Monkey Neurovirulence Research Articles

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine candidate in fetal rhesus lung cells.

A strain of primary dog kidney (PDK)-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 35-TD3) was propagated in large volumes in fetal rhesus lung (FRhL) cells to produce a candidate vaccine for evaluation in man. Production seed (FRhL p2) and candidate vaccine (FRhL p3) were subjected to rigorous safety tests to exclude contaminating microbial agents. There was no significant monkey neurovirulence of parental or PDK-passaged DEN-4 virus or of control fluid cultures. FRhL-passaged viruses retained the phenotypic characteristics: small (occasional medium) plaque; temperature sensitivity at 38.5 degrees C; and absence of plaque formation in African green monkey kidney cells, cytopathic effect in LLC-MK2 cells, and viral growth in human monocytes. FRhL p2 virus displayed low virulence for monkeys; only one of four animals was viremic and three of four developed low-titered antibody. FRhL p3 virus produced viremia in three monkeys and moderate to high hemagglutination-inhibition and neutralizing antibody titers in all animals. Virus at both passages in FRhL exhibited reduced neurovirulence in suckling mice as compared to parental DEN-4. Because of its safety and desirable monkey virulence attributes PDK 35-TD3 FRhL p3 is recommended for human phase I trial.

The standardization of vaccines: a discussion.

Many changes have been made in the World Health Organization's (WHO) requirements for the production and control of both the killed and live (oral) poliovirus vaccines since their initial formulations in 1959 and 1962, respectively. The major changes in the production of killed vaccine concern the cell substrates used from primary tissue to passaged primary tissue or even a continuous cell line such as Vero. It has been shown also that there is no longer the necessity to test for residual infectious virus by the inoculation of monkeys, since cell cultures are much more sensitive for this purpose. For the live vaccine, a more uniform test for the titration of virus content has been introduced. Furthermore, international agreement on the details of the test for neurovirulence in monkeys has been reached. The expression of the immunogen of poliovirus in eukaryotic cells has been achieved, but whether it becomes a commercial proposition replacing one or both of our present vaccines remains to be seen.

Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses.

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.

Ontogeny of yellow fever 17D vaccine: RNA oligonucleotide fingerprint and monoclonal antibody analyses of vaccines produced world-wide.

Yellow fever 17D vaccines are currently manufactured with approval of the World Health Organization (WHO) in 11 countries. These vaccines have proven highly efficacious and safe. Nevertheless, they have not been fully characterized genetically, a problem for future standardization and modernization of vaccine manufacture now being proposed by WHO. Vaccines in use are derived from two distinct substrains (17D-204 and 17DD) which represent independently maintained passage series from original 17D. In this study, all 17D vaccines produced world-wide were characterized by RNA oligonucleotide fingerprinting. Forty-two large oligonucleotides were compared, and differences from an arbitrarily selected reference strain (produced by Connaught Laboratories in the U.S.A.) were determined. With one exception (vaccine produced in South Africa), fingerprints of vaccines derived from substrain 17D-204 were identical. The South African primary seed differed in position of one oligonucleotide, reflecting a charge shift due to a single base change. This difference occurred within one egg passage; a further change in the South African vaccine occurred within one or two passages from primary seed. No antigenic differences between 17D-204-derived vaccines (including South Africa) were demonstrated by neutralization tests using monoclonal antibody. Vaccines derived from the 17DD substrain consistently differed from 17D-204 vaccines in the absence of one oligonucleotide (No. 37). This change probably occurred during 40 additional egg passages in development of the 17DD vaccines. A clear antigenic difference was shown between 17D-204 and 17DD substrain vaccines using monoclonal antibody. 17DD vaccines showed minor genotypic differences, suggesting a higher degree of genetic instability than 17D-204 vaccines. No oligonucleotide fingerprint differences were found between avian leukosis virus (ALV)-free and ALV-contaminated vaccines. No definite genomic correlate of neurovirulence was defined by fingerprinting strains with a history of encephalitic complications in man or of failure to pass monkey neurovirulence tests. Parent Asibi virus showed several oligonucleotide differences and was serologically distinct from 17D vaccine.

Isolation of a temperature-sensitive strain of enterovirus 71 with reduced neurovirulence for monkeys.

