We created a model of HIV-1 infection of conventional mice for investigation of viral replication, control, and pathogenesis. To target HIV-1 to mice, the coding region of gp120 in HIV-1/NL4-3 was replaced with that of gp80 from ecotropic murine leukemia virus, a retrovirus that infects only rodents. The resulting chimeric virus construct, EcoHIV, productively infected murine lymphocytes, but not human lymphocytes, in culture. Adult, immunocompetent mice were readily susceptible to infection by a single inoculation of EcoHIV as shown by detection of virus in splenic lymphocytes, peritoneal macrophages, and the brain. The virus produced in animals was infectious, as shown by passage in culture, and immunogenic, as shown by induction of antibodies to HIV-1 Gag and Tat. A second chimeric virus based on clade D HIV-1/NDK was also highly infectious in mice; it was detected in both spleen and brain 3 wk after tail vein inoculation, and it induced expression of infection response genes, MCP-1, STAT1, IL-1beta, and complement component C3, in brain tissue as determined by quantitative real-time PCR. EcoHIV infection of mice forms a useful model of HIV-1 infection of human beings for convenient and safe investigation of HIV-1 therapy, vaccines, and potentially pathogenesis.

Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIVexpression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.

The system of chimeric HIV-1 infection of mice permits rapid, statistically powerful, and inexpensive evaluation of antiretroviral drugs in vivo.

The biological properties of an Indian isolate (GP78) of Japanese encephalitis virus (JEV) were characterized in tissue-cultured cells and mice and these were compared with the JaOArS982 strain from Japan. The GP78 strain had a markedly extended lag phase during its growth in porcine stable kidney (PS) cells. There were no obvious defects in the penetration of GP78 into PS cells. However, viral RNA and protein synthesis were significantly delayed in GP78-infected PS cells. Fusion-from-within assays carried out in C6/36 cells indicated that GP78 was less fusogenic than the JaOArS982 strain of JEV. Moreover, maximum fusion in GP78-infected cells occurred at pH 5n5, whereas JaOArS982-infected cells showed maximum fusion at pH 6n0. These results suggested that there may be a lesion in the virus-cell fusion process. The GP78 strain also showed delayed growth in brains of 1-week-old BALB/c mice. Although JEV GP78 was as virulent as the JaOArS982 strain in these mice, the appearance of clinical symptoms of JEV infection was delayed by a day in mice infected with the GP78 strain and these animals showed an increased average survival time.Comparison of the nucleotide sequences of the GP78 and the JaOArS982 strains of JEV identified a number of amino acid substitutions in structural proteins. Of these, a Thr Met substitution at residue 76 of the envelope protein is predicted to be causally associated with the altered biology of the GP78 strain during growth.

Toll-like receptors (TLR) represent the best characterized receptor family transducing innate immune responses, the first line of defense against microbial invaders. This study was designed to investigate whether responses through TLR inhibit HIV-1 replication in its primary target cells. Primary human macrophages and lymphocytes from several different donors and HIV-1 infection in tissue culture were used exclusively in this work. We report that ligands of three different TLR: LPS, R848, and double stranded RNA, induce a common antiviral response in macrophages as assayed by measurement of HIV-1 p24 protein, gag DNA, and entry into cells. HIV-1 infection is arrested after efficient entry but prior to reverse transcription. TLR-ligand activated cells secrete antiviral factors that induce a similar restriction. HIV-1 infection of lymphocytes is not affected by exposure to TLR ligands or to antiviral factors secreted by activated macrophages. TBK1, but neither NF-κB nor JAK-STAT activity, is required in macrophages to mount this antiviral response; the combination of p38 MAPK and JNK are partially required for induction of antiviral activity. Based on transcriptional induction and inhibition, the TLR-linked antiviral activity is different from APOBEC3 A or G, interferon-β, NAMPT, or p21Cip1. The cell-type specificity, site of action, and requirement for signaling intermediates suggest that the TLR-linked antiviral activity is novel.

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus responsible for acute encephalitis in humans, with fatality rates ranging from 20% to as high as 50%. The virus is active in a vast geographic area that includes India, China, Japan, and almost all of Southeast Asia. As many as 50,000 cases of JEV infection are reported from these areas every year, of which about 10,000 result in fatality. A high proportion of survivors have serious neurologic and psychiatric sequelae (34). A mouse brain-grown, formalin-inactivated vaccine is available internationally that has many limitations; it is expensive to produce, does not provide long-term immunity, and may cause allergic reactions because of the inclusion of murine encephalogenic basic protein or gelatin stabilizer (1,20,25,27). Thus, there is a need to produce an alternate vaccine that may be safer and cheaper.JEV infection of host cells produces three structural and seven nonstructural proteins. One of these, the E protein, is the major envelope protein of the virion. This protein is believed to play an important role in a number of processes, including viral attachment, membrane fusion, and entry into the host cell. In response to JEV infection, the host produces virus neutralizing antibodies and cytotoxic T cells (CTLs). The principal target for the neutralizing antibodies is the E protein.It has been shown that protection against JEV infection is mainly antibody dependent, and virus-neutralizing antibodies alone are sufficient to impart protection (10, 18). Peptide(s) from JEV E protein that forms the virus-neutralizing epitope(s) could, therefore, be used for inducing JEV-neutralizing antibodies.Recent findings suggest that presentation of peptides in a highly ordered aggregate form can result in enhanced immune responses (12). Johnson grass m...

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