Cambios genéticos en cepas de poliovirus tipo 2 aisladas de pacientes con poliomielitis paralítica asociada a la vacuna / Genetic changes in strains of poliovirus type 2 isolated from patients with vaccine-associated paralytic poliomyelitis

Introducción. En la actualidad la poliomielitis es un padecimiento raro en países desarrollados, donde solamente parecen circular cepas vacunales, que reemplazan al poliovirus salvaje. Sin embargo, es todavía una seria enfermedad para niños de países subdesarrollados de Asia y África. Pacientes y métodos. Se analizan nueve cepas de polio virus tipo 2 aisladas de heces fecales de pacientes con poliomielitis paralítica asociada a la vacuna (PPAV), desde el inicio de las campañas de vacunación antipolio en nuestro país. Las mismas se sometieron a secuenciación de un fragmento de 114 pares de bases de la región 5'NTR (non traductional region), donde radica uno de los principales determinantes de atenuación/reversión a la neurovirulencia de los poliovirus en la posición del nucleótido 481. Resultados. Se observó un cambio en dicha posición de guanina por adenina en todas las cepas secuenciadas, de modo que coincide con la secuencia homóloga de la cepa salvaje, así como con la de cepas obtenidas de niños sanos inmunizados con la vacuna de virus vivo atenuado. Esto presupone la necesidad de que se presenten otros cambios o incidan otros factores para que se presente o no PPAV, por lo que se sugiere la secuenciación de otras regiones del genoma en busca de otros posibles cambios nucleotídicos diferenciales (AU)

Introduction. Poliomyelitis is currently a rare disease in developed countries, where only vaccinal strains seem to be in circulation, which replace wild poliovirus. Nevertheless, it is still a serious disease for children in underdeveloped countries of Asia and Africa. Patients and methods. We analysed nine strains of poliovirus type 2 isolated from the faecal matter of patients with vaccine-associated paralytic poliomyelitis (VAPP), from the beginning of anti-polio vaccination campaigns in our country. These strains were submitted to sequencing of a fragment of 114 base pairs from the 5’NTR (non-traductional region), where one of the main determinants of attenuation/reversion to the neurovirulence of poliovirus lies in the position of nucleotide 481. Results. In this position it was observed how guanine had been replaced by adenine in all the strains that were sequenced, so that it coincided with the homologous sequence of the wild strain, as well as with that of strains obtained from healthy children immunised with the live vaccine. This presupposes that other changes must occur or that other factors must be involved for VAPP to occur or not, and we therefore suggest the sequencing of other regions of the genome in search of other possible differential changes in nucleotides (AU)

[Genetic changes in strains of poliovirus type 2 isolated from patients with vaccine-associated paralytic poliomyelitis]. / Cambios geneticos en cepas de poliovirus tipo 2 aisladas de pacientes con poliomielitis paralitica asociada a la vacuna.

INTRODUCTION: Poliomyelitis is currently a rare disease in developed countries, where only vaccinal strains seem to be in circulation, which replace wild poliovirus. Nevertheless, it is still a serious disease for children in underdeveloped countries of Asia and Africa. PATIENTS AND METHODS: We analysed nine strains of poliovirus type 2 isolated from the faecal matter of patients with vaccine associated paralytic poliomyelitis (VAPP), from the beginning of anti polio vaccination campaigns in our country. These strains were submitted to sequencing of a fragment of 114 base pairs from the 5 NTR (non traductional region), where one of the main determinants of attenuation/reversion to the neurovirulence of poliovirus lies in the position of nucleotide 481. RESULTS: In this position it was observed how guanine had been replaced by adenine in all the strains that were sequenced, so that it coincided with the homologous sequence of the wild strain, as well as with that of strains obtained from healthy children immunised with the live vaccine. This presupposes that other changes must occur or that other factors must be involved for VAPP to occur or not, and we therefore suggest the sequencing of other regions of the genome in search of other possible differential changes in nucleotides.

