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Background Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. Methods Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). Results Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. Conclusions These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Influenza virus replication and the production of inflammatory cytokines are associated with symptoms, including fever, and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. Proteolytic activation of influenza viruses by serine proteases that are produced by airway epithelial cells is essential for viral entry and replication. Transmembrane protease serine S1 member (TMPRSS) 2, TMPRSS4 and TMPRSS11D have been detected certain cells, including the human alveolar epithelial cell line A549 and the surface epithelial cells of the human nasal mucosa, the trachea, the distal airways, and the lung. Several protease inhibitors, including aprotinin, reduce influenza virus replication. We previously demonstrated the following: (1) TMPRSSs (TMPRSS2, 4, and 11D) are present in primary cultures of human tracheal epithelial cells; (2) serine protease inhibitors, such as camostat and aprotinin, reduce the influenza virus replication and the release of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α into cell supernatants; and (3) camostat reduces the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. These findings suggest that serine proteases expressed by human tracheal epithelial cells induce the proteolytic activation of influenza viruses and that serine protease inhibitors may reduce viral replication and the resultant production of inflammatory cytokines. Thus, serine protease inhibitors are potential candidates for anti-influenza virus drugs Here, we review the expression of serine proteases, the role of serine proteases in influenza virus activation, and the effects of serine protease inhibitors. In this review, we aim to introduce the effects of serine proteases and their inhibitors on influenza virus infection of human airway epithelial cells by discussing the findings of previous studies performed by our group and other research groups. Furthermore, the clinical features and virulence of influenza virus infection are reviewed to clarify the association of virus replication and cytokine release with disease severity.

A total of 1372 cases of tularemia observed in Japan since 1924 were analysed. More than 90% of the cases were reported in the north-eastern part of the main island of Japan. After World War II, more than 40 cases were reported yearly for 20 years. Since 1966, however, there have been less than 10 cases per year. Ninety-three % of the cases were caused by contact with infected wild rabbits. The pattern of monthly distribution showed a peak in December and also a lower peak in May. The number of patients older than 40 years of age and the proportion of cases in females have gradually increased. In the earlier survey periods almost 70% of the cases were engaged in agriculture but at present this rate is less than 50%. The changes in the occurrence of tularemia in Japan is thought to be related to the change of life style caused by the rapid growth of the Japanese economy after World War II.

By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or -2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and -1b) and 2 (HCV-2a and -2b).

Recent field isolates of measles virus (MV) obtained by using B95-8 cells have been reported not to agglutinate African green monkey red blood cells (AGM-RBC). Vero cell-adapted, plaque-forming strains derived from three field isolates at the third passage in Vero cell cultures (T8Ve-3, T11Ve-3 and N13Ve-3) also exhibited markedly decreased binding activity, as determined by infectivity-absorption and haemadsorption tests. On the other hand, binding activity of the respective strains at the twentieth passage (T8Ve-20, T11Ve-20 and N13Ve-20) increased to practically the same level as that of the Edmonston strain, a standard strain of MV passaged long-term. A membrane immunofluorescence test revealed that the decreased binding activity to AGM-RBC of T8Ve-3, T11Ve-3 and N13Ve-3 was not due to decreased expression of the haemagglutinin (H) protein on the cell surface. The deduced amino acid sequence of the H protein synthesized in T11Ve-3-infected cells was identical to that in T11Ve-20-infected cells, although a single amino acid alteration was observed when T8Ve-3 was compared with T8Ve-20. Similarly, approximately half of the N13Ve-20-infected cells synthesized an H protein identical to that produced in N13Ve-3-infected cells, and nevertheless, exhibited markedly increased haemadsorption. The present results suggest that a viral protein(s) other than the H protein contributed to the binding activity of MV to AGM-RBC.

The mouse homologue of CD63/ME491 (Mu-CD63) was molecularly cloned and analyzed. Mu-CD63 exhibited a strikingly high similarity to CD63/ME491 and the rat homologue. Northern blot analysis revealed that Mu-CD63 mRNA was expressed strongly in the kidney of adult mice, especially in the glomerulus fraction, implying the possibility that Mu-CD63 plays an important role in maintaining normal renal function. Activated macrophages and splenocytes exhibited strong expression of Mu-CD63 mRNA, whereas cultured thymocytes barely expressed the mRNA irrespective of cell activation. Taken together, the present results suggest that Mu-CD63 expression is associated with differentiation and/or development of certain cell types, but not necessarily with cell proliferation.

With a reverse transcription-polymerase chain reaction method that utilized highly efficient primers, the NS5 region of hepatitis C virus (HCV) genome could be amplified from all of the 24 serum samples randomly obtained from Japanese patients with liver diseases. Subtype analysis on the basis of nucleotide sequence similarities of the amplified fragments revealed that 2 out of 24 Japanese patients (8.3%) were infected with the recently identified subtype HCV-3b, which had been thought to be rare in Japan.

