Joyce Poast

Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections

ABSTRACT Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the Paramyxoviridae family, Pneumovirinae subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-α/β) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-α in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-α, or directly with recombinant IFN-α showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-α than hMPV. Similarly, when the ability to directly induce IFN-α production was assessed, RSV was clearly a weaker inducer of IFN-α than hMPV, as shown by both kinetics and the absolute amount of IFN-α secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-α production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-α production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-α in the lung during infection by members of the Pneumovirinae family.

Variation in Interferon Sensitivity and Induction among Strains of Eastern Equine Encephalitis Virus

ABSTRACT Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-α, -β, and -γ generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-α/β receptors than in wild-type mice, suggesting that IFN-α/β is important in controlling replication. In contrast, IFN-γ receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-γ does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.

Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method.

While antibodies are a major extracellular tool of the highest specificity to answer important biomedical questions, the improvements in electroporation discussed below may make it feasible to also use antibodies as an intracellular deletion tool to study (a) viruses inside the cell, (b) cancer cells, (c) signal transduction, (d) genetics, (e) metabolism, and (f) other structures and mechanisms. Already, others have succeeded in depositing macromolecules, including antibodies (Abs), and nucleic acids inside cells, using many techniques, including electroporation (EP). However, EP has limitations that have precluded its widespread use, particularly its high kill rate for cells and the low percentage of cells that are able to incorporate macromolecules. If these limitations could be overcome for Abs and nucleic acids, then it would be practical to use them as highly specific probes for intracellular molecules. In our experiments using EP, we were able to largely prevent lethality for cells during EP by employing a commercially available cold-storage solution for organ transplants containing high K(+) and Mg(++) (ViaSpan, Belzer UW cold-storage solution, DuPont Pharmaceuticals). This solution decreased cell death after standard EP by an average of 50% for a number of cell lines. Viability of WISH cells after EP approached 100%. In transfection studies, ViaSpan medium strongly increased both P3HR1 cell survival as well as the total number of cells transfected with DNA for green fluorescent protein (GFP). In additional experiments with Abs, we were able to strongly increase the percent of cells that incorporated Ab by using two serial EPs. This enhanced the intracellular protection by Abs against viruses in Vero cells from 64% to a maximum of 98%. We were able to further simplify the EP technique by using unpurified antiserum in place of purified IgG. Thus, this EP technique offers multiple advantages: simplicity, high cell viability, high effectiveness, high specificity, rapid action, usefulness with adherent or non-adherent cells, and no requirement for purification of antibodies from antiserum.

Oral Transmission of Human Immunodeficiency Virus by Infected Seminal Fluid and Milk: A Novel Mechanism

Salivary transmission by the 30 million human immunodeficiency virus (HIV) carriers is rare, despite kissing, aerosolization, and dental treatment. The main protective mechanism of saliva is reported to be inactivation of HIV-transmitting leukocytes by its unique hypotonicity; however, the successful oral transmission of HIV by seminal fluid and milk is unexplained. Whether seminal fluid and milk successfully transmit HIV orally by overcoming the recipients salivary hypotonic inactivation of HIV-transmitting leukocytes was tested. Isotonic salt solution and normal donor samples of milk, colostrum, seminal fluid, and blood were studied for their ability to overcome the salivary hypotonic inactivation. All samples, in physiologic volumes, prevented the hypotonic saliva from inactivating HIV-transmitting leukocytes by providing solutes and retarding diffusion. This indicates that successful oral transmission of HIV by seminal fluid, milk, and colostrum may be due to their isotonicity, which overcomes hypotonic salivary inactivation of HIV-transmitting leukocytes.

Is Human Immunodeficiency Virus RNA Load Composed of Neutralized Immune Complexes

During acute human immunodeficiency virus (HIV) infection, both virus load (HIV RNA) and infectivity are high (10(3)-10(7) RNA copies/mL or TCID(50)/mL) until antibody is produced, which may reduce the HIV infectivity. In HIV carriers, the HIV RNA load is elevated (10(3)-10(5) copies/mL), but infectivity is low (10(0)-10(2) TCID(50)/mL). The low infectivity in carriers could be due to neutralization by antibody in serum, resulting in immune complexes (ICs). We demonstrated that ICs in plasma, prepared with protein A beads, contained HIV RNA (80%-100%) in association with immunoglobulin G (IgG). In comparison, ICs from patients with acute HIV infection and little or no antibody contained virtually no HIV RNA. Moreover, ICs prepared by ultrafiltration contained IgG and specifically and irreversibly neutralized HIV, which indicates that the ICs contained neutralizing antibody. These findings indicate that the HIV RNA in the plasma of carriers is frequently composed of antibody-neutralized HIV as ICs.

Practical Prevention of Vaginal and Rectal Transmission of HIV by Adapting the Oral Defense: Use of Commercial Lubricants

HIV is transmitted to 6.4 million human beings per year and the majority of these transmissions are sexual. Condoms are highly effective and are recommended as the primary preventive. However, the fact that there are millions of sexual transmissions each year indicates that many people do not use condoms and that additional preventives are needed. The mechanisms of natural prevention of oral transmission by saliva may be adaptable to the susceptible vagina and rectum. The objective of our study was to reduce the sexual transmission of HIV by mimicking salivas targeting of the transmitting infected leukocytes and any cell-free HIV in seminal fluid. The previously recommended anti-HIV topical microbicide, nonoxynol-9, has not prevented HIV transmission in humans, probably because it causes mucosal irritation and attracts CD4(+) cells. To identify effective preparations that are nonirritating, we studied the anti-HIV activity of commercially available, over-the-counter (OTC) lubricants and vaginal preparations that are judged safest by the U.S. Food and Drug Administration (FDA), and are nonirritating. The effect of OTC preparations on both the production of HIV by infected leukocytes and cell-free HIV suspended in seminal fluid was measured under simulated in vivo conditions. We surveyed 22 OTC vaginal preparations and excluded those with low inhibitory activity and those that were inhibitory but likely to be irritating. Three included preparations are highly active against both HIV-infected leukocytes suspended in seminal fluid and active against cell-free HIV, under in vitro conditions that simulate in vivo conditions. Since the preparations identified here as anti-HIV substances have the advantages of being widely available, inexpensive, acceptable, in the safest U.S. FDA category, and may be used by recipient women or men, they should be tested in clinical trials to help prevent sexual transmission of HIV.

