Monkey Kidney Tissue Culture Research Articles

Kyasanur forest disease virus: viremia and challenge studies in monkeys with evidence of cross-protection by Langat virus infection

Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 10 9 , and death in 82 = 100% of the animals. Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.

Kyasanur forest disease virus: viremia and challenge studies in monkeys with evidence of cross-protection by Langat virus infection

Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 10 9, and death in 82 = 100% of the animals. Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.

Case-control study of cancer among US Army veterans exposed to simian virus 40-contaminated adenovirus vaccine.

Simian virus 40 (SV40) was an accidental contaminant of vaccines produced in monkey kidney tissue cultures in the 1950s and early 1960s, including a parenteral adenovirus vaccine given to several hundred thousand US military recruits. Detection of SV40 DNA in tumor tissues by some laboratories suggests that SV40 contributes to human cancers. To determine if entry into US Army service during periods of administration of SV40-contaminated adenovirus vaccine was associated with an increased risk of cancer, the authors conducted a case-control study of cancer occurring in male Army veterans who entered service in 1959-1961. Cases of brain tumors (n = 181), mesothelioma (n = 10), and non-Hodgkin's lymphoma (n = 220) were identified through a Veterans Administration hospital discharge database, as were colon cancer and lung cancer controls (n = 221). Exposure to adenovirus vaccine was assigned on the basis of known periods of adenovirus vaccine administration and dates of Army entry obtained for cancer cases and controls. The odds ratios associated with exposure to SV40-contaminated adenovirus vaccine were 0.81 (95% confidence interval (CI): 0.52, 1.24) for brain tumors, 1.41 (95% CI: 0.39, 5.15) for mesothelioma, and 0.97 (95% CI: 0.65, 1.44) for non-Hodgkin's lymphoma. These findings do not support a role for SV40 in the development of these cancers.

Severe neonatal echovirus 17 infection during a nursery outbreak.

Severe neonatal echovirus 17 infection during a nursery outbreak.

A Search for Persistent Measles, Mumps, and Rubella Vaccine Virus in Children With Human Immunodeficiency Virus Type 1 Infection

To determine whether live measles, mumps, and rubella vaccine viruses persist after immunization of immunocompromised children with human immunodeficiency virus type 1 infection. Ten children with symptomatic human immunodeficiency virus type 1 infection who received measles, mumps, and rubella immunizations were studied. Peripheral blood mononuclear cells, polymorphonuclear leukocytes, and plasma were cocultivated with primary African green monkey kidney tissue culture cells for detection of measles, mumps, and rubella viruses. Antibody to measles and mumps was determined by indirect fluorescent antibody and rubella was determined by enzyme-linked immunosorbent assay. Children seronegative to measles were revaccinated. Neither measles, mumps, nor rubella virus was recovered from peripheral blood mononuclear cells, polymorphonuclear leukocytes, or plasma in these children. No child experienced symptoms thought to be related to the vaccines. Humoral immunity after one or two immunizations was detected in 33% of the children. After vaccination, two of 10 children had IgG to measles; four of 10, to mumps; and four of 10, to rubella. In spite of an impaired humoral immune response, persistent viremia or infection did not occur after immunization with attenuated measles, mumps, and rubella vaccine viruses. These findings suggest that immunologic response to these viruses was sufficient to prevent persistent infection in these children immunocompromised by human immunodeficiency virus type 1 infection.

Intratypic differentiation of poliovirus strains by enzyme-linked immunosorbent assay (ELISA): poliovirus type 2 and poliovirus type 3

A double-antibody sandwich ELISA was developed for the detection of antigenic differences between wild and vaccine-derived strains of poliovirus type 2 and poliovirus type 3. Antibodies were prepared in rabbits by immunization with purified antigens of vaccine strains (type 2: Sabin P712, and type 3: Sabin Leon) and wild type strains (type 2: MEF, and type 3: Pool 30). Immunoblotting analysis of all antisera demonstrated that the IgG antibodies raised in rabbits have specificity towards the main structural proteins (Vp1, Vp2 and Vp3) of poliovirus. IgG fractions were purified from antisera by affinity chromatography, on a protein A-activated Sepharose 4B column. Purified IgG antibodies were used for coating of microtest plates (catching antibodies). The same reagents labelled with horseradish peroxidase were used as conjugates, after cross-adsorption with antigens of the same type heterologous virus strains (strain-specific conjugates). 29 poliovirus type 2 strains and 73 poliovirus type 3 strains isolated from clinical samples, were differentiated intratypically, as vaccine-derived or wild types, no intermediate strains were found and all samples tested fell in two distinct (vaccine/wild) categories. As little as 40 ng of poliovirus antigens was detected in stool samples from healthy children or from polio patients cultivated in monkey kidney tissue cultures. Preparation of strain specific conjugates did not require large amounts of poliovirus antigens. The developed ELISA, which is economic and capable of (1) detection of low amounts of poliovirus antigens in cultivated clinical samples, and (2) intratypic differentiation of poliovirus antigens as either vaccine-derived or wild type, is therefore well suited for large scale screening of poliovirus isolates.