We have established two highly metastatic tumor clones, D-1 and F-3, which have the distinct features of undergoing pulmonary metastases. Both clones were derived from a skin squamous cell carcinoma (SqC-NH), which had spontaneously occurred in DS-Nh mice. F-3 was morphologically spindle-shaped in tumor mass and adherent in culture, while D-1 was round and non-adherent. In in vitro growth, there was no significant difference between them. When the clonal tumor cells were implanted intradermally into syngeneic mice, however, the growth of D-1 was slower than that of F-3. As for the metastasic ability, both D-1 and F-3 were highly metastatic compared with the original tumor. Interestingly, the pulmonary metastases of these two clones were apparently different. In F-3-bearing mice, some round nodules were detected and no tumor cells were seen in the surrounding area around the nodules (nodule-type). In contrast, D-1 cells had initially grown from the periphery of lobes, and interstitially infiltrated the lung parenchyma area without forming nodules (diffuse-type) and the appearance was similar to lymphangitis carcinomatosa. Histologically, a remarkably smaller number of metastatic foci formed around the blood vessel for D-1 as compared with F-3. It should also be noted that some localized nodules (nodule-type both visually and histologically) were seen in the lung, when D-1 cells were inoculated in the tail vein. In addition, D-1 showed markedly higher lymphatic metastasis than F-3. These observations show that the pulmonary metastases of D-1 and F-3 differ in the mechanisms underlying the processes of tumor dissemination. We suppose a possibility that D-1 may preferentially metastasize via the lymphatics and F-3 may metastasize via the bloodstream.

Patterns of antimicrobial susceptibility were determined for 155 clinical isolates of the Bacteroides fragilis group from patients with acute appendicitis in Bali, Indonesia. The transfer of drug resistance was also studied, and plasmid analyses were undertaken. Metronidazole and chloramphenicol were the most active drugs against these isolates (resistance rate, < or = 0.6%). Among the beta-lactam drugs, cefoxitin was the most active (resistance rate, 2%). Rates of resistance to tetracycline were high (16%). Resistance to clindamycin (rate, 10%) increased during the 2-year study period. Except in the case of beta-lactam agents, overall susceptibility patterns were comparable to those reported from other countries. Tetracycline resistance was more frequently transferred after tetracycline induction than without such induction (P < .05, chi 2 test). Resistance to tetracycline and clindamycin was co-transferred by five of 12 donor strains. In one of these five strains, transferability was constitutive, with a high transfer frequency (10(-5) per input donor). Plasmid analysis indicated that the transfer of resistance to tetracycline and clindamycin among the strains studied was not plasmid mediated.

Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.

Summary Genomic structure of the ME491/CD63 antigen gene and promoter activity of the 5′-flanking regulatory sequences were studied. The antigen gene consists of eight exons that span 4 kilobase-pairs. Primer extension analysis with RNA from cultured human cells identified three major transcription initiation sites. The 5′-flanking region of exon 1 has features characteristic to promoters of many house-keeping genes and growth-regulating genes. The region is highly GC rich and contains potential binding sites for transcription factors such as Sp1 and ETF, but not a TATA box. The 5′-flanking sequence exerted strong promoter activity when linked to a reporter gene. Deletion mutant analysis of the 5′-flanking sequence has strongly suggested that a potential, binding site for AP-1 plays an important role in positively regulating the gene expression.

A deletion mutant of the human melanoma-associated ME491 antigen gene starting at the first intron (lambda R31) differentially mediates the antigen expression depending on the cell type. Cryptic promoter activity residing in a 270-base-pair (bp) fragment of the first intron was examined by primer extension analysis and recombinant chloramphenicol acetyltransferase (CAT) assay. The cryptic promoter, further localized within a 153-bp fragment (fr153BN), exerted its effect in Ltk- and H-ras-transformed NIH3T3 (3T3-Hras) but not in parental NIH3T3 cells. The results suggested that the cryptic promoter was associated with a novel ras-responsive positive regulatory element, since fr153BN did not contain an AP-1-binding sequence motif, known as the ras-responsive enhancer element. The cryptic promoter activity of fr153BN was suppressed by an upstream 121-bp fragment (fr121SB) which contained a consensus sequence motif for binding of a repressor protein, GC factor, and regions showing sequence similarity with putative cis-acting repressor elements found in the vimentin gene. The degree of the suppression was greater in 3T3-Hras than in Ltk- cells. These positive and negative regulatory elements may be differentially involved in the regulation of ME491 antigen expression depending on the cell type.

