Margo A. Brinton

Replication of Flaviviruses

Flaviviruses were classified as members of the togavirus family until 1984, when the International Committee for the Nomenclature of Viruses voted to make Flaviviridae a separate family (Westaway et al., 1986). The togavirus family had originally been defined using morphological criteria. The change in classification was the result of recent research that clearly demonstrated that flaviviruses, although generally similar to alphaviruses in their morphology, differ markedly from the alpha togaviruses in their virion structure, strategy of replication, and morphogenesis.

Separation of Functional West Nile Virus Replication Complexes from Intracellular Membrane Fragments

Flaviviruses encode seven non-structural proteins for which functions have not yet been described. The identification of the viral and possible host proteins which may be involved in flavivirus replication has been impeded by the fact that the viral replication complexes are tightly associated with endoplasmic reticular membranes within infected cells and that in vitro polymerase activity is associated with large membrane fragments. To facilitate further study of flavivirus replication complexes, selected ultrapure detergents were analysed for their effect on West Nile virus (WNV) in vitro RNA-dependent RNA polymerase activity and for their ability to release functional replication complexes from partially purified intracellular BHK-21 membrane fragments. A few previous reports indicated that flavivirus in vitro polymerase activity was sensitive to detergent treatment. The present study indicates that WNV polymerase activity is variably inhibited depending on the concentration and identity of the detergent used. Of the five detergents (Tween 20, maltoside, octylglucoside, lubrol PX and sodium deoxycholate) tested, sodium deoxycholate was the most efficient at releasing functional viral replication complexes from intracellular membranes.

Comparative study of various immunomodulators for macrophage and natural killer cell activation and antiviral efficacy against exotic RNA viruses

Several immunomodulators were compared for immunomodulatory and antiviral activity in B6C3F1 female mice. Our results demonstrate that murine recombinant gamma interferon (rIFN-G), human recombinant alpha A/D interferon (rIFN-A), ampligen (a polyribonucleotide) and CL246,738 modulate nonspecific immunity and are effective antiviral agents in vivo. Administration of each of these agents 1 day before cell harvest induced high levels of splenic natural killer (NK) cell activity against YAC-1 target cells. rIFN-G was also a potent activator of peritoneal macrophages (M phi), as evidenced by high levels of antitumor activity and changes in ectoenzyme phenotype that is characteristic of tumoricidal M phi. rIFN-A, ampligen and CL246,738 induced moderate to low levels of M phi activation by these criteria. In vivo protection experiments showed that repeated therapeutic treatment with rIFN-A protected mice against i.p. infection with Venezuelan equine encephalitis (an alpha togavirus, VEE), Banzi (a flavivirus) and herpes simplex virus type 2 (HSV-2). Similar treatment with rIFN-G was effective against VEE and HSV-2, but ineffective against Banzi virus. A single prophylactic i.p. dose of ampligen 1 day before virus challenge was very effective against Banzi virus, moderately effective against HSV-2, and ineffective against VEE and Caraparu (a bunyavirus) infection. A single prophylactic oral dose of CL246,738 provided almost complete protection of mice against VEE, Banzi, and HSV-2, and also increased the mean survival time for Caraparu infected mice. Collectively, these results indicate that rIFN-A, r-IFN-G, ampligen and CL246,738 may be useful in prophylactic or early therapeutic treatment of several serious virus infections. Since these agents stimulate NK cells and M phi, their antiviral activity may result, in part, from the alterations they induce in the natural immune system.

Characterization of West Nile virus persistent infections in genetically resistant and susceptible mouse cells. I. Generation of defective nonplaquing virus particles.

Abstract Persistent infections with West Nile virus (WNV), strain E101, were readily established in SV40-transformed mouse embryofibroblast cell lines. Four separate persistently infected cultures maintained at 37° by weekly subculture, spontaneously stopped producing infectious virus as assessed by plaque assay on BHK cells by the sixteenth week. In contrast, duplicate cultures switched to 32° after the sixth subculture continued to produced virus for an extended period. However, cells that no longer produced detectable plaquing virus remained positive for WNV-specific immunofluorescence and were resistant to superinfection with WNV. Although attempts to rescue plaquing virus were unsuccessful, flavivirus-like particles were observed in these cells by electron microscopy within dense cytoplasmic vacuoles and in culture fluids. Analysis of RNA within the extracellular particles produced by these cells revealed the presence of several species of RNA all smaller than the 40 S genome RNA of wild type WNV. This RNA was demonstrated to be WNV-specific by hybridization with DNA complementary to wild type WNV 40 S RNA. The extracellular particles were able to infect BHK cells, but their ability to interfere with the replication of wild type WNV was found to be low. The frequency of generation of nonplaquing WNV mutants by the mouse cell lines indicates that these cells may exert a selective pressure for their enrichment.

Host Genes that Influence Susceptibility to Viral Diseases

Virus infections that induce permanent impairment or death in their hosts have undoubtedly exerted a selective pressure in the evolution of both plants and animals. Therefore, it is not surprising that those host alleles that fortuitously provided a reduced susceptibility to viral diseases would be maintained in the host population. A classic example is the introduction of myxomatosis virus into populations of wild European rabbits in Australia. The European rabbit had become an agricultural pest by 1950 and myxomatosis virus was introduced in an attempt to reduce the rabbit population. Initially, rabbit mortality rates were observed to be in excess of 99% [1], but a small number of rabbits recovered from infection even during the spread of the original highly virulent strain of virus. These survivors bred and, in areas where the rabbit population was reexposed to virus annually, the mortality rate had decreased to 25% by the end of 7 years [2]. In some areas attenuated virus strains arose and this provided additional opportunity for the selection of resistant animals. Thus, natural selection operated to produce an ecological balance, that ensured survival of the virus in the presence of a disease resistant host population.

