Mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype

Summary Mouse hepatitis virus (MHV) S induced typical MHV spongiform lesions in brainstem 28 days following intranasal inoculation of adult A/J, BALB/cByJ, CBA/J, C3H/HeJ and C3H/RV, but not SJL mice. In all but SJL mice, brain lesions occurred at or near the infectious dose level, based on seroconversion by the indirect immunofluorescence assay. During the acute phase of infection (day 5), lesions were limited to the nose and brain in most genotypes. Exceptions were BALB mice, which had mild hepatitis and SJL mice, which had lesions restricted to the nose. No mortality occurred in any genotype. Following intranasal inoculation of adult mice, MHV-1, -3, -A 59,-JHM and -S all caused brain lesions at 28 days after inoculation. MHV-1 and-3 caused lesions that were usually restricted to the anterior olfactory tracts, while MHV-A 59, -S and -JHM also caused more generalized and pronounced lesions involving the midbrain and pons. These studies suggest that avirulent MHV-S given intranasally to most mouse genotypes is a good model for induction of brain infection in the absence of mortality. They also confirm observations made by others in which MHV-JHM, -S and -A 59 are relatively more neurotropic than other MHV strains, such as MHV-1 and -3.With 2 Figures     

Characterization of host cell binding variants of influenza virus by monoclonal antibodies

We have previously shown that a plaque-type mutant of influenza virus A/WSN has a growth advantage in MDBK cells because its hemagglutinin (HA) has a greater affinity for host cell receptors than does the HA of the parent virus. We show here that the mutant is also less sensitive than the parent to neutralization by antibodies to epitopes in at least two regions on the HA. WSN-specific monoclonal antibodies which had higher radioimmunoassay (RIA) titers against the parent than the mutant virus also had higher plaque inhibition (PI) and hemagglutination inhibition (HI) titers. In contrast, cross-reacting antibodies bound equally well to the parent and mutant viruses as judged by RIA but those which bound to the Cb region of the HA exhibited higher PI and HI titers against the parent virus. The results suggest that preferential neutralization of the parental virus by antibodies can contribute to the selective advantage of mutants which have increased affinity for cellular receptors.     

Downstream processing of cell culture-derived virus particles

Manufacturing of cell culture-derived virus particles for vaccination and gene therapy is a rapidly growing field in the biopharmaceutical industry. The process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. For the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. Owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs. For efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. Here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy.     

Characterization of the host cell entry of filamentous influenza virus

Summary. Most laboratory-adapted strains of influenza virus exist as spheres of approximately 100 nm in diameter, which are well established to enter cells by endocytosis in a pH-dependent manner. However, influenza virus isolated from the lungs of infected individuals is believed to exist as predominantly filamentous particles, up to several micrometers in length. Here, we have attempted an initial characterization of the entry of purified influenza virus filaments into host cells – in comparison to more commonly studied spherical forms of the virus. We demonstrate that the internalization of filamentous influenza virus particles is delayed, relative to spherical particles, and that this delay is a result of morphological rather than strain differences. The filamentous influenza particles appear to retain their dependence on low-pH for entry, as demonstrated by a vacuolar-ATPase inhibitor, and viral trafficking to late endosomes, as demonstrated by the requirement for protein kinase C function. However, our data suggest that the endocytic uptake of the filamentous virus particles may be dynamin-independent, unlike spherical virions. Overall, these data provide a view of the entry of influenza virus in its filamentous morphology, demonstrating potential differences between the endocytosis of spherical virions in vitro and filamentous virions in vivo.     

Purification of cell culture-derived influenza virus A/Puerto Rico/8/34 by membrane-based immobilized metal affinity chromatography

The presented study focuses on the feasibility of immobilized metal affinity chromatography for purification of Madin Darby canine kidney cell culture-derived influenza virus particles. Therefore, influenza virus A/Puerto Rico/8/34 was screened for adsorption to different transition metal ions attached to iminodiacetic acid. Subsequently, capturing of the same virus strain using zinc-modified iminodiacetic acid membrane adsorbers was characterized regarding viral recoveries, host cell nucleic acid and total protein depletion as well as zinc-ion-leaching. In addition, the effect of the imidazole proton pump on virus stability was studied based on the hemagglutination activity. During adsorption in the presence of 1 M sodium chloride the majority of virus particles were recovered in the product (64% hemagglutination activity). Host cell nucleic acid and total protein content were reduced to approximately 7 and 26%, respectively. This inexpensive and rapid method was applied reproducibly for influenza virus A/Puerto Rico/8/34 preparations on the laboratory scale. However, preliminary results with other virus strains indicated clearly a strong strain dependency for viral adsorption.     

Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: host cell activation of serum plasminogen

Cleavage of the HA polypeptide, the largest glycoprotein of the WSN strain of influenza virus, to polypeptides HA1 plus HA2 occurs when influenza virus is grown in MDBK cells in medium containing 2% calf serum, but does not occur in serum-free medium. Addition of highly purified plasminogen in concentrations as low as 1.5 μg/ml to serum-free medium results in complete cleavage. The release of plasminogen activators by the cell is essential for the activation of the zymogen and cleavage of the HA polypeptide. Either chick or calf plasminogen can be converted to the active enzyme, plasmin, by activators produced by either infected or uninfected MDBK cells. Cleavage of the HA polypeptide by plasmin can be prevented by trypsin inhibitors from bovine pancreas (Kunitz) or soybean. Cleavage does not occur in the presence of 2% calf serum from which plasminogen has been removed specifically by affinity chromatography, indicating that plasminogen is the only serum component involved in the conversion of the HA polypeptide to HA1 plus HA2.The results obtained indicate that in the MDBK cell-WSN strain system, cleavage of the HA polypeptide can be explained by the activation of serum plasminogen by cellular activators, and that cleavage of the influenza virus hemagglutinin polypeptide is a sensitive indicator of the production of plasminogen activators by cells.     

Influence of the host cell on proteins synthesized by different strains of influenza virus.

The synthesis of influenza virus proteins has been studied quantitatively in nine different virus-cell systems, using three strains and three cell lines. The systems were remarkable for their variation. The only common properties to emerge were that NS₁ and NP were always synthesized earlier than M and HA₀ and secondly, with the exception of the P proteins, proteins were synthesized in amounts proportional to their time of appearance, i.e., the first protein to appear was present in the greatest amount. Otherwise variations were found in the order of appearance of newly synthesized proteins, in the relative amounts synthesized, and in the total synthesized. These variations were unrelated to virus strain or cell type, to the yield of infectious virus, or to its infectivity: HA ratio, and to the shut-off of host protein synthesis. We conclude that the pattern of protein synthesis in a particular virus-cell system is unique.     

Replication of influenza virus in chick embryo fibroblasts after inhibition of host cell macromolecular synthesis by frog virus 3

Influenza viruses replicated in chick embryo fibroblast cell cultures after inhibition of cellular macromolecular synthesis by heat-inactivated FV 3. Evidence is presented for the usefulness of suppression of host cell biosynthesis by FV 3 in unmasking influenza virus-specific macromolecular syntheses.     

Neutral red-labeled influenza virus loses photosensitivity during adsorption to host cells but not to erythrocytes

Summary Neutral red (NR)-labeled influenza virus is extremely photosensitive. Unlike NR-labeled picornaviruses which lose their photosensitivity only after penetrating the host cell, NR-labeled influenza virus loses most of its photosensitivity during adsorption at 4°C. We demonstrate that the underlying reaction occurs within seconds of adsorption and that it is irreversible, i.e., NR virus eluted from chick embryo cells after adsorption is hardly photosensitive anymore. In contrast to this, NR virus adsorbed to and eluted from erythrocytes retains its original photosensitivity. We suggest that the loss of photosensitivity during adsorption of NR virus to host cells reflects a conformational change in the virion which is not elicited by adsorption to red blood cells.     

Detection and identification of host cell antigens in the composition of influenza B virus

Two heterogeneous antigens, Forssman antigen and an antigen similar to human group A antigen, were detected by enzyme-immunoassay in cells of normal chick embryo CAMN and MDCK culture (dog kidney). Both these antigens were associated with purified influenza B virus preparations reproduced in these cultures since the variants under study actively reacted with antibodies both to Forssman antigen and to human A antigen. Serological differences between the variants grown in different cell systems were due to the presence in their composition of the species-specific cell component typical either of chick embryo tissues or MDCK cultures, since the viruses of chicken origin contained the specific cell antigen CAMN, whereas the same viruses grown in MDCK did not contain it but had the specific activity for dog kidney cells. The virus-specific activity of the variants under study showed no significant differences in relation to host cells.     

The pathogenesis of influenza virus infections: the contributions of virus and host factors

Influenza viruses cause acute respiratory inflammation in humans and symptoms such as high fever, body aches, and fatigue. Usually these symptoms improve after several days; however, the 2009 pandemic H1N1 influenza virus [influenza A(H1N1) 2009] is more pathogenic than seasonal influenza viruses and the pathogenicity of highly pathogenic H5N1 viruses is still higher. The 1918 influenza pandemic virus caused severe pneumonia, resulting in an estimated 50 million deaths worldwide. Several virulence factors have been identified in these virus strains, but host factors are also responsible for the pathogenesis of infections caused by virulent viruses. Here, we review the contributions of both virus and host factors to the pathogenesis of these viral infections.     

