Reiko Ohuchi

Suppressive effects of human herpesvirus-6 on thrombopoietin-inducible megakaryocytic colony formation in vitro

Two clinical observations, the association of human herpesvirus-6 (HHV-6) with delayed engraftment after stem cell transplantation and thrombocytopenia concomitant with exanthema subitum, prompted us to evaluate the suppressive effects of HHV-6 on thrombopoiesis in vitro. Different culture conditions for thrombopoietin (TPO)-inducible colonies in semi-solid matrices were examined. Using cord blood mononuclear cells as the source of haematopoietic progenitors, two types of colonies, megakaryocyte colony-forming units (CFU-Meg) and non-CFU-Meg colonies, were established. The former colonies were identified by the presence of cells with translucent cytoplasm and highly refractile cell membrane, most of which were positive for the CD41 antigen. Although the plating efficiency of both types was much higher under serum-containing conditions than under serum-free conditions, the proportion of CFU-Meg to non-CFU-Meg colonies was consistently higher under serum-free conditions. The plating efficiency of CFU-Meg colonies was doubled by adding stem cell factor to the serum-free matrix. The effects of two variants of HHV-6 (HHV-6A and 6B) and human herpesvirus-7 (HHV-7) on TPO-inducible colonies were then compared. HHV-6B inhibited both CFU-Meg and non-CFU-Meg colony formation under serum-free and serum-containing conditions. HHV-6A had similar inhibitory effects. In contrast, HHV-7 had no effect on TPO-inducible colony formation. Heat-inactivation and ultra-filtration of the virus sample completely abolished the suppressive effect. After infection of CD34(+) cells with HHV-6, the viral genome was consistently detected by in situ hybridization. These data suggest that the direct effect of HHV-6 on haematopoietic progenitors is one of the major causes of the suppression of thrombopoiesis.

Monitoring of human herpesvirus‐6 and ‐7 genomes in saliva samples of healthy adults by competitive quantitative PCR

Human herpesviruses‐6 and ‐7 (HHV‐6 and HHV‐7) are thought to be transmitted during early infancy through saliva. However, the kinetics of the virus shedding in saliva of healthy adults, from whom children are assumed to acquire the viruses, is mostly unknown. This study was conducted to determine how many copies of the genome are secreted in saliva of healthy adults and to clarify the relationship between viral DNA load and virus isolation of HHV‐6 and HHV‐7. Competitive PCR was performed using primer sets in the U42 gene of each viral genome. In saliva samples from 29 healthy adults, HHV‐6 and HHV‐7 DNA was detected in 41.4% and 89.7%, respectively. The average copy number of the HHV‐7 genome in the positive samples was higher than that of the HHV‐6 genome. Follow‐up studies of six seropositive individuals for 3 months showed that the amount of HHV‐7 DNA was constant in each individual and that “high producers” and “low producers” could be distinguished. By contrast, the amount of HHV‐6 DNA varied drastically over time in each individual. Although HHV‐6 was never isolated from the saliva of any of the six individuals during the follow‐up period, HHV‐7 was isolated from each individual several times. The amount of HHV‐7 DNA tended to be higher at the times when the virus was isolated than at the times when the virus was not isolated. These data demonstrate a striking contrast between HHV‐6 and HHV‐7 in the kinetics of genome and virus shedding. J. Med. Virol. 61:208–213, 2000.

Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation

ABSTRACT Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.

Slow Development of Measles Virus (Edmonston Strain) Infection in the Brain of Nude Mice

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 104 plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.

Mode of subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture cells. I. Detection of infectious virions from cells infected with Niigata-1 strain of SSPE virus.

By the aid of freezing and thawing, cell‐free infectious virions were detected from an apparently nonproductive Vero cell line infected with Niigata‐1 strain of subacute sclerosing panencephalitis virus. The production of infectious virions was limited in amount and such virions were detectable only during a limited period after cell subculture. The infectious virions were filtrable through a 0.65μ membrane filter and neutralized completely by an antiserum against measles virus. The virions were banded at the density of 1.132, while Edmonston strain of measles virus banded at 1.164 in potassium tartrate density gradients. Infectious virions were also released from infected Vero cells by treatment of the cells in a hypotonic solution to an amount comparable to that obtained by freezing and thawing. Infection of normal culture of Vero cells with the infectious virions readily established a virus‐cell interaction identical to that in the original infected culture from which the virions were recovered.