Bordetella Pertussis and Upper Respiratory Tract Infection Viruses in Egyptian Children with Whooping Cough

ABSTRACT Isolates of Bordetella pertussis and upper respiratory tract infection (URTI) viruses from nasopharyngeal secretions were studied in 100 Egyptian children with whooping cough. Thirteen (13...

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin.

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus I. Isolation of temperature-sensitive mutants some of which exhibit host-dependent temperature sensitivity

Influenza A/Udorn/72 virus was mutagenized to induce temperature-sensitive ( ts) and host range mutations. The mutagenized virus was tested on both rhesus monkey kidney (RMK) and Madin-Darby canine kidney (MDCK) tissue culture at permissive and restrictive temperatures. One hundred thirty-six ts mjtants were isolated from 6185 clones tested. Eighty-three of these were ts on RMK tissue and 133 were ts on MDCK. Each clone replicated at 34° on both RMK and MCDK tissue and thus failed to exhibit a difference in host range in these two tissues at permissive temperature. However, 32 clones were 1000-fold or more restricted in plaque formation on MDCK cultures compared to RMK culture at 38, 39, and/or 40°. Conversely, four clones were more restricted on RMK cultures. These clones represent temperature-dependent host range ( td-hr) mutants. The ts and td-hr mutants may be useful in exploring certain biological, genetic, biochemical, and pathogenic properties of influenza A virus.

NEW ENZYME IMMUNOASSAYS FOR MEASUREMENT OF INFLUENZA A/VICTORIA/3/75 VIRUS IN NASAL WASHES

Nasopharyngeal (NP) washes from 12 patients experimentally infected with influenza A/Victoria/3/75 wild-type virus were tested in 2 newly developed enzyme-immunoassay (EIA) systems. Results were compared with infectivity titres of the NP wash specimens in rhesus monkey kidney-tissue culture. EIA detected A/Victoria/3/75 hæmagglutinin antigen for more days than did culture. EIA is also rapid and easy to do in large numbers; hence it may have advantages over culture methods for the diagnosis of influenza infections and the investigation of influenza epidemiology.

Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses

Rhesus and cynomolgus monkey kidney tissue cultures and two continuous lines, Madin-Darby canine kidney (MDCK) and LLC-MK2, were compared in titrations and isolations of influenza and parainfluenza viruses. Tube cultures were inoculated with laboratory virus strains or stored patient specimens and observed for hemadsorption. Trypsin was added to the medium of the continuous lines to increase sensitivity. All four tissue cultures gave similar titers of influenza A/USSR (H1N1), A/Texas (H3N2), and B/HK, but lower titers of parainfluenza 1, 2, and 3 were observed with MDCK. Cynomolgus kidney was the best single tissue culture for reisolation of the six viruses, but foamy-virus contamination of many lots was a serious problem. Reisolation of influenza viruses was as successful with MDCK as with primary monkey kidney. LLC-MK2 was similar to rhesus kidney but less successful than cynomolgus kidney. For reisolation of parainfluenza viruses, LLC-MK2 was superior to rhesus monkey kidney and similar to cynomolgus kidney. MDCK was less useful for parainfluenza viruses. Thus, LLC-MK2 would be an acceptable single tissue alternative to primary monkey kidney. The combination of MDCK and LLC-MK2 would provide optimal sensitivity for isolation of all six viruses.