Persistence of vaccine-derived poliovirus following a mass vaccination campaign in Cuba: implications for stopping polio vaccination after global eradication.

BACKGROUND: With substantial progress made toward polio eradication, developing the appropriate strategy for discontinuing global oral poliovirus vaccine (OPV) after global eradication becomes increasingly important. At issue is the theoretical risk of independent circulation of potentially virulent OPV-derived strains. Because Cuba uses OPV only in mass campaigns, it represents an ideal site to assess vaccine-derived poliovirus persistence. METHODS: Infants born after the 1997 biannual mass campaigns were evaluated for past (neutralizing antibody) or current (virus excretion) evidence of vaccine-derived poliovirus exposure. We obtained sera and/or stool specimens from 861 infants; a second serum from 218 infants. RESULTS: All stool specimens were poliovirus negative. Of 762 infants, 113 (14.8%) had initially detectable poliovirus type 1 antibody, 193 (25.3%) type 2, and 94 (12.3%) type 3. A precipitous antibody decline occurred in initially positive sera. CONCLUSIONS: Our results suggest that in a country with high population immunity, vaccine-derived virus is unlikely to establish ongoing circulation.

[Rapid detection of Enterovirus by a direct method of polymerase chain reaction]. / Detección rápida de Enterovirus mediante un método directo de reacción en cadena de la polimerasa.

For the detection of Enterovirus, we devised a direct economical method of polymerase chain reaction which does not require a previous extraction of ribonucleic acid and uses infected cell culture supernatants. The system was developed on the basis of universal primers of Enterovirus and specific primers of vaccinal strain Sabin 1. The achieved results proved that the non-existence of methods of RNA extraction and purification prior to the reaction does not affect the susceptibility and specificity of the system, in the rapid detection of Enterovirus genomes and identification of vaccinal strains of poliovirus.

Eradication of poliomyelitis in Cuba: a historical perspective.

The eradication of poliomyelitis in Cuba, for which effective vaccines had to be acquired, is reviewed in this article. The strategy for eradication was based on mass immunization campaigns for the annual delivery of two doses of trivalent Sabin oral poliovirus vaccine (OPV). Except during the first campaign in 1962, the ages of the children for immunization were determined through national serological surveys of the entire country, including rural and urban areas. The interruption of wild virus transmission had been suspected since 1967 in Cuba, and since 1970 no studies have detected any wild virus. The important role of political and social organizations in the success of the programme and in the execution of the mass immunization campaigns is underscored. Countries that have successfully interrupted poliovirus circulation should maintain high immunization coverage for as long as there are other countries in the world where poliovirus still exists.

[Evaluation of a new technology for the detection of enterovirus in sewage]. / Valoración de una novedosa tecnología para la detección de enterovirus en aguas negras.

This paper reported the appraisal of a novel technology for the detection of enterovirus in sewage based on a viral recovery method using polyethylenglycol as a concentrating agent and on the combined use of viral isolation and polymerase chain reaction as viral detection and identification techniques. It was also confirmed that the viral recovery method is highly efficient since it allows to recover all viruses present in sewage and to preserve their infective capacity. It was proved that the polymerase chain reaction (PCR) is less sensitive than cell culture for detecting enteroviruses in sewage. It is also possible to use guadinine isothiocyanate, whose components can be prepared in the lab, as an alternative method for taking out and purifying nucleic acids instead of using the conventional TRIZOL method which is the one recommended in these cases by the World Health Organization.

[Possible factors associated with epidemic neuropathy in Cuba]. / Posibles factores asociados con la neuropatía epidémica en Cuba.

