Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is de- sirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bron- chial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive im- mune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live at- tenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of vi- ruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Cleavage of Influenza A Virus Hemagglutinin in Human Respiratory Epithelium Is Cell Associated and Sensitive to Exogenous Antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 (“cleavage from within”) and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions (“cleavage from without”), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases

An improved reverse genetics system for mammalian orthoreoviruses

AbstractMammalian orthoreoviruses (reoviruses) are highly useful models for studies of double-stranded RNA virus replication and pathogenesis. We previously developed a strategy to recover prototype reovirus strain T3D from cloned cDNAs transfected into murine L929 fibroblast cells. Here, we report the development of a second-generation reovirus reverse genetics system featuring several major improvements: (1) the capacity to rescue prototype reovirus strain T1L, (2) reduction of required plasmids from 10 to 4, and (3) isolation of recombinant viruses following transfection of baby hamster kidney cells engineered to express bacteriophage T7 RNA polymerase. The efficiency of virus rescue using the 4-plasmid strategy was substantially increased in comparison to the original 10-plasmid system. We observed full compatibility of T1L and T3D rescue vectors when intermixed to produce a panel of T1L×T3D monoreassortant viruses. Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use

Detection of Viruses in Human Adenoid Tissues by Use of Multiplex PCR▿

By PCR, we detected a high frequency of viruses in adenoids obtained from children without acute respiratory symptoms. Our results suggest that persistent/latent viral infection in the respiratory tract confounds interpretation of the association of pathogen detection by PCR with acute respiratory infection in these sources

Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

El objetivo es presentar categorías de la Teoría Histórico-Cultural de la Actividad como caja de herramientas para construir indagaciones e intervenciones vinculadas al trabajo de Psicólogos y otros Agentes en escenarios educativos. En el marco de problemas cruciales de agenda psicoeducativa, como el de las unidades de análisis, se introduce la Teoría de la Actividad en su primera, segunda y tercera generación y “más allá”, como Engestrom caracterizó su desarrollo histórico. Se presentan similaridades y diferencias con conceptualizaciones de autores de perspectivas socio-cultural e histórico–cultural, originadas en el pensamiento de Vygotsky, como Cole, Rogoff, Wertsch, Chaiklin, Daniels y Edwards, tanto en relación a la Psicología del Desarrollo como al giro relacional en la experticia. Se despliega la Teoría como artefacto mediador para construir investigaciones psicoeducativas en curso sobre violencias en escuelas desde la perspectiva de los actores y participación de psicólogos en configuraciones de apoyo entre Educación Especial y Escuela Común. Finalmente, conceptualizaciones y figuras diseñadas por Engestrom y Yamazumi, en la post-generación de la Teoría, permiten analizar procesos de vinculación e intercambio entre Universidad y Escuelas para la inclusión y calidad educativas, en los que participan psicólogos en formación y formadores de la Universidad Nacional de La Plata.The aim is to present categories of Cultural-Historic-Activity-Theory as a tools cage for the building of research and interventions related to Psychologists and other Agents´ work in educational stages. In the frame of central problems of the psycho-educational agenda, as the units of analysis, the Activity Theory is introduced in its first, second and third generation and beyond, as Engestrom depicted its historical development. Similarities and differences are presented between Activity Theory and conceptualizations of different authors of socio-cultural and cultural-historic perspectives, both founded on Vygotsky thought, linked to Development Psychology and to the relational shift in expertice, as Cole, Rogoff, Wertsch, Chaiklin, Daniels and Edwards. The Activity Theory is displayed as a mediating artifact for building current psycho-educational research about violences at school from the perspective of social actors and about psychologists and other agents´ participation in supportive configurations between Special Education and Common Schools. At last, figures and conceptualizations drawn by Engestrom and Yamazumi in the post-generation of the Theory, allow to analyse processes of linking and exchange between University and Schools for educational enhancement and inclusion, in which psychologists in modeling and tutors of La Plata National University are participating.Facultad de Psicologí