By reverse transcription-PCR, measles virus (MV) mRNA was detected in the brain, kidney, spleen, liver, and lung tissues obtained from 23 (45.1%) of 51 autopsy subjects, with the detection rates of each tissue ranging from 8 to 20%. Sequence analysis revealed frequent mutations in the corresponding viral protein. These results suggest that MV mutants commonly persist in apparently healthy individuals.

Sixteen cases of tularemia transmitted by arthropods are used to characterize arthropod-borne tularemia in Japan. Arthropod-borne tularemia accounted for 1.2% of a total of 1,374 cases of tularemia observed between 1924 and 1996. The number of cases reported in Japan was low when compared with the number of cases reported in the United States. Arthropod-borne tularemia, however, is increasing and has reached 10.1% incidences during the last 16 yr. No arthropod-borne cases were reported prior to 1951. The occurrence of tularemia infection caused by contact with diseased hares was diphasic with the higher peak occurring during the winter, whereas the occurrence of arthropod-borne tularemia was common from spring to autumn. Among the 16 cases that we studied, 5 were initiated by tick-bites, 5 by the crushing of ticks found on domestic dogs, and 1 by an unidentified insect. In the remaining 5 cases, the vectors were not identified although arthropod bites were confirmed. These arthropod-borne cases were observed exclusively in the northeastern area of Honshu, the main island of Japan, and the age of patients ranged from 23 to 74 yr. The arthropod vectors associated with tularemia and factors influencing incidence of arthropod-borne tularemia in Japan are discussed.

An avirulent mutant of Sendai virus, Ohita-MVC11 (MVC11), was generated from a highly virulent field strain, Ohita-M1 (M1), through successive passages in LLC-MK 2 cell cultures (M. Itoh, Y. Isegawa, H. Hotta, and M. Homma, J. Gen. Virol. 78:3207-3215, 1997). In LLC-MK 2 cells, MVC11 induced a high degree of apoptotic cell death that was demonstrated by chromatin condensation of the nucleus and DNA fragmentation, and production of MVC11 declined markedly after prolonged culture. On the other hand, M1 did not induce prominent apoptosis and maintained high virus titers. In primary mouse pulmonary epithelial cell cultures, M1 replicated rather slowly to reach maximum level of virus production at 3 days postinfection, and high levels of virus production were maintained thereafter without causing apoptosis. In contrast, MVC11, which produced 20 times more progeny virus than M1 at 1 day postinfection, induced a high degree of apoptotic cell death before the virus replication cycle was completed. Accordingly, the production of progeny virus was strongly inhibited thereafter. In the lungs of mice infected with MVC11, virus antigens and signals of DNA fragmentation detected by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique colocalized in bronchial epithelial cells, clearly demonstrating that infection by MVC11 triggered apoptosis in vivo as well as in vitro. These results suggest the possibility that induction of apoptosis by MVC11 plays an important role in attenuation of mouse pathogenicity by restricting progeny virus production in the lung. The C protein was shown to have the capacity to induce apoptosis, and the increased level of the C protein in MVC11-infected cells was considered to account partly, if not entirely, for the induction of apoptosis.

Based on phylogenetic and restriction fragment length polymorphism analyses of the hemagglutinin and nucleoprotein gene sequences, measles virus strains obtained in western Japan were divided into two types. Type 1 isolates have largely replaced type 2 isolates during the last 10 years in the area surveyed.

By means of RT-PCR, a portion of measles virus (MV) mRNA encoding nucleoprotein (NP) could be detected in 11 (18%) of 61 brain tissue samples obtained from administrative autopsy cases, who apparently had not suffered from subacute sclerosing panencephalitis (SSPE)-like central nervous system disorders. Most of the brain-derived NP sequences showed significant asynonymous nucleotide substitutions when compared with wild-type MV isolates and SSPE virus. Our present results suggest that MV commonly persists in the human brain without causing apparent clinical symptoms, probably due to decreased virus replication.

A recently identified subtype of hepatitis C virus, subtype 1d, was found to be common in Indonesia, being isolated from 4 (20%) of 20 and 11 (34%) of 32 patients with chronic hepatitis and liver cirrhosis, respectively. A new group of sequence variants was also identified, although its prevalence ratio was not as high in the area surveyed.

Hepatitis C virus (HCV) is classified into at least 7 genotypes, HCV-I through -VI and African-type HCV, on the basis of sequence variation of the viral genome. Core and envelope regions of the HCV genome were amplified from sera of Indonesian patients using reverse transcription-polymerase chain reaction (RT- PCR). Sequence analysis of the amplified fragments identified a novel sequence variant (Td-6) that was distinct from all of the reported genotypes. RT-PCR for the NS5 region using genotype-specific primers for HCV-V and -VI further supported the above observation. Also found was another variant (Td-17) that was closely related to, but distinct from HCV-III and -IV on the basis of core region sequence. It thus appears that at least two novel sequence variants exist in HCV in Indonesia, although their prevalence rates may be relatively low. The present results also revealed HCV-II to be the most prevalent HCV genotype in Indonesia.