Control of Biological Activities of Influenza Virus Hemagglutinin by Its Carbohydrate Moiety

Influenza A and B viruses have two envelope spike proteins, hemagglutinin (HA) and neuraminidase (NA). HA mediates viral entry into cells; that is, after the binding of HA to sialoglycan on the cell surface, the virus is internalized through receptor-mediated endocytosis, and then HA induces fusion of the viral envelope with the endosomal membrane, thus allowing delivery of the viral genome into the cytoplasm. NA releases the progeny virus from the infected cell by destroying the sialoglycan viral receptor so as to help the virus spread (14, 28). Study on functions of the carbohydrate moiety of HA was originated by Nakamura and Compans (16, 17), who showed that the oligosaccharide side chains should protect the HA protein from proteolytic degradation. The most dramatic instance how the oligosaccharide of HA could affect the biological activity was reported by Kawaoka et al ( 1 1). They demonstrated that the loss of an oligosaccharide near the cleavage activation site of HA made the site easily accessible to ubiquitous cellular proteases, which made the virus pantropic. Consequently, the virus caused a serious outbreak of fowl plague in Pennsylvania in 1983. In this minireview, we will focus on other functions of the carbohydrate moiety of HA and demonstrate how the oligosaccharide side chains work to regulate the biological activities of HA and thereby help the virus adapt to various environments.

Phenotypic mixing with recombinant haemagglutinin of high cleavability mediates multi-cycle replication of human influenza virus in cell culture

When CV-1 cells expressing haemagglutinin (HA) of fowl plague virus A/FPV/34/Rostock(H7) (FPV) from an SV40-based recombinant vector were superinfected with the human influenza virus A/FM/1/47(H1N1)(FM1), phenotypically mixed progeny virus was observed. It contained cleaved FPV HA and uncleaved FM1 HA, was infectious without trypsin treatment and its infectivity was neutralizable by anti-FPV serum. When superinfection of H7 HA-expressing CV-1 cells was performed at a low multiplicity of infection, multi-cycle replication occurred. Control cells preinfected with an SV40-based recombinant not expressing FPV HA did not allow multi-cycle replication. Multi-cycle replication of FM1 virus was also observed when cells were preinfected with a vector expressing a highly cleavable mutant of influenza virus A/Port Chalmers/1/73(H3) HA carrying an insert of four arginine residues at the cleavage site. This was not the case when cells expressing uncleaved wild-type H3 HA were used. The results show that by phenotypic mixing with recombinant HA of high cleavability, a human influenza virus can be obtained in infectious form from cells lacking a suitable protease to activate this virus.

Protective Effect of Measles Virus Inoculation on Subacute Sclerosing Panencephalitis Virus‐Infected Mice

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti‐measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus‐infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.

Proteolytic Activation of Influenza Viruses: Substrates and Proteases

Like many other viral glycoproteins, the hemagglutinin (HA) of influenza viruses is activated by proteolytic cleavage. Cleavage which is necessary for the fusion activity of the hemagglutinin and thus for the infectivity of the virus is exerted by host cell proteases, and the presence of an appropriate enzyme determines whether infectious virus is made in a given cell. Proteolytic activation is therefore indispensable for effective virus spread in the infected host and has been found to be a prime determinant for virus pathogenicity. This concept has been derived mainly from studies on avian influenza viruses. The pathogenic strains of these viruses are activated by ubiquitous proteases and cause therefore systemic infection mostly leading to rapid death of the animal, whereas activation of the apathogenic strains occurs only in epithelial cells of the respiratory or the enteric tract resulting in local infection of these organs. The mammalian influenza viruses, including the human ones, resemble the apathogenic avian strains in possessing also hemagglutinins of restricted cleavability and in causing usually local infection of the respiratory tract.1