Seroepidemiological survey on dengue and Japanese encephalitis virus infections in Asian monkeys.

To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomolgus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys. The frequency of antibody to one or more types of dengue virus were 87.2, 49.5, 34.3, 34.2 and 14.9% in Malaysian, Vietnamese, Cambodian, Indonesian and Filipino cynomolgus monkey sera, respectively. None of the Indian bonnet monkey sera neutralized type 1 dengue virus which was the only virus type examined with this monkey species. Monkey sera collected in Japan where dengue virus infection had not been known since 1944 did not significantly neutralize dengue viruses. JE virus antibody was detected at 29.7, 9.0, 8.6, 2.7, 1.4 and 0% in Japanese, Cambodian, Vietnamese, Indonesian, Filipino and Malaysian monkey sera respectively.

Isolation of cowpox virus clones deficient in production of type A inclusions: relationship to the production of diffusible LS antigen.

Cowpox virus clones (A- clones) deficient in production of type A inclusions were isolated from two cowpox strains, Amsterdam and 53. These clones did not differ from their parents in major markers such as pock morphology in chorioallantoic membranes and pathogenicity in the rabbit skin. However, the LS antigens induced by A- clones developed precipitin lines in agar gel diffusion tests, while the antigens from their parents failed to precipitate. Immunofluorescence and agar gel diffusion tests revealed that antigens detectable by antisera against purified type A inclusions and LS antigens were closely related to each other. These findings suggest that the A- clones might be variants of cowpox virus which have lost the ability to assemble LS antigens into type A inclusions.

Strain-specific aggregation of enterovirus by dextran sulfate.

Dextran sulfate aggregates several enteroviruses depending not only on the pH, the ionic strength of the medium, but also on the protein content of the fluids and on strain specificities of the viruses. The aggregation effect was measured by filtration experiments, by sedimentation in the ultracentrifuge and by electron microscopy. The well known inhibiting effect of dextran sulfate on plaque formation may be due to its aggregating effect: A very strong inhibition of the release of matured virions from the infected cells is observed in medium containing dextran sulfate, whereas the adsorption process is inhibited much less compared with PBS controls. The maximal effect on virus aggregation, plaque size and virus release is observed at the same concentration of dextran sulfate.

A new candidate type of human enterovirus (Drilon strain) isolated in the Philippines.

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl2 against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect on primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.

Antiviral effects of atropine and caffeine.

The antiviral activities of atropine and caffeine were investigated. Atropine inhibited the multiplication of enveloped viruses and caffeine suppressed th growth of polio, influenza, herpes simplex and vaccinia viruses but not Japanese encephalitis virus, Newcastle disease virus and type 2 adenovirus.

Structural proteins of hand, foot and mouth disease viruses.

Coxsackievirus A16 (CA16) and enterovirus 71 (E71), which cause hand, foot and mouth disease, were compared with respect to their polypeptide composition by PAGE. Only three proteins were resolved for CA16, whereas the four characteristic structural proteins of E71, (VP1, VP2, VP3 and VP4) were separated. The distribution of labeled proteins suggested that the molecular weights of VP2 and VP3 of CA16 were very similar. The smallest protein, VP4, of CA16 and E71 had the same molecular weight, but the other proteins of the two serotypes were different in their molecular weights.

Virion polypeptides of poxviruses.

Structural polypeptides from a number of poxviruses were analysed by SDS-polyacrylamide gel electrophoresis. The patterns obtained with orthopoxviruses were generally quite similar to one another, but variola, monkeypox, cowpox and vaccinia viruses could be distinquished by their profiles in the molecular weight (mol. wt.) region around 30,000 to 40,000; some additional variation was found amongst cowpox and vaccinia strains. Whitepox virus was shown to have structural polypeptides indistinguishable from those of variola virus. The structural polypeptides of poxviruses belonging to other genera were different from those of the orthopoxvirus, except those of mol. wt. about 122,000 and 97,000, which were common to all viruses irrespective of genus. A polypeptide of mol. wt. about 25,000 was also observed in all cases, though its position varied slightly with the individual virus.

Paralytic poliomyelitis in a child with agammaglobulinemia.

This paper gives the clinical, immunological and virological data on a patient with agammaglobulinemia who developed paralytic poliomyelitis. The patient was a 3 year-old boy who had a typical B-cell defect without a T-cell defect. He had profound hypogammaglobulinemia and defective plasma cells and had repeated pyogenic infections which were controlled by gammaglobulin replacement therapy. At 3 years of age, he was admitted to our hospital with suspected meningitis. He had fewer, tremor and neck stiffness for 3 days and subsequently developed paralysis in his left arm and right leg. There was lymphocytosis in the cerebrospinal fluid. A non vaccine-like strain of poliovirus type 2 was isolated from the stool.

Orthopoxvirus strains defective in surface antigen induction.

Various strains of vaccinia, variola, whitepox, monkeypox and cowpox viruses were examined for their capacity to induce a specific early antigen detectable on the surface of infected cells. The Elstree strain of vaccinia, two strains of variola minor and white variants of cowpox and monkeypox viruses lacked the capacity to induce the antigen. Variation of the parent cowpox and monkeypox viruses to white variants was always accompanied by the loss of the antigen-inducing capacity.

Plaque formation by a host range mutant of vaccinia virus in non-permissive cells co-infected with Yaba virus.