Treatment of a mouse macrophage cell line Mk1 with pokeweed mitogen (PWM) either before or during but not after virus inoculation resulted in an enhancement of dengue virus (DV) infection. The infection enhancement was primarily due to an increase in the number of DV-infected cells but not to increased virus production in a cell. These results suggested that PWM treatment mediated increased DV binding and/or penetration to Mk1 cells, thereby resulting in the infection enhancement. N-acetylglucosamine (GlucNAc) did not suppress PWM-mediated enhancement of DV infection when added to Mk1 cells after PWM treatment was done, although GlucNAc clearly suppressed the effect of PWM when added simultaneously with PWM. The results implied the possibility that the PWM-mediated increase in viral binding/penetration was not due to a cross-linking by PWM between DV and a cell-surface receptor, but due to another mechanism, presumably exposure of a masked DV receptor(s). The DV receptor, unidentified as yet, involved in the PWM-mediated infection enhancement appeared to have no relation with IgG Fc receptors that are known to be involved in antibody-mediated enhancement of DV infection.

Hepatitis C virus (HCV) is currently classified into 6 major types, HCV-1 through -6, each of which can be further divided into a few subtypes, e.g., HCV-1a, -1b, -1c, etc., on the basis of sequence variation of the viral genome. The core and E1 envelope regions of HCV genome were amplified from sera of Indonesian patients using reverse transcription-polymerase chain reaction. Sequence analysis of both core and E1 regions followed by molecular evolutionary phylogenetic analysis identified a novel sequence variant of HCV-1 (Td-6). Antibodies in the serum from which Td-6 was isolated reacted only marginally to the core protein of HCV-J, a representative strain of HCV-1b, despite strong antibody response against a mixture of the core, NS3 and NS4 proteins of HCV-1a. The possible mechanism for the diminished reactivity of the antibodies in the serum to the core protein of HCV-J is discussed.

Five acriflavine agglutination test-positive (acf+) colonies and five negative (acf–) colonies were isolated from each of the four strains (Ebina, CMB2, N9, and Schu) of Francisella tularensis, and the correlation between the virulence in experimental mice and the entry and intracellular multiplication in cultured mouse fibroblast cells (L-929 cells) was examined. All of the acf– colonies derived from the Ebina and CMB2 strains were highly virulent in mice, readily entering and growing well in the cells, while all of the acf- colonies from N9 and Schu strains were of low virulence and neither entered nor grew in the cells effectively. On the other hand, regardless of their parent strains, the acf+ colonies were low virulent and most of those colonies did neither enter nor grow in L-929 cells. In addition, two acf- colonies, one from the N9 and the other from the Schu strain, gained virulence through several passages in mice, and in parallel, their entry and multiplication also improved. However, two acf+ colonies from the Ebina strain and one acf+ colony from the N9 strain showed a moderate degree of the entry and multiplication although they were all low virulent. The overall results indicate that the entry and multiplication in cells are important factors regulating the virulence of F. tularensis. The results also showed, however, that they were not sole factors to elucidate the virulence of the bacterium in mice.

When treated with fungal polysaccharide schizophyllan, mice survived otherwise lethal Sendai virus infection. Both intraperitoneal and oral administrations were effective when sonicated schizophyllan with a relative molecular mass (M(r)) of 4.6 x 10(5) was used. Antiviral antibody in the serum could be detected at an earlier time after virus infection and virus spread in the lung was more efficiently inhibited in schizophyllan-treated mice than in untreated controls. Schizophyllan also augmented protective immune responses induced by low doses of a live Sendai virus vaccine that were insufficient to confer complete protection against challenge infection with a virulent strain. On the other hand, schizophyllan did not influence interferon production in mice whether or not infected with Sendai virus. The present results suggest that schizophyllan confers better protection against virus infection through augmentation of antiviral immune responses and can be used as an immune enhancer.