Of the 115 patients who suffered from epidemic neuropathy and received attention from March 16 de April 30, 1993, at the Center for Medical and Surgical Research (CMSR), in Havana City, 114 controls were selected at random in the community. In the univariate analysis it was found a significant association mainly with the smoking habit (odds ratio = 3.45 [95% confidence interval 1.85 to 6.35)] and with the consumption of edible fat of uncertified procedence (odds ratio = 2.77 [95% confidence interval 1.34 to 5.8]). All were ratified in the multivariate logistical regression analysis. Association (odds ratio = 8.8 [95% confidence interval 2.58 to 30.55] with the presence of antibodies against the Coxsackie virus A-9 strain 47 was also found among the 182 individuals who had serological test. It seems that the toxic factor derived from the smoking habit, together with the deficiency of some nutrients due to the changes occurred in the diet, as well as the contact with the isolated virus in one of the patients facilitated the clinical expression of this disease and therefore in the origin of the epidemics.

[Outbreak of viral meningoencephalitis. Comparison of 2 biological systems used for isolation of the virus]. / Estudio de un brote de meningoencefalitis viral. Comparación de 2 sistemas biológicos empleados para el aislamiento.

An increase of the cases of viral meningoencephalitis was reported in our country from October to December, 1995. 23 faeces specimens were received from 2 provinces aimed at obtaining the viral isolation by their inoculation in cell culture and in newborn mice at the same time in order to compare the sensitivity of both systems in isolation. A higher number of isolations was obtained in cell culture. The cell culture from kidney of a normal adult green African monkey (Vero) proved to be more sensitive. 18 isolations were obtained and identified as Coxsackievirus B5. An anatomical and pathological study was conducted in inoculated mice and injuries compatible with the enteroviral infection were observed.

[Eradication of measles in Cuba]. / La eliminación del sarampión en Cuba.

The vaccine against measles came into use in Cuba in 1971. During the seventies, a new early strategy for measles control was established, and it was followed by further efforts in the early eighties. Despite improvements to the control program, disease outbreaks continued to occur. In 1986, after examining the experience acquired through the control initiatives that were already in place, a new measles vaccination strategy was adopted. In time, the new vaccination strategy against measles came to have three main components: first, a single vaccination "catching-up" campaign targeting children 1 to 14 years of age. Second, efforts were made to achieve and maintain high vaccine coverage through mandatory vaccination services for 12-month-old children ("maintenance vaccination"). Finally, periodic "follow-up" campaigns were carried out for children 2 to 6 years of age. Steps were taken, for the purpose of monitoring the progress made so far toward eliminating measles, to strengthen disease surveillance systems, including the screening of suspected cases. The "catching-up" and "follow-up" campaigns both achieved greater than 98% coverage within targeted age groups. The routine vaccination program has also maintained high coverage. The high population immunity against measles that has been attained through these vaccination strategies has resulted in a rapid decrease in the incidence of the disease. From 1989 to 1992, less than 20 laboratory-confirmed cases were reported annually. In Cuba, the last case confirmed through serologic screening was reported in July 1993. Cuba's strategy for measles elimination has interrupted disease transmission and kept the causal virus from circulating on the island. Cuba's experience with measles elimination suggests that if an appropriate vaccination strategy is applied, measles can be globally eradicated.

[The intratypic characterization of the poliovirus by the polymerase chain reaction technic]. / Caracterización intratípica de Poliovirus por la técnica de reacción en cadena de la polimerasa.

The polymerase chain reaction techniques was introduced for the intratypic characterization of Poliovirus. Primers were used only to promote the amplification of the Sabin vaccine strains proved by electrophoretic run of the amplified DNA products (Sabin 1-97 pb, Sabin 2-71 pb, Sabin 3-44 pb) and whose specificity was satisfactorily verified. 23 Cuban poliovirus strains isolated and identified at the Laboratory of Enterovirus of the "Pedro Kourí" Tropical Medicine Institute from 1993 to 1994 were studied by this technique. All of them were of the vaccine type. It was observed how the Sabin vaccine poliovirus may be the cause of viral meningoencephalitis as a milder neurological complication. This study provided one more evidence about the non circulation of the wild poliovirus in Cuba.