Postinfection therapy of arbovirus infections in mice.

Most antiviral agents are efficacious prophylactically in vivo, and a few are efficacious for postinfection (p.i.) therapy. To explore possibilities for p.i. therapy of encephalogenic Banzi virus (BZV) and Semliki Forest virus infections in mice, we evaluated candidate antiviral therapies after development of the first clinical signs of infection. The earliest clinical indication of BZV viremia in mice is a rise in core body temperature beginning on day 3 p.i. BZV-infected mice showing elevated core body temperatures (greater than or equal to 37.3 degrees C) on days 3 and 4 p.i. were treated intraperitoneally with the interferon inducer poly(ICLC) (80 micrograms per mouse) and/or specific antiserum. Combined therapy on day 3 of a BZV infection protected over 75% of mice showing clinical evidence of viral disease before treatment. Protection against early brain infection must occur on day 4 p.i., since by that day BZV has started multiplying in the brains of the mice. Significant protection occurred with antiserum alone and increased with poly(ICLC). Similar protection was obtained during Semliki Forest virus viremia, but this infection is so rapid that the first clinical signs are reliably detectable only after viremia. Images

Interferon protects mice against inhalation anthrax.

Interferons (IFNs) play a role in innate immunity during many viral, bacterial, and protozoal infections. With the increasing threat of bioterrorist attacks with Bacillus anthracis, its high lethality, and the limited effectiveness of antibiotics, alternative treatments are being studied. Antibodies to protective antigen (PA) are promising, as is IFN. During many bacterial infections, production of and protection by IFNs has been reported, including B. anthracis in vitro. In vivo, we find that (1) the type I IFN inducer, Poly-ICLC, strongly and rapidly protects mice; (2) the protection is IFN-mediated since recombinant murine IFN-beta can protect, and protection by Poly-ICLC is abrogated in IFN type I receptor knockout mice. The greatest protection by Poly-ICLC was conferred by intranasal treatment. A delay in death was observed with the intramuscular route alone, but was not significant. Together, the results suggest the IFN defense could protect mice, up to 60%, against lethal inhalational anthrax, and thus have important medical implications for therapy of human anthrax.

Poly I:CLC Induction of the Interferon System in Mice: An Initial Study of Four Detection Methods

Induction of a large number of the components of the interferon (IFN) system (IFN genes, their mRNAs, IFN proteins, IFN receptors, IFN signaling molecules, the IFN response genes, and their effector proteins) has been studied. Less well studied is the comparative induction of these components in vivo. Induction of IFN by double-stranded RNA (dsRNA) treatment mimics certain aspects of viral infection and induces the components of the IFN system. To determine the comparative sensitivity of detection of induction in mice, we initially studied the limiting concentrations of polyribinosinic-polyribocytidylic acid, polylysine complex (poly I:CLC, a synthetic dsRNA preparation), for induction of four representative components of the IFN system: (1) IFN in serum, (2) the IFN response gene mRNA ISG54 in spleen and liver, (3) the IFN-beta mRNA in spleen, and (4) resistance of mice to Banzi viral infection. The results of this initial study showed that resistance to infection was 7-fold more sensitive for detection of the IFN response than was ISG54 mRNA and 70-fold more sensitive than either IFN-beta mRNA or IFN production in serum. In comparison, mouse cells in vitro treated with poly I:CLC were 3-10-fold less sensitive to its antiviral action than is the mouse. The results demonstrate that in the four tests in mice, the most sensitive indicator of poly I:CLC induction of the IFN system was protection against Banzi viral infection, followed by ISG54 mRNA levels, IFN-beta mRNA, and IFN protein levels. It is hypothesized that the highest sensitivity of mouse protection may be due to priming by the initial poly I:CLC-induced IFN of the subsequent Banzi virus-induced IFN, resulting in rapid and high concentrations of IFN at the local site of viral replication. Future studies are needed to study other molecular components of the IFN system to identify those that cause the unanticipated high sensitivity of mice to protection against Banzi virus.

Vertebrate brains contain a broadly active antiviral substance

Brain tissue extracts from vertebrates were examined for non-specific, broad-spectrum virus inhibitors, previously identified and characterized from other body tissues and fluids. An antiviral activity found in human, bovine, ovine, porcine, lapine, murine and piscine brain tissues shares some properties with a contact blocking-virus inhibitor, which was previously found only in cell culture supernatants. The inhibitor was active against (in order of sensitivity to inhibitor) Banzi, Sindbis, Bunyamwera, Newcastle disease, herpes simplex I, Semliki forest, polio I, mengo, vaccinia and vesicular stomatitis viruses. It is approximately 4000 kDa and possesses a complex structure containing protein, carbohydrate and lipid moieties. The inhibitor does not directly neutralize virus or induce an antiviral state in cells, but appears to act early in the replication cycle, most likely by preventing virus attachment to target cells. Its occurrence in concentrations sufficient to reduce virus yield in cell cultures at least 30-fold may indicate a role in limiting viral infections of the central nervous system.