A study was made to elucidate the effect of host cells on the HANA protein of Sendai virus. Two strains of Sendai virus were isolated from an epidemic in an animal laboratory by inoculating the lung homogenate of a moribund mouse either into LLC-MK2 cells (Oh-L) or into the allantoic cavity of embryonated eggs (Oh-E). Oh-E agglutinated chicken red blood cells at 37 degrees (HA37+), while Oh-L did not (HA37-). When Oh-L was passaged in eggs, conversion of the HA37- virus to the HA37+ virus readily occurred. A single point mutation was recognized on the HANA protein of the HA37+ virus either at position 525 (Gln----Arg) or at position 198 (Leu----Phe). Hl test with monoclonal antibody revealed conformational changes around the receptor binding site. Neuraminidase activity was also affected by these mutations. The changes in these biological activities of the HANA protein seemed to allow the HA37+ virus to replicate in eggs. On the contrary, the HA37+ virus replicates as efficiently as the HA37- virus in LLC-MK2 cells and no reversion to the HA37- virus was observed. The overall results indicate that the passage of Sendai virus in eggs resulted in selection of viruses possessing a specific mutation on the HANA protein. The pneumopathogenicity in mice was not significantly different between the HA37- virus and the HA37+ virus, suggesting the existence of genes other than the HANA gene that determine mouse pathogenicity.

A gelatin particle agglutination (PA) test for Japanese spotted fever has been developed. Gelatin particles were sensitized with a sonicated causative rickettsia and used as antigens. The antibodies by PA test were detected as early as days 4-7 after the onset, whereas those by indirect immunoperoxidase (IP) test were detected after days 8-11. In addition, PA titers were higher than IP titers before days 20-23. The agglutinins detected by PA test were proven to be IgM because they were all sensitive to dithiothreitol. PA test was, however, less specific than IP test, giving a little nonspecific reaction to the sera from patients with scrub typhus and from individuals unrelated to those two rickettsioses. Nevertheless, PA test, which is simple, rapid, and easy to interpret the results, is useful for the early serodiagnosis of Japanese spotted fever.

Proteolytic activation of Sendai virus in the lungs of mice is necessary to cause pneumopathogenicity. Using Sendai virus-infected lung block cultures, protease inhibitors were tested for their antiviral effect by examining inhibition of proteolytic activation. Among the inhibitors tested, a serine protease, aprotinin, was shown to be most effective. In vivo protection experiments demonstrated that aprotinin, when administered intranasally, could confer protection on mice against lethal Sendai virus pneumonia through the same mechanism as observed in the in vitro system. The present study provides an experimental basis for the use of protease inhibitors as antiviral drugs.

The prevalence of influenza C virus among pigs in Hyogo Prefecture, Japan, was investigated by serological techniques. Out of 240 sera tested, 45 (19%) showed haemagglutination inhibition (HI) to influenza C virus. Pig sera with high HI titres also scored high in neutralization tests and ELISAs. When fractionated by sucrose density gradient ultracentrifugation, the HI/ELISA reactivities corresponded to antibodies of the IgM and IgG classes. Radioimmunoprecipitation tests revealed that some, but not all, of the pig sera with high HI activities precipitated HEF glycoprotein of influenza C virus. These results suggested that the HI activities of pig sera in Hyogo Prefecture were due to the presence of antibody to influenza C virus. Sera with IgM class antibody to influenza C virus were found throughout the year. However, the question of whether or not pigs serve as a natural reservoir for human influenza C virus still remains to be solved.

A protease-activation mutant of Sendai virus, TCs, was isolated from a trypsin-resistant mutant, TR-5. TCs was activated in vitro by both trypsin and chymotrypsin. TCs was, however, less sensitive to trypsin and chymotrypsin than were the wild-type virus and TR-5, respectively. F protein of TCs had a single amino acid substitution at residue 114 from glutamine to arginine, resulting in the appearance of the new cleavage site for trypsin and the shift of the cleavage site for chymotrypsin. Activation of TCs in the lungs of mice occurred less efficiently than that of the wild type, and TCs caused a less severe pneumopathogenicity than did the wild-type virus, which supports our previous view that the in vitro trypsin sensitivity of Sendai virus can be a good indication of pneumopathogenicity in mice.

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination-inhibition (HI), hemolysis-inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement-requiring NT (C-NT) and complement-requiring hemolysis (C-HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C-NT and C-HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgG1 isotype having neither C-NT nor C-HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement-mediated immunological responses such as immune virolysis but also antibody-dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved, play an important role in the protection of mice from Sendai virus infection.

Dengue type 2 virus (D2V) infection in cultured human monocytes was studied. D2V permissiveness of the monocytes was enhanced when the cells were inoculated with D2V in the presence of either polyclonal or type-specific monoclonal anti-dengue antibody. The enhancement of D2V permissiveness mediated by the antibodies was more clearly demonstrated when the monocytes had been treated with trypsin before virus inoculation, though treatment of the cells with trypsin alone decreased D2V permissiveness. The enhancement of infection by type-specific neutralizing monoclonal antibody suggests that the D2V particles possess at least two antigenic determinants closely associated with virus infectivity. Infectious center assays revealed that the infection enhancement in the presence of the antibodies was due primarily to an increase in the number of D2V-infected cells, and that only a small proportion of the monocyte population supported D2V replication. The virus-permissive monocytes did not bear HLA-DR antigens on their cell surface. The presence of nonadherent lymphocytes in the monocyte cultures before D2V inoculation did not affect the D2V permissiveness of the monocytes. Treatment of cultured monocytes with the synthetic adjuvants N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and its lipophilic derivative, [B30]-MDP, did not significantly affect the D2V permissiveness of the cells.