Control of in Vitro and in Vivo Spread of Coxsackievirus B4 Infection by Sensitized Spleen Cells and Antibody

The effects of antibody and immune spleen cells of mice on the spread of coxsackievirus B4 in monkey kidney tissue cultures and mice were investigated. The spread of the virus in vitro was decreased both by antibody and by sensitized spleen cells, and the antiviral effect persisted even when the immune serum or immune cells were separated from the virus-infected monolayer by agar. The antiviral effect of the spleen cells was blocked by treatment of cells with antiserum to mouse IgG, but treatment of cells with antiserum to thymocytes had no effect. Non-glass-adherent cells were more capable of limiting the spread of coxsackievirus in cell cultures than were glass-adherent cells. Migration of sensitized splenic leukocytes in vitro was inhibited by specific antigen beginning three or four weeks after various doses of coxsackievirus B4. Adoptive transfer of antiserum protected 80%-100% of recipient mice, but spleen cells from only some of the mice protected 20%-66% of recipient mice against challenge with coxsackievirus B4. Thus, immunized mice develop sensitized T (thymus-derived) and B (bone marrow-derived) cells in the spleen. The B cells elaborate antibody to coxsackievirus, which can act locally to control the spread of infection. These results suggest that antibody plays a critical role in the control of the spread of coxsackievirus infection.

Effects of low- and high-passage influenza virus infection in normal and nude mice.

A human isolate of type A Hong Kong influenza virus (H3N2) was adapted to mice by serial passage. Lung homogenates from mice who received low passage levels contained about the same quantity of virus (10(6.2-6.95) 50% tissue culture infective doses/ml) as those from mice who received high passage levels (10(5.95-6.45) 50% tissue culture infective doses/ml); however, death occurred only in animals given high-passage virus. Passage 3 (P3) and passage 9 (P9) viruses were selected as representative of low-passage and high-passage viruses, respectively. Although minimal differences were detected in infectivity for rhesus monkey kidney tissue cultures and mice, P9 virus was at least 10,000 times more lethal for mice (mean lethal dose = 10(4.2)). Infection with P3 virus was accompanied by minimal bronchitis and bronchiolitis only, whereas P9-infected animals exhibited marked bronchitis, bronchiolitis, and pneumonia. Striking thymic cortical atrophy was also demonstrable in the P9-infected animals and, although virus was more commonly recovered from thymuses from these animals, immunofluorescent studies revealed only a few cells containing influenza virus antigens. To further explore the participation of thymus-derived lymphocytes in influenza, athymic nude mice and furred immunocompetent littermates were given 500 50% mouse infectious doses of P9 virus. Nude mice exhibited an increased survival time and, in contrast to the extensive lung pathology seen in furred littermates, manifested minimal cellular infiltration and no tissue destruction in lungs. Brains from nude mice exhibited encephalomalacia with lymphocytic perivascular cuffing, which was not seen in furred animals. Virus was recovered from brains of 6 of 13 nude mice and 1 of 10 furred animals. The contrasting models suggest that thymus-dependent cells play a significant role in the inflammatory response to influenza virus infection and should prove useful for probing host-virus interactions which characterize influenza virus virulence.

VIROLOGIC CONSIDERATIONS ON ANTI-DENGUE VACCINE

Possibilities of developing an anti-dengue vaccine are discussed based on our experimental findings with a dengue virus type 1 (DEN-1) Mochizuki strain. The Mochizuki strain virus was attenuated for human beings in the course of several passages through mouse brains since its isolation from the blood of a dengue patient in the 1943 epidemic in Nagasaki, Japan. This attenuated virus in the living state induced no abnormal symptoms after injection into human volunteers or monkeys, yet it stimulated production of specific anti-DEN-1 NT and HI antibodies and induced a prophylactic effect on the human beings against virulent dengue viruses (in the form of patient's serum) given experimentally. The immunogenic effects of the virus was maintained after passages in primary monkey kidney tissue cultures. Using the tissue culture virus, together with the 17D yellow fever (YF) vaccine, a combined immunization of human beings against DEN and YF was possible; production of the antibodies of significant titers against both viruses was unequivocally demonstrated. The partially purified virus was similarly effective in stimulating production of the anti-DEN antibodies, and the same statements could be made as to the viral components obtained by sucrose density gradient centrifugation. The virus was shown to multiply in the WI-38 cell line cultures, and this suggests application of this cell line to produce a live tissue culture vaccine.Conclusions of this review are : (1) the Mochizuki virus is completely attenuated; (2) yet it stimulates production of specific anti-DEN antibodies and induces protection against DEN-1 infection by virulent human virus; and (3) that possibility of developing a live tissue culture vaccine is suggested; this also makes possible a combined DEN-YF vaccination on the experimental basis. It is stated in the connection that the purified virus or its components can be used for the above purposes.Problems regarding the implication of “hypersensitivity hypothesis” of dengue and dengue hemorrhagic fever-shock syndrome in anti-DEN vaccination are in addition discussed.