Varying role of alpha/beta interferon in the antiviral efficacy of synthetic immunomodulators against semliki forest virus infection

The question of whether interferon alpha/beta is the common mechanism of antiviral action of synthetic immunomodulators was investigated in B6C3F1 mice infected with Semliki Forest virus. Mice were treated with various concentrations of normal sheep serum or potent anti-alpha/beta interferon antiserum, inoculated with the immunomodulators, and infected 24 hours later with virus. Three patterns emerged. The antiviral action of the pyrimidinone (ABMP) and the oral interferon inducer (CL246,738) appeared to be mediated primarily by interferon alpha/beta; their protective ability was almost completely abrogated by treatment with low levels of anti-alpha/beta interferon antiserum. The antiviral action of two other immunomodulators, a mismatched polyribonucleotide (Ampligen) and a polyanionic copolymer (MVE-2) at least partially involved interferon. Activity of these compounds was reduced, but not consistently eliminated by treatments with high doses of antiserum. The antiviral activity of another polyribonucleotide, polyriboinosinic-cytidylic acid complexed with lysine carboxymethylcellulose (poly ICLC), was not affected by treatment with even the highest amount of antiserum (two injections of 100,000 neutralizing units each). Almost complete protection by poly ICLC was observed despite the fact that this high concentration of antiserum, when given alone, caused a decrease in natural resistance to Semliki Forest virus infection. Taken together, these results indicate that induction of interferon alpha/beta does not appear to be the major common mechanism of antiviral activity among these diverse synthetic immunomodulators.

A replication-efficient mutant of West Nile virus is insensitive to DI particle interference

A previous report described the isolation of a mutant of West Nile virus (WNV) from culture fluid obtained from persistently infected genetically resistant C3H/RV mouse cells that replicates significantly more efficiently in cultures of C3H/RV cells than does the parental virus. This replication-efficient mutant, designated RE-WNV, has now been found to be insensitive to interference by WNV defective interfering (DI) particles. This characteristic was demonstrated by several means. The RE-WNV mutant was able to superinfect persistently infected cultures that were no longer producing detectable parental virus, while the parental virus was not. Good yields of the mutant virus were produced during six serial undiluted passages of RE-WNV in both resistant C3H/RV and congenic susceptible C3H/HE cells. In contrast, during passage of parental virus in C3H/RV cells, progeny virus could not be detected after the third passage, due to an enhanced interference by WNV DI particles with standard virus replication in these cells. The RE-WNV was also insensitive to interference by a pool of parental virus enriched for DI particles. Analysis of the mutant genome by oligonucleotide fingerprinting indicated that the genome RNA of the mutant differs by two unique spots from the parental RNA. The relevance of this mutant to the eventual understanding of the mechanism by which C3H/RV and C3H/HE cells manifest their flavivirus-specific difference in the efficiency of progeny virus production is discussed.

Characterization of murine Caraparu Bunyavirus liver infection and immunomodulator-mediated antiviral protection.

A rapid, peripheral disease model utilizing the Bunyavirus, Caraparu, was established in mice for the evaluation of antiviral therapy with immunomodulators. 4-6-week-old B6C3F1 female mice, inoculated intraperitoneally with virus, developed coagulative liver necrosis and died between 4-6 days after infection. This Caraparu disease model was relatively resistant to treatment with immunomodulators, such as ABMP, Ampligen, alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta). However, a significant increase in median survival time (MST) was consistently observed upon treatment with gamma-interferon (IFN-gamma). The nucleoside analog--ribavirin--was highly effective against Caraparu virus in repeated treatment schedules begun on either day -1, day 0, or day +1 of infection. Ribavirin gave little protection when initiation of treatment was delayed until day +2. However, combined treatment with IFN-gamma, starting on day 0 and ribavirin starting on day +2, significantly reduced mortality.

The 3′ terminus of lactate dehydrogenase-eVirus genome RNA does not contain togavirus or flavivirus conserved sequences

Abstract Lactate dehydrogenase-elevating virus (LDV) is currently considered to be an unclassified togavirus. The 3′ terminus of the genome RNA of the C-strain of LDV was cloned and sequenced. A synthetic DNA oligomer complementary to the 3′ portion of this cloned sequence was then used to prime dideoxy sequencing from the LDV-C genome RNA as well as from the genome RNAs of three additional LDV isolates. A high degree of sequence conservation was observed in the 3′ terminal region among the four LDV isolates analyzed. Comparison of the LDV 3′ sequence with those of the alpha togaviruses, rubella virus, and the flaviviruses showed that the LDV genome does not contain conserved 3′ sequences characteristic of these viruses.

Detection of viral-specific nucleic acid and intracellular virions in ventral horn neurons of lactate dehydrogenase-elevating virus infected C58 mice

C58 mice which have been immunosuppressed by treatment with cyclophosphamide (200 mg/kg) one day prior to infection with the C strain of lactate dehydrogenase-elevating virus (LDV-C) develop poliomyelitis. Using in situ hybridisation, we found that some ventral horn neurons in these mice contain cytoplasmic viral-specific nucleic acid. Viral-specific nucleic acid was also found within a few small cells located near inflammatory foci. In addition, mature virus particles were observed by electron microscopy in some ventral horn neurons, indicating that these cells are productively infected in C58 mice. Neither viral nucleic acid nor virions were found in the ventral horn neurons of poliomyelitis-resistant mouse strains or C58 mice that were not immunosuppressed prior to infection. Ventral horn neurons which contained viral nucleic acid or virions within cytoplasmic vesicles generally were normal in appearance and were not located within poliomyelitis inflammatory foci. Our data are consistent with the hypothesis that infected neurons first replicate virus and subsequently are attacked and cleared by inflammatory cells.