Innate gastrointestinal immunity: characterization of broadly active viral inhibitors.

Innate viral inhibitors that are broadly active have been characterized in the serum and the nervous system, but incompletely characterized in the gastrointestinal (GI) tract. GI preparations from porcine gastric mucosa, mouse intestine, and in neuramide (a pharmaceutical product), were examined for broad antiviral activity, molecular size and mechanism of action for comparison with the previously characterized, innate inhibitors in the serum and nervous system. The GI inhibitors were found to be active in high titers against RNA and DNA viruses, resistant to proteolysis, glycolysis, lipid extraction and possessed differing mechanisms of action. The mouse intestinal inhibitor prevented virus attachment to cells, and neuramide acted at an early post-attachment stage of virus multiplication. The porcine mucosal inhibitor acted as late as 6 h after initiation of the multiplication cycle. These broadly active GI inhibitors differed from the previously described serum inhibitor (UTI beta) high density lipoproteins (HDL) and the nervous system (NS) inhibitor by being smaller (600 +/- 400 kDa) and resistant to proteinase K, glycosidases and organic solvents. The mouse intestinal inhibitor acts similarly to UTI beta and NS inhibitor by preventing attachment of virus to the cells. In comparison, the neuramide and the porcine mucosal inhibitor, like HDL, acted after attachment to the target cells. The innate nonspecific, broadly-active virus inhibitors, based on high titers and location, are considered important initial immune defense mechanisms against viral infections and thus potentially useful in medical applications.

Innate antiviral defenses in body fluids and tissues.

Innate, non-specific, resistance mechanisms are important barriers to pathogens, particularly delaying virus multiplication at the onset of infections. These innate defense mechanisms include a series of mechanical barriers, pre-existing inhibitory molecules, and cellular responses with antimicrobial activity. The antiviral activities of these innate inhibitors reside in a variety of partly characterized substances. This review presents the innate antiviral inhibitors in cell cultures, urine, serum, the gastrointestinal tract, the nervous system, tissues of crustaceans, and saliva. Medical adaptation of the innate antiviral defense mechanisms may be useful for prevention and treatment of viral infections.

Broad antiviral activity in tissues of crustaceans.

Innate antiviral substances occur in vertebrates and may function as host defenses. Virus infections are common among invertebrates, but little is known about the ability of invertebrates to control viral infections. Pre-existing antiviral substances may be particularly important, since invertebrates lack the antiviral defense conferred by specific immunity. In our study, we found that tissue extracts of blue crab (Callinectes sapidus), shrimp (Penaeus setiferus), and crayfish (Procambarus clarkii) contained antiviral activities that inhibit a variety of DNA and RNA viruses, i.e. Sindbis virus (SB), vaccinia virus (VAC), vesicular stomatitis virus (VS), mengo virus (MENGO), banzi virus (BANZI) and poliomyelitis (POLIO). The concentration of inhibitory activity was relatively high, ranging from 102 to 216 U/g tissue for Sindbis virus, using the various tissue extracts. The other viruses were somewhat less sensitive to the inhibitor. The main antiviral activity in the inhibitor preparation from blue crab resided in an approximately 440 kDa fraction. It was inactivated significantly by lipid extraction, but not by proteinase K or glycosidases. The antiviral mechanism of the inhibitor from the blue crab was inhibition of virus attachment to eukaryotic cells, as evidenced by inhibitory activity at 4 degrees C. These studies are among the first to show the existence of broadly active antiviral activities in aquatic crustaceans. These antiviral substances may function as innate host defenses in these species that lack specific antibody immunity and, therefore, merit further study.

Lipoproteins account for part of the broad non-specific antiviral activity of human serum.

