Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors

No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.     

The expression of essential components for human influenza virus internalisation in Vero and MDCK cells

MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.     

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.     

The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors

The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. The recent transmissions of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian influenza viruses can directly infect humans without an intermediate host, such as pigs. In this study, we used two strains of influenza A virus (A/PR/8/34, which preferentially binds to an avian-type receptor, and A/Memphis/1/71, which preferentially binds to a human-type receptor) to probe the receptor specificities in host cells. Epithelial cells of both quail and chicken intestines (colons) could bind both avian- and human-type viruses. Infected cultured quail colon cells expressed viral protein and allowed replication of the virus strain A/PR/8/34 or A/Memphis/1/71. To understand the molecular basis of these phenomena, we further investigated the abundance of sialic acid (Sia) linked to galactose (Gal) by the alpha2-3 linkage (Siaalpha2-3Gal) and Siaalpha2-6Gal in host cells. In glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells, there were some bound components of Sia-Gal linkage-specific lectins, Maackia amurensis agglutinin (specific for Siaalpha2-3 Gal) and Sambucus nigra agglutinin (specific for Siaalpha2-6Gal), indicating that both Siaalpha2-3Gal and Siaalpha2-6Gal exist in quail and chicken colon cells. Furthermore, we demonstrated by fluorescence high-performance liquid chromatography (HPLC) analysis that 5-N-acetylneuraminic acid was the main molecular species of Sia, and we demonstrated by multi-dimensional HPLC mapping and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis that bi-antennary complex-type glycans alpha2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues in both the chicken and quail. Taken together, these results indicate that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

Evolving complexities of influenza virus and its receptors

Sialic acids (Sias) are regarded as receptors for influenza viruses and are usually bound to galactose (Gal) in an alpha2-3 or alpha2-6 configuration. The detection of these Sia configurations in tissues has commonly been through the use of plant lectins that are able to identify which cells contain Siaalpha2-3- and Siaalpha2-6-linked glycans, although other techniques for receptor distribution have been used. Initial experiments indicated that avian versus human influenza virus binding was determined by either Siaalpha2-6 or Siaalpha2-3 expression. In this review, we suggest that the distribution and detection of these terminal Siaalpha2-3- and Siaalpha2-6-linked receptors within the respiratory tract might not be as clear cut as has been reported. We will also review how other viral and receptor components might act as determinants for successful viral replication and transmission. Understanding these additional components is important in comprehending the infection and the transmission of both existing human influenza viruses and newly emerging avian influenza viruses.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.     

Low growth ability of recent influenza clinical isolates in MDCK cells is due to their low receptor binding affinities

Madin Darby canine kidney (MDCK) cells have generally been used to isolate influenza viruses from patients. However, in recent years, most fresh isolates of the H3N2 subtype have shown poor growth in MDCK cell cultures. Such low-growth viruses were often converted to high-growth viruses after several passages through MDCK cell cultures. In the present study, viruses were found to lose a potential glycosylation site near the receptor-binding pocket of hemagglutinin (HA), at the same time as they acquired the high-growth property. The growth curves of viruses in MDCK cell cultures revealed that multi-cycle replication did not function well in the low-growth viruses. However, the production of progeny viruses within a single cycle of growth did not differ much between the low- and high-growth viruses. The high-growth viruses showed higher infection efficiency in MDCK cell cultures than the low-growth viruses. The HA genes of both low- and high-growth viruses were separately cloned into the SV40 vector to compare their receptor binding affinities. The HA of high-growth viruses showed a much higher receptor binding affinity than that of low-growth viruses, when assayed by hemadsorption and the release kinetics of erythrocytes with bacterial neuraminidase. Reverse genetics studies demonstrated that HA was a crucial determinant for multi-cycle replication in MDCK cell cultures. Taken together, these results demonstrate that inefficient multi-cycle growth of fresh isolates is due to their low receptor binding affinities.     

Comparison of Different Cell Substrates on the Measurement of Human Influenza Virus Neutralizing Antibodies

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log₂ lower) or superior (>1 log₂ higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.     

