High Hemagglutinin Research Articles

Acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology.

Membrane fusion is not spontaneous. Therefore, enveloped viruses have evolved membrane-fusion mediating glycoproteins that, once activated, refold, and release energy that fuses viral and cellular membranes. The influenza A virus hemagglutinin (HA) protein is a prototypic structural class I viral fusion glycoprotein that, once primed by proteolytic cleavage, is activated by endosomal low pH to form a fusogenic “leash-in-grooves” hairpin structure. Low-pH induced HA protein refolding is an irreversible process, so acid exposure in the absence of a target membrane leads to virus inactivation. The HA proteins of diverse influenza virus subtypes isolated from a variety of species differ in their acid stabilities, or pH values at which irreversible HA protein conformational changes are triggered. Recently, efficient replication of highly pathogenic avian influenza (HPAI) viruses such as H5N1 in avian species has been associated with a relatively high HA activation pH. In contrast, a decrease in H5N1 HA activation pH has been shown to enhance replication and airborne transmission in mammals. Mutations that alter the acid stabilities of H1 and H3 HA proteins have also been discovered that influence the amantadine susceptibilities, replication rates, and pathogenicities of human influenza viruses. An understanding of the role of HA acid stability in influenza virus biology is expected to aid in identifying emerging viruses with increased pandemic potential and assist in developing live attenuated virus vaccines. Acid-induced HA protein activation, which has provided a paradigm for protein-mediated membrane fusion, is now identified as a novel determinant of influenza virus biology.

Improving influenza virus backbones by including terminal regions of MDCK-adapted strains on hemagglutinin and neuraminidase gene segments

Reverse genetics approaches can simplify and accelerate the process of vaccine manufacturing by combining the desired genome segments encoding the surface glycoproteins from influenza strains with genome segments (backbone segments) encoding internal and non-structural proteins from high-growth strains. We have developed three optimized high-growth backbones for use in producing vaccine seed viruses for group A influenza strains. Here we show that we can further enhance the productivity of our three optimized backbones by using chimeric hemagglutinin (HA) and neuraminidase (NA) genome segments containing terminal regions (non-coding regions (NCRs) and coding regions for the signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) from two MDCK-adapted high growth strains (PR8x and Hes) and the sequences encoding the ectodomains of the A/Brisbane/10/2010 (H1N1) HA and NA proteins. Viruses in which both the HA and NA genome segments had the high-growth terminal regions produced higher HA yields than viruses that contained one WT and one chimeric HA or NA genome segment. Studies on our best-performing backbone indicated that the increases in HA yield were also reflected in an increase in HA content in partially purified preparations. Our results show that the use of chimeric HA and NA segments with high-growth backbones is a viable strategy that could improve influenza vaccine manufacturing. Possible mechanisms for the enhancement of HA yield are discussed.

A Hemagglutinin from Northeast Red Beans with Immunomodulatory Activity and Anti-proliferative and Apoptosis-inducing Activities Toward Tumor Cells

A 64-kDa hemagglutinin from a Phaseolus vulgaris cultivar, the northeast red bean, was purified by a protocol composed of three chromatographic steps involving affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The purified hemagglutinin appeared as a single 32-kDa band in SDS-PAGE indicating its dimeric nature. The N-terminal amino acid sequence of the hemagglutinin resembled the sequences of lectins and hemagglutinins from a number of Phaseolus species. The hemagglutinin manifested moderate thermostability and pH stability. It retained full activity up to 65 °C and in the pH range 2-12. It did not interact with simple sugars such as glucose, mannose and galactose. The hemagglutinin exerted immunostimulatory effects by upregulating the expression of cytokines like interferon-γ and tumor necrosis factor-α. It also exhibited antiproliferative activity on a number of tumor cells including MCF7 (breast cancer), HepG2 (liver cancer), CNE1 and CNE2 (nasopharyngeal cancer) cells, with stronger activity toward MCF7 and CNE1 cells. The hemagglutinin induced phophatidylserine externalization, mitochondrial depolarization and DNA condensation in MCF7 cells, indicating initiation of apoptosis. However, at high hemagglutinin concentrations, severe damage to the MCF7 cells was detected.

