV-MAD-infected MAVS-/- mice had elevated serum neutralizing antibodies, splenic germinal center B cells, plasma cells and effector T cells. We found that when rechallenged with the normally lethal WNV-TX, MAVS-/- mice previously infected with WNV-MAD were protected from disease. Thus, protective humoral and cellular immune responses can be generated in absence of MAVS. Mice with a conditional deletion of MAVS only in CD11c+ dendritic cells phenocopied MAVS whole body knockout mice in their humoral responses to WNV-MAD, displaying elevated virus titers and neutralizing antibodies. Conversely, a B cell-specific deletion of MAVS had no effect on immune responses to WNV-MAD compared to WT controls. Thus, MAVS in dendritic cells is required to control WNV replication and thereby regulate downstream humoral immune responses.