Several antiviral substances have been detected in human serum but few have been shown to possess broad antiviral activity. These broadly active antiviral molecules could be of significance as innate defense mechanisms. We have previously identified and characterized a broadly antiviral glycoprotein, UTI3, which accounts for 50 antiviral units/ml of human and mammalian sera. In addition there are reports of antiviral activity of human serum apolipoprotein A-1 (apo A-1), an important constituent of high density lipoprotein (HDL), against human immunodeficiency virus (HIV) and herpesvirus. Therefore we investigated (1) whether HDL is broadly antiviral, (2) how much of the broad antiviral activity of serum is due to HDL, and (3) the mechanism(s) of HDL's antiviral action. In this paper we report that (1) HDL does have broad antiviral activity, (2) HDL accounts for a modest but significant portion of the antiviral activity of serum, and (3) HDL acts by preventing virus penetration. Overall, HDL may be one of the broadly antiviral defences in the bloodstream.

A host defense role for a natural antiviral substance in the nervous system.

The pathogenesis of virus infections of the nervous system (NS) is regulated by host defenses. The defensive role of a major constitutive antiviral substance was studied by determining its distribution in the human nervous system, its concentration and the ability of this viral inhibitor to protect mice against viral infection. The 4000 kDa inhibitor complex in the human nervous system was detected in brain gray and white matter, spinal cord, and sciatic nerve but not in human cerebrospinal fluid. The inhibitor was found in the extracellular medium incubated with minced murine brain. The inhibitory titer ranged from approximately 50 to 200 antiviral units per gram against polio 1, Semliki Forest, Banzi, mengo, Newcastle disease and herpes simplex 1 viruses. The inhibitor is composed of lipid and essential protein and carbohydrate moieties as determined by enzymatic inactivation. Protection of inhibitor-treated mice was demonstrated against both an alphavirus and a picornavirus. Thus a natural defensive role for the broadly antiviral inhibitor is suggested by its constitutively high concentration, wide distribution in nervous system tissues, presence in extracellular fluid and its ability to provide protection in infected mice.

Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila.

A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Maximum toxin yield was obtained with the pTRX vector. Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector systems. The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography-based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies. Purified mature toxin migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously. Binding of the toxin to erythrocytes was temperature dependent, with no binding occurring at 4 degrees C. However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min. It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy. Calcium chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol. With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated. None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act. The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis. As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited.

Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin from Aeromonas hydrophila.

We report herein the DNA sequence analysis of the heat-labile cytotonic enterotoxin gene (alt) from Aeromonas hydrophila and the biological function of the purified hyperproduced toxin (Alt). One large open-reading frame (ORF), comprised of 1104 bp, was detected at positions 804 to 1907 bp on a 4.0-kb Sa/l DNA fragment from Aeromonas. This ORF encodes for a protein having 368 amino acids (aa) with a computed molecular weight of 38 kDa. The aa sequence of the first 15 NH2-terminal residues of the mature native Alt from A. hydrophila matched with the DNA-derived aa sequence of the alt gene expressed in E. coli starting at position 19, which was leucine. The first 18aa residues of the Alt represented a putative signal sequence with alanine at its carboxy terminus. A BLAST search of the entire database showed 45-51% identity of the Alt, starting at position 158 with the carboxy half of the phospholipase C (PLC) and lipase from A. hydrophila; however, the purified Alt had no lipase/PLC activity. The alt gene was hyperexpressed using gene fusion expression vector systems, and the recombinant Alt exhibited a size of 35-40 kDa. The pure recombinant Alt elongated Chinese hamster ovary cells and elicited fluid secretion in rats ligated intestinal loops, indicating its enterotoxicity. Immunization of mice with recombinant Alt resulted in a reduced fluid secretory response when challenged with Aeromonas. The biological activity of the recombinant Alt in E. coli was about 10-fold less, compared to native Alt from Aeromonas, indicating differential processing of the toxin. The antibodies to native Alt neutralized the biological activity of the recombinant toxin, and these antibodies reacted with the same specificity to the native and recombinant Alt in immunoblots. The role of cyclic adenosine monophosphate and prostaglandins in causing a fluid secretory response by Alt also was demonstrated.

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.

Treatment of intracranial alphavirus infections in mice by a combination of specific antibodies and an interferon inducer.