Assessment of viral replication in eggs and HA protein yield of pre-pandemic H5N1 candidate vaccine viruses

H5N1 infection and the potential for spread from human to human continue to pose a severe public health concern. Since vaccination remains the most effective way to prevent a potential H5N1 pandemic, the World Health Organization (WHO) Collaborating Centers (CCs) and Essential Regulatory Laboratories (ERLs) engineered and developed a panel of H5N1 pre-pandemic vaccine viruses for pandemic vaccine preparedness as well as production of antigen potency testing reagents (reference antigen and reference anti-serum) for vaccine standardization. To develop a strategy utilizing a number of biochemical methods for the characterization of the viral growth properties and protein yield in eggs, we have selected eight H5N1 pre-pandemic viruses and determined the viral Egg Infectious Dose 50 (EID50), total protein yield, hemagglutinin (HA) to nucleoprotein (NP) ratios (HA:NP), and HA1 content of each virus. Our results showed that all the tested H5N1 vaccine viruses grew to high titers in eggs. The total viral protein yield varies within a narrow range, whereas there were greater differences in the HA:NP protein ratios among the eight viruses. The RP-HPLC based HA1 content analysis demonstrated that the viruses A/Anhui/1/2010, A/Hubei/1/2005, and A/goose/Guiyang/337/2006 contained higher HA contents than other five viruses including A/Vietnam/1203/2003. Our approach for analyzing virus growth and protein yield will allow us identify optimal vaccine virus in a timely manner. In addition, we successfully purified the HA proteins of H5N1 vaccine viruses by optimizing bromelain cleavage conditions. Our studies on the HA protein purification may improve the quality control of the production of influenza vaccine test reagent.

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

Higher Antigen Content Improves the Immune Response to 2009 H1N1 Influenza Vaccine in HIV-Infected Adults: A Randomized Clinical Trial

The immunogenicity of a high hemagglutinin (HA) dose or a second dose of influenza vaccine in human immunodeficiency virus (HIV)-infected individuals has not been fully explored. One hundered ninety-two HIV-infected individuals aged 18-64 years were stratified by CD4 cell count (<200 cells/mL or ≥200 cells/mL) and randomized to receive 2 doses of 15 μg or 30 μg HA 2009 H1N1 vaccine 21 days apart. Hemagglutination inhibition (HAI) and microneutralization (MN) antibodies were measured on days 0, 10, 21, 31, 42, and 201. Recipients of 30 μg HA had significantly higher HAI geometric mean titers (GMTs), compared with recipients of 15 μg HA on days 10 (139.0 vs 51.9; P = .01), 21 (106.7 vs 51.9; P = .001), and 31 (130.0 vs 73.7; P = .03) but not on days 42 (91.8 vs 61.6; P = .11) and 201 (43.0 vs 27.0; P = .08). When analyzed by CD4 cell count stratum, HAI GMTs were significantly higher among 30 μg HA recipients than among 15 μg HA in the CD4 cell count <200 cells/mL stratum on days 21 and 31 and the MN GMTs on days 10, 21, 31, and 42 (P < .05). In the CD4 cell count ≥200 cells/mL stratum, MN GMTs were significantly higher among recipients of 30 μg HA than among recipients of 15 μg HA on day 10 (P = .03). Increasing the HA dose of the 2009 H1N1 vaccine improves the vaccine's immunogenicity in HIV-infected individuals. NCT00992433.

Comparative immunogenicity of recombinant adenovirus-vectored vaccines expressing different forms of hemagglutinin (HA) proteins from the H5 serotype of influenza A viruses in mice

Recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 viruses in poultry and their subsequent transmission to humans have highlighted an urgent need to develop preventive vaccines in the event of a pandemic. In this paper we constructed recombinant adenovirus (rAd)-vectored influenza vaccines expressing different forms of H5 hemagglutinin (HA) from the A/Vietnam/1194/04 (VN/1194/04) virus, a wild-type HA, a sequence codon-optimized HA and a transmembrane (TM) domain-truncated HA. Compared to the rAd vectors expressing the wild-type HA (rAd-04wtHA) and the TM-truncated form of HA (rAd-04optHA-dTM), the rAd vectored vaccine with the sequence codon-optimized HA (rAd-04optHA) showed a tendency to induce much higher hemagglutinin inhibition (HI) antibody titers in mice immunized with a prime-boost vaccine. Furthermore, administration of the rAd-04optHA vaccine to mice could elicit cross-reactive immune responses against the antigenically distinct HK/482/97 virus. Additionally, we constructed another vector containing the codon-optimized HA of the A/Hong Kong/482/97 (HK/482/97) virus. Administration of a bivalent immunization formulation including the rAd-04optHA and rAd-97optHA vaccines to mice induced a stronger immune response against HK/482/97 virus than the monovalent formulation. Taken together, these findings may have some implications for the development of rAd-vectored vaccines in the event of the pandemic spread of HPAI.