A host-dependent conditional lethal mutant of vaccinia virus strain DIs was rescued to form plaques in a non-permissive cell line JINET co-infected with Yaba virus, a poxvirus serologically distant from the rescued virus. The efficiency of plaque formation under optimal conditions in this system was comparable to that in Vero cells which were permissive for the mutant. The plaque number of the mutant in JINET cells was influenced greatly by the multiplicity of Yaba virus, the interval between inoculations with DIs and Yaba virus and the maintenance medium for pre-infection of cells with Yaba virus. The plaque formation by DIs in JINET cells was suppressed or inhibited when the duration of pre-infection with high multiplicities of Yaba virus was prolonged. Thus, Yaba virus possessed both rescuing and inhibitory activities on DIs and the plaque number of DIs in doubly infected cells was thought to be determined by the balance between the two activities. Ultraviolet light-inactivated Yaba virus retained rescuing activity on DIs in JINET cells.

Variation in susceptibility of HeLa cell lines to coxsackievirus A9.

In the course of serial passages for several years a line of uncloned HeLa cells (A line) showed a gradual decrease in plaquing efficiency by coxsackievirus A9 (CA9 virus), while subcultures prepared from the same line kept frozen at an early passage level (A original line) did not show any change. However, it was observed later that the plaque-forming ability of the A original line (A orig. line) also decreased after serial passages as was observed with the A line. Comparing the characteristics of the same cell line at two different passage levels, it was found that the efficiency of adsorption of virus to cells was nearly the same, while virus yield at 8 hours after infection was different. The activity of alkaline phosphatase of cells was also different between these two passage levels, suggesting that a high enzymatic activity is associated with the susceptibility of cell cultures to CA9 virus. Magnesium chloride at 25 mM enhanced plaque formation by CA9 virus in highly passaged less susceptible cell cultures, and a possible role of the chemical as a stabilizer of alkaline phosphatase was discussed.

Studies on Cotia virus--an unclassified poxvirus.

This paper is a report of studies on Cotia virus; this had been first isolated in 1965 in Brazil and was subsequently shown to be a poxvirus. Cotia virus grew in a wide range of cell cultures and on the chick chorioallantois (CAM), Its growth characteristics are similar to those of other poxviruses. Microscopy showed virus factories or type B inclusions appearing before infectious progeny virus could be demonstrated. Type A inclusions appeared later, after development of progeny virus; these were shown by electron microscopy to differ from the type A inclusions of cowpox and other poxviruses and they have been termed Cotia bodies. Immunofluorescent staining also showed ring structures which appeared before the development of Cotia bodies. The growth of Cotia virus in human embryo lung (HEL) cells was sensitive to inhibitors of DNA and protein synthesis but was resistant to a concentration of rifampicin which inhibited vaccinia virus. Sharing of antigens between the Cotia virus and vaccinia virus was shown by gel precipitation tests and immunofluorescent staining. There was no cross neutralization between Cotia virus and vaccinia virus nor did anti-Cotia sera neutralize representatives of other poxvirus groups.

Aggregation of enterovirus small plaque variants and polioviruses under low ionic strength conditions.

Virion aggregation in low ionic conditions was observed with small plaque variants of Coxsackievirus type B3 and Echovirus types 4 and 11 by sedimentation and filtration methods. Inclusion of salts or DEAE-dextran into the media prevented or reversed virion aggregation. The effect of pH on aggregate formation in low ionic strength solutions was also investigated with various strains of poliovirus. Type I Sabin strain formed aggregates even at high pH, while Mahoney strains did so only below pH 6.5. Type 2 virus, Sabin and MEF1 strains, and type 3 virus, Sabin, Saukett and Suwa strains, showed an intermediate behaviour between the two type 1 strains, except MEF1-LB strain, a clone obtained from MEF1 strain under acidic overlay, which showed little tendency to aggregate. These results were compared with the degree of the d character of the strains. Besides the effect of inhibiting virion aggregation, the inclusion of DEAE-dextran into a sucrose gradient slowed the sedimentation of some of the viruses in low ionic strength solutions.

Acute gastroenteritis among schoolchildren associated with reovirus-like agent.

During early summer 1975 and spring 1976, outbreaks of acute gastroenteritis were reported from primary schools and other institutions in several districts of Japan. Outbreaks occurred in an explosive manner resembling mass food poisoning from a school lunch. The majority of patients were in the age group 6-14 years. Clinical features were generally mild, consisting of vomiting and/or diarrhea, often with low-grade fever. Reovirus-like agents in the feces were found in 27 (44%) of 62 patients. The virus found in feces of schoolchildren with acute gastroenteritis (SCGV) was related morphologically as well as serologically, not only to the agent found in infantile gastroenteritis (IGV), but also to neonatal calf diarrhea virus (NCDV). A slight difference in antigenicity between SCGV and IGV as suggested by cross complement fixation (CF) remains to be elucidated. About one-half of paired sera from 54 patients showed a significant rise in CF antibody against SCGV and/or NCDV. The pattern of neutralizing (NT) antibody against NCDV in patients' sera was similar to that of CF antibody. Most children studied had a titer of 1:4 or greater of CF and/or NT antibodies to SCGV and NCDV in acute sera. The relationship between acute gastroenteritis associated with reovirus-like agent in infants and that in schoolchildren is discussed.