Finding an effective treatment for viral infections that cause encephalitis remains an important problem. A model of human alphavirus infections, Semliki Forest virus, causes lethal encephalitis in weanling mice. Mice are viremic within 24 hr of an intraperitoneal challenge with the equivalent of three 75% lethal doses of Semliki Forest virus. Virus reaches the brain by 48 hr, and mortality results in all mice in 5-7 days. Introduction of virus intracranially accelerates the course of the infection. Neither anti-Semliki Forest virus hyperimmune serum nor the potent interferon inducer poly I:CLC given intraperitoneally are protective when used therapeutically after an intracranial virus infection, but a combination of 1,000 U hyperimmune serum and 80 micrograms/mouse of poly I:CLC results in a 50% survival rate. This combination treatment of intracranial Semliki Forest virus infection eliminates detectable viremia and reduces virus load in the brain over the course of the infection. These data show that when combined, specific antibody and an interferon inducer can interact synergistically to protect mice from alphavirus infections of the central nervous system even when given after the virus is replicating in the target organ.

HIV recovery from saliva before and after dental treatment: inhibitors may have critical role in viral inactivation.

Unstimulated whole saliva was collected from 21 HIV-positive men and women before and after dental treatment. The frequency of HIV detection did not increase after dental treatment. Infectious HIV was recovered from only one patient. Study findings raise the possibility that, in most cases, salivary inhibitors render the virus non-infectious.

Generalized occurrence of the broadly antiviral substance UTI beta in mammalian sera.

This study compares the characteristics of naturally occurring antiviral activities in nonhuman mammalian sera to UTI beta (University of Texas virus inhibitor beta), an innate, non-specific viral inhibitor found in human serum. The antiviral agent in sera from four different species appears to possess properties similar to those of UTI beta, e.g. molecular weight of 60 +/- 10 kDa, broad spectrum antiviral activity, glycoprotein structure with antiviral oligosaccharide moiety(s) attached to a carrier protein, and inhibition of most viruses by preventing their attachment to target cells. These findings suggest that UTI beta or UTI beta-like virus inhibitors are a normal component of the non-specific immune defenses of mammals.

Further characterization of a broad-spectrum antiviral substance in human serum.

A broadly active antiviral glycoprotein (UTI beta) occurs naturally in human sera at an average antiviral titer of 50 U/ml. This inhibitor is active against all virus families tested to date, including representative poxviruses, herpesviruses, enteroviruses, paramyxoviruses, alpha-viruses, flaviviruses, bunyaviruses, and rhabdoviruses. It is a glycoprotein of approximately 60,000 +/- 10,000 Da, which is stable at pH 2 to 10 and at 80 degrees C for up to 10 min. Mild oxidation with NaIO4 and treatment with glycosidases inactivates the material. Proteolytic degradation of the inhibitor molecule releases small active components of < 1000 Da, which retain antiviral activity. This activity of the small components has increased heat stability (120 degrees C for 15 min) and is inactivated by glycosidases. The antiviral activity thus appears to reside mainly in the oligosaccharide moiety of the glycoprotein. The inhibitor does not neutralize virions, but prevents attachment of most viruses to cells. These properties occur also in highly purified preparations. These findings indicate that human serum contains significant concentrations of a broadly active antiviral glycoprotein, which is distinct from interferon and other antiviral substances naturally found in human body fluids and tissues.

The interferons. Mechanisms of action and clinical applications.

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

The effect of an arenavirus infection on liver morphology and function.

Patients with severe Lassa fever have high serum levels of liver enzymes. Studies of the histology of the liver have shown only minor alterations, seemingly insufficient to account for death. Pichinde virus is an arenavirus which causes severe illness similar to Lassa fever in strain 13 guinea pigs, but does not cause severe illness in man. This can serve as a relatively safe model for studying the pathology and pathophysiology of fatal arenaviral infection. We used this infection to evaluate the effect of arenavirus on liver morphology and function. When guinea pigs were infected with Pichinde virus, all developed severe disease and died within 14 days of infection. The animals lost large amounts of weight. Higher levels of virus were detected in the liver than in serum. Aspartate aminotransferase and alanine aminotransferase were elevated late in the course of the disease; no elevations were seen in gamma glutamyl transpeptidase or bilirubin. Alkaline phosphatase, initially high in these growing animals, was markedly decreased early in infection. Prothrombin time and activated partial thromboplastin time were increased late in the disease, and decreased levels of Factors VIII and IX were seen relatively early. Fatty metamorphosis, indicating problems in lipid processing, occurred by day 11, but necrosis was minor and occurred late. Pichinde virus infection results in significant alterations in the metabolic and synthetic capacities of the hepatocytes early in infection in the absence of significant necrosis.