Differences in Lateral Membrane Organization in Fibroblasts Expressing Low and High Levels of the Influenza Viral Protein Hemagglutinin

Lateral organization in cell membranes is crucial for biological processes such as endocytosis, signaling, protein transport, membrane trafficking and viral infection. Hemagglutinin (HA) is an influenza viral envelope transmembrane protein which has been shown to be associated with the liquid ordered (lo) phase. Fibroblasts that constitutively express HA, referred to as HAb2 cells, were used to characterize the lateral organization of HA. Since HAb2 cells have cell-to-cell variability in the membrane density of HA, cells with low and high expression levels of HA containing more consistent densities of HA were also used. Fluorescence correlation spectroscopy (FCS), confocal microscopy and fluorescence photoactivation localization microscopy (FPALM) were used to characterize membrane organization after labeling cells with fluorescent probes and/or transfecting with either EGFP-HA or Dendra2-HA. Preliminary FCS results show that the diffusion of the liquid-disordered (ld) phase probe Lissamine Rhodamine DOPE is similar in cells with high and low HA expression levels i.e. the amount of HA present does not influence the diffusion time. Confocal microscopy was used to study the effect of HA expression level on the extent of phase separation observed after blebbing was induced by DMSO treatment. FPALM was used to obtain details about membrane organization at the nanometer length scale.

Using gamma‐cytokine complexes to improve antigen specific CD8 T cell responses in tumor‐bearing mice

In the absence of inflammatory signals, the exposure of T cells to tumor antigen in the periphery results in tolerance. Remaining tumor specific T cells are usually of low avidity or anergic. Immunotherapy aims at reversing this tolerance to induce an efficient T cell response. STAT5 Cytokines of the interleukin‐2 family signal through STAT5 and are candidates to increase T cell anti‐tumor response. The effects of these cytokines are greatly increased when the cytokine is complexed with a specific antibody.We have studied the potential of cytokine complexes to serve as adjuvants for CD8 cells activation in a murine model, RIP‐Tag2‐HA, in which pancreatic insulomas expressing the influenza hemagglutinin (HA) as a surrogate antigen develop spontaneously. HA specific CD8 cells become effector after 3 days of treatment with IL‐2 complexes and antigen in vivo. This differentiation leads to the elimination of endogenous tumors by high avidity HA specific CD8 cells. Survival of CD8 cells is increased by using the TLR ligands polyIC or by extending the length of treatment with IL‐2 complexes. Under such conditions, even low avidity HA specific CD8 cells eliminate the tumors. We further show that cytokine complexes also increase survival of adoptively transferred, ex‐vivo activated HA specific CD8 cells. These results indicate that IL‐2 complexes are strong adjuvants that greatly enhance tumor vaccines or immunotherapy.

Higher polymerase activity of a human influenza virus enhances activation of the hemagglutinin-induced Raf/MEK/ERK signal cascade

Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production.

Baculovirus-expressed influenza vaccine

Effectiveness of the influenza vaccine in persons with high-risk conditions needs to be improved. In this paper, the authors review various vaccination strategies, including repeated doses of the vaccine or the use of higher hemagglutinin (HA) content vaccines that have been shown to result in improved immunogenicity. A recombinant HA vaccine produced in insect cells using the baculovirus vectors system presents the possibility for safe and expeditious vaccine production. The high purity of the antigen enables administration at much higher doses without a significant increase in side effects in human subjects. An overview of the use of this production system for the development of alternative influenza vaccine targets is also provided, such as neuraminidase and possibly M2. However, the role of M2 may be more appropriate as an adjuvant vaccine in combination with standard HA vaccine supplement and needs further evaluation. The conclusion that the insect cell–baculovirus production technology is a modern solution for rapid viral or parasitic antigen production is made and that this technology is particularly suitable for influenza where annual adjustment of the vaccine is required. In addition, a highly purified recombinant protein vaccine results in an improved influenza vaccine response in those with high-risk medical conditions.