[The antigenic characteristics of the viruses isolated from the cerebrospinal fluid of patients with epidemic neuropathy]. / Características antigénicas de los virus aislados del líquido cefalorraquídeo de pacientes con neuropatía epidémica.

During the epidemic of neuropathy occurred in Cuba from 1992 to 1994 viral agents antigenically related to the Coxsackie viruses were isolated from the cerebrospinal fluid of patients. To establish a function of these viruses in the etiopathogenesis of the disease, strains 47/93 IPK identified as Coxsackie A9 and the strain 44/93 of light cytopathic effect were selected to conduct a study of their antigenic characteristics by Western Blot. It was proved that there is an antigenic relationship between both strains and it was also demonstrated the absence of structural proteins in their native form among the agents of light cytopathic effect. According to these results, it is stated the possibility that persistence may be a mechanism through which these viruses take part in the etiopathogenesis of epidemic neuropathy in Cuba.

[New evidence of the antigenic relation between the viruses of epidemic neuropathy and the human nervous system. A study of the immune cellular response in patients with epidemic neuropathy and controls. A review of the topic]. / Nuevas evidencias de la relación antigénica entre los virus de la neuropatía epidémica y el sistema nervioso humano. Estudio de la respuesta inmune celular en pacientes con neuropatía epidémica y controles. Revisión del tema.

A series of experiments was made at the Virology Department of the "Pedro Kourí" Institute of Tropical Medicine aimed at obtaining new evidences on the possible antigenic relations existing between the viruses isolated from patients with epidemic neuropathy and the structures of the human nervous system. According to the results it may be finally inferred that the persistence and/or autoimmunity may be considered as mechanisms through which the studied viruses participate in the etiopathogenesis of the epidemic neuropathy in Cuba. For future experiments it is very important to identify the possible viral epitopes involved in the molecular mimicry that are responsible for the probable autoimmune mechanisms or for the viral persistence.

[Epidemic neuropathy. An etiopathogenetic hypothesis]. / Neuropatía epidémica. Hipótesis etiopatogénica.

An etiopathogenic hypothesis is explained taking into consideration the most significant results of the research performed on neuropathy as well as the latest knowledge about the infections produced by Enterovirus. The hypothesis allows to make a logical interpretation of these results; however, the new aspects included make it controversial in the light of the present knowledge to pathogeny by Enterovirus. Further research is needed to confirm this hypothesis.

[The genomic characterization of the Cuban strain with a light cytopathic effect]. / Caracterización genómica de la cepa cubana de efecto citopático ligero.

The genomic characterization of the strain of light cytopathic effect by nucleotide sequence is presented. It was possible to sequence 90% of the strain of light cytopathic effect. A close relationship with Coxsackie A9 was observed. The greatest mutations occurred in the region of the genome that codifies for the structural proteins. Therefore, it may be considered as a variant of Coxsackie A9 never reported before.

Meningoencefalitis virales por Enterovirus en Cuba en el período 1990-1995 / Viral meningoencephalities due to enteroviruses in Cuba during 1990-1995