Small- and large-plaque-producing viruses were selected from the enterovirus 71 (E71) prototype, BrCr. The small-plaque virus was a temperature-resistant (tr) strain which could multiply at 39.5 degrees C as well as 35 degrees C. The large-plaque virus was temperature-sensitive (ts) and could not grow at 39.5 degrees C. It was shown that the ts strain was much less neurovirulent for monkeys than the tr strain. Both tr and ts strains reacted with homologous and heterologous antibodies in cross-neutralization tests. While no difference was found between tr and ts strains in the size of virion polypeptides, the VP1 polypeptide could be differentiated by isoelectric focusing. Genomic differences between the two strains were found by oligonucleotide mapping.

Monkey neurovirulence of live, attenuated (Sabin) type I and type II poliovirus vaccines

This paper presents a survey of neurovirulence tests on type I and type II poliovirus vaccines and reference viruses performed at this Institute since 1961. The results show that there is no general increase in neurovirulence, for cynomolgus monkeys inoculated by the intraspinal route, with cell culture passage of either type I or type II polioviruses, in contrast to type III polioviruses which have been found to show a significant increase in monkey neurovirulence with passage. Occasional batches of both type I and type II vaccines showed unexplained increased monkey neurovirulence. However, a similar variation was seen with the reference viruses, particularly the type I reference, so that the significance of this finding is not clear.

Comparative monkey neurovirulence of sabin type III poliovirus vaccines

The paper presents a retrospective review of all monkey neurovirulence tests on type III oral poliovirus vaccines tested at this Institute since 1961. The results indicate that although occasional batches of vaccine from different manufacturers showed relatively high levels of neurovirulence for cynomolgus monkeys, when inoculated intraspinally, there was no consistent overall difference between vaccines from different manufacturers at SO+3 passage level. There was however a significant increase in neurovirulence with cell culture passage. The desirability of establishing a homotypic type III reference virus, of proven safety in the field, is emphasized.

Neurovirulence in cynomolgus monkeys of enterovirus 71 isolated from a patient with hand, foot and mouth disease

Six cynomolgus monkeys were inoculated subcutaneously with enteroviurs 71 (E71), isolated from the stools of a patient with hand, foot and mouth disease (HFMD). Clinical symptoms were observed in three of the six monkeys. One monkey showed complete paralysis of the lower extremities and two animals showed weakness in the hind limbs 4 to 7 days after inoculation. Lesions were found in the central nervous system (CNS) of all monkeys. Mild to moderate vascular lesions, perivascular cuffings, degeneration and disappearance of the neurons and meningial lymphocytic infiltration were observed in the grey and/or white matter of the spinal cord, medulla oblongata, cerebral cortex and brain stem. No virus was recovered from the CNS or liver of any of the six monkeys. However, serum neutralizing antibody titers had risen in monkeys inoculated with E71.

Chronic Progressive Poliomyelitis Secondary to Vaccination of an Immunodeficient Child

We investigated an immunodeficient child in whom chronic progressive poliomyelitis developed after she had received live oral poliovirus vaccine. Poliovirus, Type II, was isolated from throat and stool during life and from several sites within the brain at autopsy. The brain isolate was classified as vaccine-like on the basis of temperature sensitivity and antigenic markers. However, in the monkey neurovirulence test, the brain isolate produced moderately severe lesions throughout the spinal cord and brainstem and appeared nonvaccine-like. Thus, the brain isolate demonstrated a dissociation between the antigenic and neurovirulence markers. Our observations suggest that, under unusual circumstances, such as immunodeficiency, attenuated poliovirus can produce a chronic progressive neurologic disease. This case also emphasizes the need to diagnose immunodeficiency as early as possible, so that live-virus vaccines will not be administered.

Aluminum-marker of wild type 1 poliovirus strains.