Functional interactions of the influenza virus glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.

Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation.

Respiratory tract infections with viruses and Pasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 x 10(3) to 1.2 x 10(7) PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 x 10(5) and 2.3 x 10(9) per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV and Pasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.

Interleukin-7-enhanced cytotoxic T lymphocyte activity after viral infection in marrow transplanted mice.

Lethally irradiated BALB/c mice were reconstituted by syngeneic bone marrow transplantation (BMT), and injected with recombinant interleukin 7 (rIL-7), recombinant interleukin 2 (rIL-2), or saline 10 days post-transplantation. Intranasal infection with A/PR8/34 influenza virus 2 weeks after BMT was associated with the highest survival rate in the rIL-7 treated group. The protective mechanism elicited by rIL-7, as manifested by very low virus titers in the lung, involves T and B cell functions. High hemagglutinin inhibition antibody levels were observed on days 7 and 12 post-challenge in the rIL-7 mice. Moreover, the anti-influenza cytotoxic T lymphocyte activity was induced primarily by rIL-7, leaving the effect of rIL-2 on the same level as that of the control. Thus, rIL-7 promotes both T cell-mediated function and B cell production during the immunodeficient state after BMT. This cytokine may prove a potential immunotherapeutic modality in BMT recipients.

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

Molecular Changes in virulent mutants arising from avirulent avian influenza viruses during Replication in 14-day-old embryonated eggs

The emergence of virulent avian influenza viruses in poultry is unpredictable. To gain insight into the mechanism for thisevent, we sought to identify the molecular changes in virulent mutants that occur during replication in 14-day-old embryonated chicken eggs. After three passages in 14-day-old eggs, avirulent H5 viruses with the K/R-K-K/T-R sequence at the hemagglutinin (HA) cleavage site became virulent in chickens, concomitantly acquiring high HA cleavability, whereas those with the R-E-T-R sequence did not. None of the test viruses converted to a virulent phenotype when passaged in 10-day-old eggs. Nucleotide sequence analysis indicated that the virulent mutants either lost a glycosylation site near the HA cleavage site or acquired an additional arginine at the latter. Avirulent viruses that became virulent after passage in older eggs included an H5N2 avian strain with the R-K-T-R sequence that was isolated in 1993, indicating that viruses with this sequence motif, which are currently circulating in bird populations, should be considered potentially virulent. Failure to generate virulent mutants from viruses with R-E-T-R at the HA cleavage site underscores the pathogenic heterogeneity among avian influenza viruses.

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.

Cytotoxic effect of two legumes in epithelial cells of the small intestine

The previously known high toxicity and hemagglutinin concentration found in Escumite bean (Phaseolous acutifolius), widely consumed in the south-east of Mexico, induced us to study the cytotoxic effect of these beans on the epithelial cells of the small intestine of rats. Another, less toxic leguminosae, chick-pea (Cicer arietinum), was used for comparison. In vitro tests showed that the viability of the cells was much more affected by the Escumite bean extract than by the chick-pea extract (P<0.001). The cellularity was much diminished when the epithelial cells were treated with Escumite extract (P<0.001), but not with chick-pea extract. In vivo experiments confirmed these findings. The present results suggest that the deleterious effect of some toxicants present in the raw legume is due to an interaction with intestinal epithelium, which, in turn, may cause a reduced absorption of nutrients.

PFC formation utilizing adoptive transfer of lymphoid cells in embryonic and baby chick hosts

Embryos and newly hatched chicks appear unresponsive to mouse erythrocyte (MRBC) immunization as measured with the hemagglutinin (HA) and plaque-forming cell (PFC) assays. The competence for PFC formation first detected in the two day chick increased progressively with age. After grafting appropriate lymphoid cells into chick embryos or newly hatched chicks, PFC were observed consistently in the host spleens. Among embryo hosts grafted with blood leucocytes, spleen cells, thymus cells or bone marrow cells from antigen-primed donors, high PFC counts were obtained only in those that received either leucocytes or spleen cells. However, when the hosts were allowed to complete development and immunized with MRBC at hatching, moderate to high splenic PFC counts and HA production occurred in all groups, including those that received thymus or bone marrow cells. The data suggested that the immunologic immaturity may be due not to an inadequacy of the embryonic splenic microenvironment but to the deficiency of immunocompetent cells, particularly the thymus-derived helper cells.

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.