An epidemic of picornavirus and adenovirus conjunctivitis.

A pandemic of acute haemorrhagic conjunctivitis occurred in south-east Asia during 1970 and 1971, and became epidemic in Lucknow in May 1971. From the conjunctival swabs adenovirus-like agents were isolated in monkey kidney tissue culture; one was typed as adenovirus 2. In paired sera rising antibody titres were found against an adeno-like agent isolated and the picornavirus (EC2/71) isolated in Singapore. The epidemics in south-east Asia were caused by a picornavirus while in Lucknow both adenovirus and picornavirus appeared to be implicated.

Subnuclear redistribution of DNA species in confluent and growing mammalian cells.

About 20 to 25 percent of the nuclear DNA from cultured cells of the African green monkey, Cercopithecus aethiops, consists of a homogeneous, highly repetitive fraction designated C. aethiops component α DNA. Use of in situ hybridization techniques reveals component α at the centromeres of chromosomes from both diploid and heteroploid African green monkey kidney (AGMK) tissue culture cells. — Component α DNA comprises 47 percent of the nucleolar DNA in actively growing primary AGMK cells, but only 31 percent of the nucleolar DNA in confluent cells which show density-dependent growth inhibition. Further, there is a pronounced shift of both main band and component αDNA from euchromatin to heterochromatin when growing cells attain confluency. Thus, the relative subnuclear distributions of component α and main band DNA's are different in growing and confluent cells. — In situ hybridization techniques indicate that component α sequences aggregate in clumps in nuclei of growing cells and show a diffuse distribution in nuclei of confluent cells. This suggests that centromeric regions of the various chromosomes or groups of chromosomes aggregate and disaggregate reversibly as the culture changes from density-dependent growth inhibition to active cell division. — Hypotonic citrate treatment of primary AGMK cells causes nucleoli of confluent cells to disperse: this dispersion following citrate treatment was not seen in growing AGMK cells or in confluent or growing heteroploid cells. Similarly, this nucleolar dispersion was seen in confluent diploid mouse and human cells but not in growing diploid cells or in confluent or growing heteroploid cells.

ISOLATION OF INFECTIOUS MEASLES VIRUS IN MEASLES ENCEPHALITIS

A 40-year-old man developed encephalitis 3 days after onset of acute measles. He died 6 weeks later. Histopathological findings were consistent with late type of post-infectious measles encephalitis. Direct inoculation of a homogenate of fresh brain tissue obtained at necropsy into susceptible tissue cultures produced no infectious virus. However, co-cultivation of cells derived from this brain tissue with those of a continuous line of African green monkey kidney tissue culture yielded an infectious virus that was identified as measles.

The isolation of influenza viruses.

Of 129 strains of the Hong Kong variant of influenza A2 virus isolated from 176 respiratory infections, 105 (81.4%) were isolated in tissue culture, the remainder being detected only by the inoculation of fertile hen's eggs. Organ cultures of chick embryo trachea were at least as sensitive as monkey kidney tissue culture for the isolation of this virus but organ cultures of human embryonic ciliated epithelium were much less efficient. The reverse was true for strains of influenza B virus studied in 1970 and 1971 when eggs and chick embryo tracheal organ culture were of little value and organ cultures of human embryonic ciliated epithelium and monkey kidney tissue cultures were the systems of choice.

Aspects of influenza C virus replication.

Influenza C virus antigen could be detected by immunofluorescence in chick amniotic epithelium 4 hr. after inoculation with undiluted amniotic fluid. Antigen could be detected in the nucleus of rhesus monkey kidney tissue cultures 10 hr. after infection. Only cytoplasmic fluorescence was observed 17-21 hr. after infection.The pattern of replication was similar to that reported for group I myxoviruses, indicating that the virus is correctly classified as an influenza virus.

Production and Evaluation of a Formalin-Killed Chikungunya Vaccine

Abstract Production methods and immunogenic characteristics of a formalin-inactivated Chikungunya vaccine prepared in bank-frozen green monkey kidney tissue culture were described. The total absence of untoward reactions or side effects and the excellent immunogenic response in volunteers attested to the safety and immunogenicity of this vaccine.