Aluminum (A)-marker of 14 wild type 1 poliovirus strains (4 from the central nervous system of fatal cases, 10 from feces of paralytic cases) were studied. These strains were isolated in Japan from 1957 to 1961 before the implementation of the mass vaccination campaign with Sabin live poliovaccine. While an attenuated strain LSc,2ab, and a virulent strain, Mahoney, were shown to be typically A− and A+ respectively, only one of the wild strains examined was A+, 3 strains were A− and the remaining 10 were apparently A−, being thermoresistant in the presence or absence of Al3+ ions, when an ordinary stock virus was used. After three passages in primary monkey kidney cell cultures maintained with l-cystine-free synthetic medium No. 199 most of these strains became more or less thermosensitive in the absence of, but stabilized in the presence of Al3+, The results of the monkey neurovirulence test by intrathalamic inoculation with 2 A− strains indicated that they are almost as neurovirulent as the reference A+ Mahoney strain. These results suggest that the A-marker is an attribute inherent to each strain of type 1 poliovirus without any correlation with neurovirulence in monkeys.

Yellow fever vaccine. II. Antigenicity and neurovirulence of a vaccine seed free from avian leukosis virus.

Avian leukosis virus (ALV)-free candidate primary and secondary seed lots were indistinguishable from corresponding ALV-contaminated lots with respect to (i) potency as measured by titration in newborn and weanling mice and in the MA-104 plaque system, (ii) degree of viscerotropism as measured by viremia in monkeys, (iii) neurotropism as determined by the monkey neurovirulence test, and (iv) potency as determined by antibody response in monkeys inoculated by the intracerebral route.

Neurovirulence of polioviruses isolated from sewage and stool of healthy children. I. Studies on neurovirulence, antigenic marker and RCT-40 mrker of type 2 strains.

Antigenic and rct/40 markers were examined of 66 strains of type 2 poliovirus, 39 of them originating from stools of healthy children, and 27 from sewage samples. Seven of the strains were also tested for monkey neurovirulence. There was no satisfactory correlation between the twoin vitro markers. The 7 strains tested for neurovirulence were shown to occupy an intermediate position between highly neurovirulent “wild” strains and highly attenuated “oral vaccine-like” strains. The correlation between neurovirulence and rct/40 marker was better, although not absolute, than between neurovirulenee and antigenic marker. The sudden reappearance of type 2 in the second half of 1965 is discussed.

Yellow Fever Vaccine. II. Antigenicity and Neurovirulence of a Vaccine Seed Free from Avian Leukosis Virus

Avian leukosis virus (ALV)-free candidate primary and secondary seed lots were indistinguishable from corresponding ALV-contaminated lots with respect to (i) potency as measured by titration in newborn and weanling mice and in the MA-104 plaque system, (ii) degree of viscerotropism as measured by viremia in monkeys, (iii) neurotropism as determined by the monkey neurovirulence test, and (iv) potency as determined by antibody response in monkeys inoculated by the intracerebral route.

Intratypic serodifferentiation of polioviruses by comparison of neutralization indices and correlation of the antigenic marker, temperature marker and monkey neurovirulence.

A description is given of a simple method for the intratypic serodifferentiation of polio type 1 viruses, based on a modified determination of neutralization index with specific anti-LSc-2ab sera. The principal modification is a very short incubation of the virus-serum mixtures before they are inoculated into tissue cultures of monkey kidney cells. The results of this method agree very well with those of the Wecker technique. The method has been used to study the antigenic character of virus strains excreted after oral vaccination. It appears that the antigenic marker is not stable during the intestinal passage of the virus. In the majority of cases the isolated strains gradually lose their antigenic relationship to the parent virus. The temperature marker has proved to be relatively stable. No correlation could be found between antigenic and temperature marker. During multiplication of the virus in the intestinal tract a slight increase of monkey neurovirulence may occur, which is, however, not correlated with the loss of antigenic relationship to the vaccine virus. The applicability of the method is restricted because no differentiation can be made between a natural wild strain and a vaccine-derived strain which has lost its relationship to the parent virus. The above mentioned results are discussed and compared with those of other authors.

The properties of a new attenuated type 3 poliovirus

The results of two simultaneously performed monkey neurovirulence tests with USOL-D virus are presented, as performed in two laboratories. As a reference, OP2 virus was used, which is Sabin's attenuated type 1 poliovirus of German origin.

Histological studies of the monkey neurovirulence of group B arboviruses. I. A semiquantitative grading scale.