Nucleotide and amino acid sequence of lymphocyte-derived corticotropin: endotoxin induction of a truncated peptide.

To determine the degree of similarity between pituitary and lymphocyte proopiomelanocortin, the lymphocyte mRNA was reverse transcribed, cloned, and sequenced. Murine lymphocyte mRNA was first purified by oligo(dT)-cellulose affinity chromatography and was reverse transcribed by using a selective 3' antisense oligonucleotide primer directed at the boundary between the translated/nontranslated region on the 3' end of exon 3. This cDNA was then amplified in a polymerase chain reaction with selective primers containing Sal I and Kpn I restriction endonuclease sites. Amplified cDNA was then directionally ligated into M13mp18 and M13mp19 bacteriophage and was sequenced. The nucleotide sequence encoding this peptide was identical to that of mouse pituitary corticotropin (ACTH). Elevated levels of lymphocyte immunoreactive ACTH were then induced with bacterial lipopolysaccharide and the peptide(s) was purified by antibody affinity chromatography and reverse-phase high-performance liquid chromatography. The predominant immunoreactive ACTH species was approximately 3 kDa and its sequence was identical to pituitary ACTH(1-25). These results conclusively demonstrate that lymphocytes produce authentic ACTH and harbor its mRNA.

Prosimian hemoglobins--V. The primary structures of the alpha-I, alpha-II and beta-hemoglobin chains of Hapalemur griseus, with a note on the classification of Microcebus.

1. The duplicated adult hemoglobins were isolated from a mature Hapalemur griseus and the constituent chains prepared. Sequence analysis of the isolated alpha-globins showed that the alpha-I and alpha-II chains differed by a glycine for lysine substitution at position 15. 2. The complete amino acid sequence of the single adult beta-globin of Hapalemur griseus was determined. The beta-globin sequence of Hapalemur griseus clusters with those of other authentic lemurs, and is clearly separated from the sequences characteristic of lorisiform primates. 3. Partial sequence analysis of the beta-globin of Microcebus murinus showed only a single amino acid difference when compared to the Hapalemur globin. 4. Partial sequence analysis of the alpha-globin of Microcebus murinus showed only three amino acid residues that are not found in other lemuriform alpha-globins; two of these are unique to Microcebus among all known prosimian alpha-globin sequences. 5. The Microcebus alpha- and beta-globins are more similar to the homologous lemuriform sequences than they are to lorisiform sequences.

Arenavirus infection in the guinea pig model: antiviral therapy with recombinant interferon-alpha, the immunomodulator CL246,738 and ribavirin.

Human arenaviral infections have a high mortality, and are dangerous to work with in the laboratory. There is a need for good antiviral agents to treat these infections. Pichinde virus infection of the inbred strain 13 guinea pig is a relatively safe, good animal model for human arenavirus infections. Mortality is consistently 100% between days 12 and 25 (mean 14.8) days after infection. When infected animals were treated with recombinant human interferon alpha A, or with CL246,783, an immunomodulator known to induce interferon, no beneficial effect was noted. When animals received ribavirin, 25 mg/kg/day for the first 14 days of infection, the course of infection was prolonged, with death occurring a mean of 22.5 days after infection. If ribavirin was administered for 28 days, mortality was reduced to 25%, with those animals dying a mean of 21.0 days after infection. These results confirm the studies that indicate that ribavirin is a useful agent for treating arenaviral infections. However, treatment with this agent must be prolonged. They also demonstrate the potential usefulness of Pichinde virus infection in strain 13 guinea pigs as an animal model of human disease.

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.