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.     

Glycomic Analysis of Human Respiratory Tract Tissues and Correlation with Influenza Virus Infection

The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.     

Specific biochemical features of replication of clinical influenza viruses in human intestinal cell culture

Influenza A viruses isolated from the respiratory tract of patients with influenza were cultured in human intestinal epithelium cells (CACO-2 line). The CACO-2 cells were found to be 100-fold more susceptible to the clinical viruses than MDCK cells and chicken embryos. On passaging in CACO-2 cells, clinical isolates of the subtype H3N2 retained the original "human" phenotype and agglutinated human but not chicken erythrocytes, whereas on passaging in MDCK cells the virus phenotype changed to the "avian" one. On comparison with laboratory strains (grown in chicken embryos or MDCK cells), the clinical viruses were characterized by higher stability of the anti-interferon protein NS1 but had a reduced synthesis of the matrix protein M1, and this could facilitate the virus adaptation and escape of the infected cells from immune attack in the human body. The increased tropism to the human CACO-2 cells correlated with higher adsorption of the clinical viruses on cellular receptors. However, in the CACO-2 and MDCK cells the ratio of sialyl-containing glycoreceptors of the 2-3 and 2-6 type was similar. These observations indicated that not only sialic acid residues were involved in the adsorption and penetration of the clinical viruses into human cells, but also the protein moiety of the cellular receptor itself and/or an additional cellular coreceptor. Thus, clinical influenza viruses are shown to possess a specific mechanism of sorption and entry into human epithelial cells, which is responsible for their higher tropism to human cells and is unlike such a mechanism in canine cells.     

流感病毒感染诱导MDCK细胞凋亡的研究

用荧光染色、DNA凝胶电泳等方法检测了A型流感病毒株A1／京防86－1和B型流感病毒株B/沪防93－1诱导狗肾体代细胞(MDCKcells)的凋亡情况,并采用MTT法和流式细胞仪比较了这2株病毒对MMCK细胞的毒力和凋亡诱导能力水平。结果显示：病毒感染6h后,细胞DNA发生断裂,病毒感染12h后,可见明显的染色质凝聚;在一定范围内,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。     

High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.     

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells

Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells.     

Human-specific regulation of alpha 2-6-linked sialic acids

Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids (Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch from alpha 2-3 linkage preference during adaptation from animals to humans. This correlates with the expression of alpha 2-6-linked Sias on ciliated human airway epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins, which are unable to inhibit virus binding. Given several known differences in Sia biology between humans and apes, we asked whether this pattern of airway epithelial Sia linkages is also human-specific. Indeed, we show that since the last common ancestor with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia expression between airway epithelial cell surfaces and secreted mucins. This can explain why the chimpanzee appears relatively resistant to experimental infection with human Influenza viruses. Other tissues showed additional examples of human-specific increases or decreases in alpha 2-6-linked Sia expression and only one example of a change specific to certain great apes. Furthermore, while human and great ape leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression during human evolution represent another example of a human-specific change in Sia biology. Because the data set involves multiple great apes, we can also conclude that Sia linkage expression patterns can be conserved during millions of years of evolution within some vertebrate taxa while undergoing sudden major changes in other closely related ones.     

Properties of reassortants of human and avian influenza viruses. Reproduction in MDCK cells at suboptimum temperatures

A series of reassortants has been constructed by crossing of UV-inactivated avian influenza virus of H3N8 subtype and live human influenza virus of H1N1 subtype, adapted to growth in continuous canine kidney cell line (MDCK). The analysis of RNA duplexes has shown that the reassortants contain HA gene of avian influenza virus whereas the other genes belong to human parent virus. The reassortants were efficiently reproduced in MDCK cells at low temperature (limiting for the avian parent virus). The data suggest that the avian virus HA gene does not hamper the reproduction of reassortant viruses in mammalian cells under the conditions unfavorable for the multiplication of avian influenza subtype H3N8 viruses.