A device was made to analyze the pneumotropism of Sendai virus in mouse. Minced lung blocks were prepared from the mouse intranasally infected with Sendai virus for 2 hours and cultured in a CO2 incubator. This culture system provided a suitable in vitro model of Sendai virus infection in mice in terms of the distribution of the viral antigens and histopathological findings. The progeny virus recovered from the lung culture was already activated and was accompanied by the cleavage of F glycoprotein into F1 and F2. This fact demonstrates that the activating mechanism is reversed in the lung culture as found in vivo infection of mouse lung. The viral activation and the cleavage of F glycoprotein were simultaneously inhibited by tosyllysylchloromethylketone, leupeptin, soybean trypsin inhibitor and antipain, but not by tosylamidophenylethylchloromethyl-ketone, chymostatin, pepstatin, iodoacetamide, phenylmethylsulfonylfluoride and p-chloromercuribenzoate. These results show that the activating enzyme of Sendai virus found in the lung culture was similar to trypsin. The existence of the activating enzyme may support the replication of Sendai virus in mouse lung in multiple-step and also result in the lung pathology.

Polypeptides of egg-borne Sendai virus (egg Sendai), which is biologically active on the basis of criteria of the infectivity for L cells and of hemolytic and cell fusion activities, were compared by polyacrylamide gel electrophoresis with those of L cell-borne (L Sendai) and HeLa cell-borne Sendai (HeLa Sendai) viruses, which are judged biologically inactive by the above criteria. Densitometer profiles on the stained gels of egg Sendai resolved six polypeptides (virion protein [VP] 1 to VP6), in which VP2 and VP4 were identified as glycoproteins by PAS stain. Comparative electropherograms of both L Sendai and HeLa Sendai revealed that there were significantly larger amounts in the VP2 region of these viruses but VP4 was present only in greatly reduced amounts as compared to egg Sendai. It was also found that VP2 of L Sendai and HeLa Sendai consisted of two components, VP2a and VP2b, but the one of egg Sendai consisted of only VP2a. A mild trypsin treatment which converts both L Sendai and HeLa Sendai to a biologically active form selectively removed VP2b from these viruses and increased concomitantly the amounts of materials in the VP4 region. The same treatment of egg Sendai affected neither its biological activities nor its electropherogram. Consequently, gross polypeptide profiles on the stained gels of L Sendai and HeLa Sendai after trypsin treatment became favorably comparable to that of egg Sendai. Electrophoresis of labeled L Sendai and HeLa Sendai with a 3 H-amino acids mixture and 14 C-glucosamine resolved at least three glycoproteins, GP1, GP2, and GP3, each corresponding to VP2a, VP2b, and VP4, respectively. The trypsin treatment of these viruses removed almost all the radioactivity of GP2 and simultaneously increased the radioactive counts of GP3 and raised small amounts of rapidly moving heterogeneous glycoprotein, GP4. A possible relationship between the biological modification and the above characteristic polypeptide patterns of Sendai virus was discussed.

When purified HANA glycoproteins of Sendai virus were centrifuged in a sucrose density gradient in the presence of Triton X-100, neuraminidase activity was distributed into two peaks, a rapidly sedimenting peak H and a slowly sedimenting peak L. The analysis of polypeptides under nonreducing condition showed that peak H contained HANA glycoproteins exclusively in the form of tetramer whereas peak L was in the form of dimer, which suggests that the different sedimentation rate is due primarily to the difference in the mode of intermolecular association of HANA glycoproteins by disulfide linkage. A glycoprotein designated C-HANA with a molecular weight of ∼55,000 was also involved in the solubilized HANA preparation and found as tetramer and dimer in peaks H and L, respectively, when analyzed under nonreducing condition. The C-HANA protein was produced only after solubilization of Sendai virions with Triton X-100. The C-HANA was found more in the preparation from egg-grown virions than from LLC-MK2 cell-grown virions, indicating that production of C-HANA is host cell-dependent. Trypsin treatment of the intact HANA oligomers resulted in the formation of oligomers of the size similar to the C-HANA oligomers. These results suggest that host cell-derived protease(s) present in the purified virus preparation is responsible for the cleavage. In contrast with the intact HANA oligomers, the C-HANA oligomers could neither reconstitute large multivalent structures nor exhibit hemagglutinating activity when centrifuged in sucrose density gradients in the absence of Triton X-100. Though the C-HANA oligomers could adsorb to chicken red blood cells, they were released rapidly from the cells even at a low temperature like monovalent forms of the intact HANA oligomers, suggesting that the formation of multivalent structure is essential for HANA glycoproteins to continue to adsorb to the cells and the C-HANA protein loses the hydrophobic part required for the reconstitution of HANA glycoprotein. In the light of these observations the previous result obtained by several authors who demonstrated an apparent dissociation of hemadsorbing activity from neuraminidase activity of paramyxoviruses can be reasonably explained now.

