Jean Lindenmann

Mx protein: Constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.

Inheritance of Resistance to Influenza Virus in Mice.

Summary F1, F2, first, second, and third backcross generation mice resulting from crosses of A2G with A, and F1 F2 and first backcross generation mice resulting from crosses of A2G with C3H were challenged with neurotropic influenza A virus, strain NWS. The results were compatible with the hypothesis that a single dominant autosomal allele was responsible for resistance to the virus. The symbol Mx (myxovirus-resistant) is tentatively proposed for the allele involved.

Studies on vaccinia virus plaque formation and its inhibition by interferon: III. A simplified plaque inhibition assay of interferon☆

Abstract A plaque inhibition assay of interferon using vaccinia virus as challenge has been developed. No adsorption period of either interferon or virus was required; instead, both interferon and virus were added simultaneously to chick embryo cell monolayers in bottles without an agar overlay. After 44–48 hours of incubation at 37 ° the monolayers were stained with crystal violet and the plaques were counted. The dose-response relationship remained unaffected by variations in the amount of challenge virus between 50 and 19,000 PFU per monolayer. When the percentage plaque inhibition was plotted against the logarithm of the dose, different interferon preparations yielded parallel curves. The dose-response curves were sigmoidal and could be subjected to probit analysis. The method could be adapted to other tissues and other inhibitors.

Studies on vaccinia virus plaque formation and its inhibition by interferon. I. Dynamics of plaque formation by vaccinia virus.

Abstract Plaques of vaccinia virus in chick embryo cell monolayers without agar emerged into visibility according to a normal sigmoid curve. The distribution of plaque diameters could be approximated by a normal Gaussian curve, with a variable proportion of plaques hidden below the threshold of visibility (infraplaques). Between 32 and 48 hours, and possibly up to 57 hours after inoculation, all plaque diameters increased linearly with time at the same rate. The total plaque area increased between 32 and 48 hours at an almost exponential rate. Strains of vaccinia virus differ in the rate of appearance of primary and secondary plaques.

Studies on vaccinia virus plaque formation and its inhibition by interferon. II. Dynamics of plaque formation by vaccinia virus in the presence of interferon.

Abstract The effect of interferon on the dynamics of vaccinia virus plaque formation in chick embryo cell monolayers was investigated. Preincubation of cells with interferon only slightly affected inhibition as compared to simultaneous addition of interferon and challenge virus. Within limits, a linear relationship was found when the following parameters were plotted against log interferon concentration: (a) Plaque counts; (b) average plaque diameters; (c) logarithm of total plaque area. Plaques in interferon-treated cultures emerged at a slower rate into visibility and took longer to reach a plateau in numbers than did controls. As a consequence, the amount of inhibition as estimated from plaque counts decreased after primary control plaques had reached their maximum number. The growth rate of treated plaques was reduced but remained independent of plaque size or time. Some of the infected cells seemed to be permanently prevented from yielding plaques.

Further studies on the resistance of mice to myxoviruses.

Mice from 16 inbred strains were challenged with neurotropic influenza A virus, strain WSA, in an effort to secure other examples of the occurrence of the genemx. All strains, with the exception of Z/Gw, were fully susceptible. Z/Gw mice showed some degree of resistance probably unrelated tomx. A2G mice were challenged with 12 different virus strains. All influenza viruses used (3 strains of fowl plague, MEL, FM-1, A2/Ann Arbor/2/60 and B/Ann Arbor/3/62) were definitely less virulent for A2G than for control (C3H, A, ICR) mice. Two strains of Newcastle disease virus, and one strain each of rabies, vesicular stomatitis and encephalomyocarditis viruses were of similar virulence for A2G and control mice. The possible bearing of these findings on the epidemiology of influenza is discussed.

Host gene influence on interferon action in adult mouse hepatocytes: specificity for influenza virus.

Abstract Primary monolayer cultures of hepatocytes isolated from adult mice either genetically resistant (bearing the autosomal dominant allele Mx ) or genetically susceptible toward influenza virus infection were tested for susceptibility to three viruses: a hepatotropic influenza A virus, vesicular stomatitis virus, and herpes simplex type I virus. Hepatocytes of both genotypes spontaneously released equal amounts of interferon during the first day of culture. Spontaneous interferon was sufficient to protect the cells markedly against vesicular stomatitis virus and marginally against herpesvirus infection. With respect to influenza virus, only Mx -bearing hepatocytes lost their initial susceptibility. Treatment with anti-interferon antibodies during the first 24 hr of culture rendered Mx -bearing cells as permissive for all three viruses as non- Mx -bearing cells. In such anti-interferon-treated cultures the antiviral resistance could be restored with highly purified mouse type I interferon. The antiviral state induced by exogenous interferon displayed the same characteristics as that induced by spontaneous interferon: it was highly efficient in inhibiting influenza A virus replication in Mx -bearing cells and inefficient in non- Mx -bearing cells. Inhibition of vesicular stomatitis virus or herpesvirus was independent of the Mx genotype.

Fowl plague virus adapted to human epithelial tumor cells and human myeloblasts in vitro

A strain of avian influenza A virus, originally isolated from an epidemic among turkeys, was adapted to human cells by serial passages. Four passage levels of the virus were compared with respect to their growth potential in human cell monolayers and their plaque morphology in chick embryo fibroblasts and in HeLa cells. The original virus, never passed in human cells before, was already able to replicate in human cells. Serial passages in human epithelial cells increased this ability. Further serial passages in human myeloblasts resulted in the selection of an apparently stable mutant which grew well in human malignant epithelial cells and even better in diploid human fibroblasts.

Viral oncolysis with host survival.

Summary Ehrlich ascites tumors grew equally well in ICR and inbred A2G mice. ICR mice were sensitive, A2G mice were fully resistant to WSA influenza virus given intracerebrally. When mice bearing Ehrlich ascites tumors were inoculated intraperitoneally with WSA virus, rapid oncolysis ensued. In ICR mice this invariably led to death of the animals, while around 80% of A2G mice survived. Of these, some later succumbed to slowly growing subcutaneous tumors developing at the site of primary tumor implantation. This occurred frequently when tumors were either treated early with large doses of virus, or late with small doses. With other combinations of timing and dosage, some mice developed subcutaneous tumors which later regressed. Mice surviving the primary tumor inoculation for 1 to 4 months were resistant to challenge with the same tumor.

Allotypes of anti-alloantibodies

Abstract Rat antibodies directed at the two known rat light-chain allotypes, characterizing strains DA and Lewis, were trace-labeled with 125 I. With these reagents, the allotypes of alloantibodies DA anti-Lewis and Lewis anti-DA could be determined in a sandwich technique involving rat red cells coated with alloantibody and topped with radioactive anti-allotype. When rat red cells were doubly coated with matching alloantibody and the corresponding anti-alloantibody [produced in (Lewis × DA)F 1 hybrid rats], both radioactive anti-allotype reagents were fixed to a significant extent. This suggests that the anti-alloantibody is an immunoglobulin produced by the F 1 hybrid and not a complex between the originally injected alloantibody and antigen.