Journal Article HISTOLOGICAL STUDIES OF THE MONKEY NEUROVIRULENCE OF GROUP B ARBOVIRUSES: I. A SEMIQUANTITATIVE GRADING SCALE Get access NEAL NATHANSON, NEAL NATHANSON Search for other works by this author on: Oxford Academic PubMed Google Scholar DAVID GOLDBLATT, DAVID GOLDBLATT Search for other works by this author on: Oxford Academic PubMed Google Scholar INDERJIT S. THIND, INDERJIT S. THIND Search for other works by this author on: Oxford Academic PubMed Google Scholar MILES DAVIS, MILES DAVIS Search for other works by this author on: Oxford Academic PubMed Google Scholar WINSTON H. PRICE WINSTON H. PRICE Search for other works by this author on: Oxford Academic PubMed Google Scholar American Journal of Epidemiology, Volume 82, Issue 3, November 1965, Pages 359–381, https://doi.org/10.1093/oxfordjournals.aje.a120556 Published: 01 November 1965 Article history Received: 03 August 1965 Published: 01 November 1965

Relationship between Guanidine-dependence and Neurovirulence of Poliovirus

GUANIDINE-resistance1 and guanidine-dependence can be induced in vitro in poliovirus2,3. We have recently demonstrated that both characters can be easily conferred and withdrawn in in vitro experiments4,5. Moreover, we have demonstrated that guanidine-dependent viruses are not neurovirulent for monkeys6. In the present report we show that neurovirulence (of the type producing paralysis) for monkeys and guanidine-dependence in vitro are two strictly related viral properties.

POLIOVIRUS: GUANIDINE DEPENDENCE AND LOSS OF NEUROVIRULENCE FOR MONKEYS.

Guanidine-dependent polioviruses are obtained in vitro by subculturing Brunenders, Mahoney, and Sabin strains in the presence of increasing concentrations of guanidine. Mahoney viruses dependent on guanidine lose virulence (as indicated by paralysis) for monkeys inoculated intramuscularly or intracerebrally. Protection against virulent Mahoney viruses is induced by treatment with guanidine-dependent strains, and serum antibodies against the virulent strains are present in the treated animals

CHARACTERS OF GUANIDINE RESISTANT MUTANTS OF TYPE 1 POLIOVIRUS.

Following exposure of type 1 poliovirus (Mahoney and LSc 2ab strains) to guanidine hydrochloride, the presence of mutants resisting the action of the inhibitor could be demonstrated by means of the plaque method. Triple exposure to guanidine in increasing concentrations (70–150–250 μg/ml.) resulted in the isolation of highly resistant mutants, as compared to the parental strains, while a single exposure to 20–40 μg/ml. led to the demonstration of strains displaying an intermediate degree of resistance. Quantitative investigations of the resistance to 35 μg/ml. guanidine hydrochloride (i. e. to a concentration which still permits comparison with the parental strains) have shown the difference of susceptibility between the clonal strains obtained by the first method and the respective parental strains to be of the order of 106 in mutants of Mahoney virus and of the order of 105 in mutants of the LSc 2ab strain. The high degree of resistance of these mutants was further demonstrated by the fact that the maximum guanidine concentrations tolerated by the host cells (175–225 μg/ml.) still allowed the above mentioned clonal strains to display a plaque forming capacity of 10−1.2–10−4 as compared to that of the initial population, whereas similar resistance rates were displayed by the parental population only at concentrations as low as 10–25 μg/ml. Theg − character (guanidine resistance) proved to be stable in the course of 10 passages in the absence of the inhibitor, and the virus reconstituted from the infectious RNA extracted fromg − mutants maintained this character. A study of the correlation between guanidine resistance on the one hand, and the markersrct 40°, MS, E, as well as neurovirulence in monkeys, on the other, has shown that the presence of theg − character is not associated with any change in these markers, as compared to their pattern in theg + parental strains. This shows that guanidine resistance is controlled by a distinct portion of the genetic material and does not display the phenomenon of covariation with neurovirulence.

Characteristics of Sabin Type I Poliovirus After Gastrointestinal Passage in Newborn Infants: I. Monkey Neurovirulence and Temperature Marker Findings

Characteristics of Sabin Type I Poliovirus After Gastrointestinal Passage in Newborn Infants: I. Monkey Neurovirulence and Temperature Marker Findings