A study was performed to determine if type C influenza infection could be established in dogs as a model for human cases. Mongrel dogs were infected with the Ann Arbor/1/50 strain of type C influenza virus and were examined for clinical symptoms, virus isolation and antibody response. After the first exposure to the virus, all infected animals developed nasal discharge and some of them also showed swelling of the eyelids, and suffusion of the eyes with tears and eye mucus, within 1 to 4 days. The animals showed an increase in hemagglutination-inhibiting (HI) serum antibody, and recovery of the agent from the nasal swabs was successful. The symptoms lasted for as long as 10 days in most infected dogs, which was comparable to our human cases reported previously (Katagiri, S., Ohizumi, A., and Homma, M. 1983. J. Infect. Dis. 48: 51-56). After the second and third virus exposures at intervals of 50 days, all animals developed the same symptoms as those described above and the rise in antibody titer was evident. The virus could be recovered from four of the six dogs 2 to 5 days after the second exposure and from one dog as late as 10 days after the third exposure. Increases in antibody titer in the IgM fraction were observed after every infection. In control dogs which were mock-infected with UV-inactivated virus, no symptoms were evident and recovery of the virus was not successful although an increase in HI serum antibody titer was seen. These results show that mongrel dogs are sensitive to type C influenza virus and that repeated infections characteristic of human influenza C can be experimentally produced in dogs.

Our previous study has shown that, although a trypsin-resistant mutant of Sendai virus, TR-2, replicates only in a single cycle in mouse lung with a negligible lesion, the animal acquires a strong immunity against lethal infection with wild-type Sendai virus, suggesting that TR-2 could be used as a new type of live vaccine (M. Tashiro and M. Homma, J. Virol. 53:228-234, 1985). In the present study, we investigated the immunological response elicited in TR-2-infected mice, particularly with respect to cell-mediated immunity. Analyses of cytotoxic activities of spleen cells with 51Cr release assays revealed that Sendai virus-specific T lymphocytes (CTL), in addition to natural killer activity and antiviral antibodies, were induced in DBA/2 and C3H/He mice infected intranasally with TR-2. Proteolytic activation of the fusion glycoprotein F was required for the primary induction of CTL, though not necessarily for stimulation of natural killer and antibody responses. Memory of the CTL induced by TR-2 was long-lasting and was recalled in vivo immediately after challenge with wild-type Sendai virus. In contrast to TR-2, immunization with inactive split vaccine failed to induce the CTL response, but it elicited a high titer of serum antibody and a low level of natural killer activity.

Mice were immunized with dengue type 2 virus (DEN 2) under a schedule favoring the production of IgE antibody. The antibody obtained could sensitize peritoneal resident mast cells both in vitro and in vivo so that the sensitized cells were degranulated and released histamine on challenge with the DEN 2 antigen. It was also demonstrated that the antibody was cytophilic and heat-labile. The above observations suggest that the present experimental system can be used to detect anti-DEN 2 IgE antibody in mice.

Summary The major virus-specific proteins (HA, NA, NP, NS1 and M) of five different isolates of influenza B virus (B/Lee/40, B/Osaka/2/70, B/Yamagata/1/73, B/Aomori/1/76 and B/Yamagata/26/77) were compared by limited proteolysis with Staphylococcus aureus V8 protease and subsequent polyacrylamide gel electrophoresis. The peptide patterns of matrix (M) proteins from all five strains were virtually identical. The nucleoproteins (NP) as well as the non-structural proteins (NS1) were also very similar among strains although the peptides of B/Lee/40 could be distinguished from those of the strains isolated from 1970 to 1977. In contrast, the peptides from haemagglutinin (HA) glycoproteins were largely different even among the strains isolated later than 1970. It therefore appears that the HA glycoproteins of influenza B virus are more changeable than any of the non-glycosylated proteins. Furthermore, it was found that the maps of HA1 were markedly different among strains while the maps of HA2 were very similar, which suggests that the structural changes in the HA polypeptide occur preferentially in the HA1 portion. The neuraminidase (NA) glycoproteins also showed strain-dependent differences in their mapping patterns.

A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethyl-enediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.