Detection of influenza C virus but not influenza D virus in Scottish respiratory samples

BackgroundA newly proposed genus of influenza virus (influenza D) is associated with respiratory disease in pigs and cattle. The novel virus is most closely related to human influenza C virus and can infect ferrets but infection has not been reported in humans.ObjectivesTo ascertain if influenza D virus can be detected retrospectively in patient respiratory samples.Study design3300 human respiratory samples from Edinburgh, Scotland, covering the period 2006–2008, were screened in pools of 10 by RT-PCR using primers capable of detecting both influenza C and D viruses.ResultsInfluenza D was not detected in any sample. Influenza C was present in 6 samples (0.2%), compared with frequencies of 3.3% and 0.9% for influenza A and B viruses from RT-PCR testing of respiratory samples over the same period. Influenza C virus was detected in samples from individuals <2 years or >45 years old, with cases occurring throughout the year. Phylogenetic analysis of nearly complete sequences of all seven segments revealed the presence of multiple, reassortant lineages.ConclusionWe were unable to detect viruses related to influenza D virus in human respiratory samples. Influenza C virus was less prevalent than influenza A and B viruses, was associated with mild disease in the young (<2 years) and old (>45 years) and comprised multiple, reassortant lineages. Inclusion of influenza C virus as part of a diagnostic testing panel for respiratory infections would be of limited additional value.     

Infectivity of Chlamydia trachomatis serovar LGV but not E is dependent on host cell heparan sulfate

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity of Chlamydia trachomatis serovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody to C. trachomatis by flow cytometry. Results showed no binding of the heparan sulfate antibody to C. trachomatis serovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.     

Attenuation of a human H9N2 influenza virus in mammalian host by reassortment with an avian influenza virus

Summary. In order to develop a surrogate virus strain for production of an inactivated influenza vaccine against a human H9N2 virus, A/Hong Kong/1073/99 (HK1073: H9N2) was co-infected in embryonated chicken eggs with an apathogenic avian influenza virus, A/Duck/Czechoslovakia/56 (Dk/Cz: H4N6), for gene segment reassortment. Multiple-gene reassortants obtained were examined for replication in mammalian hosts in vitro and in vivo by infecting MDCK cells and by intranasal administration to hamsters, respectively. A 2–6 gene reassortant with both surface glycoproteins of HK1073 origin and the rest of Dk/Cz origin, HK/CZ-13, was shown to replicate poorly in the mammalian hosts both in vivo and in vitro comparing with HK1073, although this reassortant replicated as efficiently as each parental strain in embryonated eggs. No sequence difference was observed in the HA1 region between HK1073 and HK/CZ-13, indicating that the reassortant would be equivalent in its immunogenicity to the parental HK1073 strain when it is used as an inactivated vaccine. A virus strain with attenuation in mammalian hosts is preferable for production of an H9 vaccine, since it should reduce the risk of manufacturing-related infections of employees during the vaccine production. HK/CZ-13 can therefore be a surrogate strain for production of an inactivated vaccine as well as diagnostic antigens in case of a possible future pandemic caused by an HK1073-like H9 influenza virus.     

Biological properties of a hemagglutinin mutant of influenza virus selected by host cells

Chick embryo fibroblast (CEF)-grown stocks of the WSN strain of influenza A(HINI) contain two variants which were designated F and C for fuzzy and clear plaque morphology on Madin-Darby bovine kidney (MDBK) cells. During growth in MDBK cells plaque-isolated F virus was completely replaced by C virus (L. Noronha-Blob and I. T. Schulze (1976), Virology69, 314–322). The parental (F) and the mutant (C) viruses contain hemagglutinins which differ in their ability to bind to host cells. In addition, the host cells from which the purified viruses are obtained affect their binding properties. Thus, as compared to MDBK-grown F virus (F_BK), MDBK-grown C virus (C_BK) produced high amounts of mRNA and high virus yields in MDBK cells. C_BK had greater affinity for SAα2,3Gα1 and SAα2,6Ga1 linkages on derivatized human erythrocytes than did F_BK, independent of whether neuraminidase was present on the virions. C_BK was also resistant to components of calf serum which inhibited F_BK hemagglutination at 37°. As compared to F_BK, C_BK had increased ability to bind to both MDBK cells and CEF at 37° in the presence or absence of an inhibitor of neuraminidase. In addition, when cells with virus bound at 0° were transferred to 37°, C_BK remained cell associated whereas about 80% of FBK dissociated from both cells. Thus, mutation from F to C increased the ability of the virus to associate with MDBK cell receptors. Studies carried out with F and C viruses from both cells indicated that the expression of the mutation depended in part on the host cells in which the virus was grown and in part on the cells used to measure the binding properties. A model relating these observations to selection of HA variants in nature is presented.