Infection of a subline of L cells adapted to grow in suspension (Ls) with Fushimi strain of HVJ (HVJ-F) resulted in a virus carrier state. Ls cells, when cultured in monolayer, showed morphological changes following infection of HVJ-F and were detached from the glass wall. However, when the detached cells were transferred to a new environment of suspension culture within 5 days after infection, the carrier state was again established. HVJ-F caused only lethal infection in L cells maintained exclusively in monolayer (Lm). On the other hand, both Ls and Lm, irrespective of their culture conditions, were lethally infected by Nagoya 1–60 strain of HVJ. The overall results showed that culture condition as well as the kind of host cells or virus strains is an important factor regulating the establishment and maintenance of the virus carrier state.

A cloned DNA fragment (lambda R31) containing the human gene for melanoma-associated ME491 antigen was transfected into mouse fibroblast cell lines and the antigen expression was studied. Our preliminary observation of higher expression of the antigen in more malignant Ltk- cells and weaker expression in less malignant NIH3T3 cells tempted us to investigate the antigen expression in Harvey(H)-ras-transformed NIH3T3 cells. It was observed that malignant transformation of the lambda R31-transfected NIH3T3 cells by H-ras oncogene enhanced the antigen expression to some extent. Northern blot analysis suggested that the enhancement occurred at the transcriptional level. Nucleotide sequence analysis of the 5'-regulatory region of the ME491 antigen gene in lambda R31 identified a number of consensus sequence motifs for binding of transcription factors such as Sp1, AP-2 and polyomavirus enhancer binding proteins 2 and 3. A consensus sequence motif for binding of AP-1, known as a ras-responsive element, was not found in that region. The significance and possible involvement of the transcription factors in the enhancement of ME491 antigen expression are discussed.

Interaction of human erythrocytes was studied morphologically with intact nonhemolytic Sendai virus and an artificially induced hemolytic one which was obtained by freezing and thawing of the former virus. Fusion of the viral envelope with the erythrocyte membrane was observed with both viruses. In the nonhemolytic preparation, virions were uniformly spherical in shape and completely devoid of uranyl acetate in negative staining. During the whole period of the envelope-membrane fusion, the integrated viral envelope appeared to be intact, and the release of hemoglobin into the extracellular space was never observed even after the incubation period of 1 hr at 36°. On the contrary, in the hemolytic preparation, virions had damages on their envelopes so that they were permeable to uranyl acetate stain. The release of hemoglobin occurred immediately after formation of the envelope-membrane fusion and was complete by the incubation period of 10 min at 36°. The intact nonhemolytic virion also could become hemolytic after treatment with the antiviral antibody and complement. The results indicate that Sendai virus hemolysis is caused exclusively by virions having injured envelopes but not by virions having intact envelopes. The study also revealed that the envelope-membrane fusion was followed by dispersal of the viral envelope antigens on the erythrocyte membrane, migration of the nucleocapsids into cytoplasm, and cell-cell fusion of the erythrocytes.

The RNA genomes of sixteen human strains of influenza C virus isolated in Japan between 1964 and 1983 were compared by SDS-polyacrylamide gel electrophoresis and oligonucleotide fingerprinting. A high degree of genetic variation was observed among the strains analysed. However, there were some strains with the genomes closely related to one another, and they could be divided into two groups. The first group consists of C/Shizuoka/79, C/Kanagawa/1/81 and four strains of C/Yamagata/81. The 1981 strains of this group were all isolated in March of the year. The second one consists of C/Kyoto/41/82, C/Nara/82 and C/Hyogo/1/83 that were isolated between February 1982 and December 1983. Little or no difference was observed in the genomes of the same group, while the difference was evident between two groups. The Aichi/1/81 strain isolated in November 1981 had a genome distantly related to either of these two groups. Thus three different types of influenza C virus were isolated during the period of 12 mth from March 1981 to February 1982, suggesting that multiple influenza C viruses with distant genetic relationship were circulating at the same time in Japan.

Summary Growth of influenza B virus was found to be greatly restricted in Vero cells compared with that in MDCK cells. The analysis of protein synthesis in infected cells showed that the synthesis of M protein is selectively inhibited in abortive infection. Cell fractionation experiments demonstrated that the viral glycoproteins, HA and NA, migrate from rough membranes via smooth membranes to plasma membranes in abortive as well as in productive cells. These results suggest that intracellular migration of influenza B virus glycoproteins occurs independently of the synthesis of M protein although M protein synthesis appears to be required for the formation of virus particles.

Summary Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cDNA encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg → Ile), the cleavage site of the F protein, and 109 (Asn → Asp). Two trypsinsensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from Ile to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendai virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to Ile is responsible for the acquisition of resistance to trypsin.