Se describen los resultados del estudio de enterovirus como agentes causanes de meningoencefalitis viral (MEV) en Cuba, desde 1990 hasta 1995. En este período fueron estudiados 586 muestras de heces, 108 líquidos cefalorraquídeos y 1095 sueros pareados para un total de 1789 muestras, procedentes de 1458 pacientes diagnosticados clínicamente con esta patología. Las muestras para el aislamiento viral se inocularon en dos sistemas celulares diferentes, encontrándose 225 muestras positivas a enterovirus que representan el 32,42 o/o del total; el mayor número de aislamientos (217) fue a partir de heces, en células diploides de fibroblastos de pulmón humano (PHuE-1). Las determinaciones de anticuerpos se realizaron por prueba de neutralizacion en micrométodo, enfrentándolos con 10 antígenos de enterovirus (Echovirus 4, 6, 9, 11 y Coxsackievirus B1-6) y, en períodos epidémicos, además con el virus aislado. En los años estudiados se produjeron tres brotes epidémicos por Coxsackievirus A9 (1990-91), Echovirus 30 (1994) y Coxsackievirus B5 (1995). Es de señalar que desde 1970 los Coxsackievirus A9 y Echovirus 30 se vincularon en nuestro país, por primera vez, con epidemias de MEV. En los sueros pareados se obtuvo 66,84 o/o de positivos, siendo la mayor positividad a los Echovirus 6 y 11. Al considerar en conjunto la positividad por aislamiento y serología, más del 80 o/o de los casos estudiados pudieran tener ua explicación por algún enterovirus, lo uqe demuestra la importancia de estos agentes como causantes de MEV en Cuba (AU)

Meningoencefalitis virales por Enterovirus en Cuba en el período 1990-1995 / Viral meningoencephalities due to enteroviruses in Cuba during 1990-1995

Se describen los resultados del estudio de enterovirus como agentes causanes de meningoencefalitis viral (MEV) en Cuba, desde 1990 hasta 1995. En este período fueron estudiados 586 muestras de heces, 108 líquidos cefalorraquídeos y 1095 sueros pareados para un total de 1789 muestras, procedentes de 1458 pacientes diagnosticados clínicamente con esta patología. Las muestras para el aislamiento viral se inocularon en dos sistemas celulares diferentes, encontrándose 225 muestras positivas a enterovirus que representan el 32,42 o/o del total; el mayor número de aislamientos (217) fue a partir de heces, en células diploides de fibroblastos de pulmón humano (PHuE-1). Las determinaciones de anticuerpos se realizaron por prueba de neutralizacion en micrométodo, enfrentándolos con 10 antígenos de enterovirus (Echovirus 4, 6, 9, 11 y Coxsackievirus B1-6) y, en períodos epidémicos, además con el virus aislado. En los años estudiados se produjeron tres brotes epidémicos por Coxsackievirus A9 (1990-91), Echovirus 30 (1994) y Coxsackievirus B5 (1995). Es de señalar que desde 1970 los Coxsackievirus A9 y Echovirus 30 se vincularon en nuestro país, por primera vez, con epidemias de MEV. En los sueros pareados se obtuvo 66,84 o/o de positivos, siendo la mayor positividad a los Echovirus 6 y 11. Al considerar en conjunto la positividad por aislamiento y serología, más del 80 o/o de los casos estudiados pudieran tener ua explicación por algún enterovirus, lo uqe demuestra la importancia de estos agentes como causantes de MEV en Cuba

[Factors associated with the incurability of epidemic neuropathy in Cuba]. / Factores asociados a la no curabilidad de la neuropatía epidémica en Cuba.

56 patients were surveyed and reexamined at the Center for Medical and Surgical Research (CMSR) a year after the onset of the neuropathy epidemic. The possible factors associated with the occurrence were investigated to be studied together with the disease persistence, including the antibody titre of 33 of them, who had a previous serology against the biological agent (strain 47/IPK) isolated from the cerebrospinal fluid of a sick patient. A fourth of the patients (15 cases) were still ill. The loss of weight and the smoking habit were stressed as the factors most associated with the persistence of the clinical manifestations of epidemic neuropathy. In spite of the fact that the small number of patients who underwent the checkup limited the study, this is a basic document about the existent situation a year after the problem happened.

[Outbreak of Coxsackievirus B5 meningoencephalitis in Cuba, 1995]. / Brote de meningoencefalitis por coxsackievirus B5 en Cuba, 1995.

An increase in the reported cases of viral meningoencephalitis (VME) was detected in October and November, 1995, compared with the same period of 1994. 43 stock specimens from children with this diagnosis were received at the Laboratory of Enterovirus from the "Pedro Kourí" Institute of Tropical Medicine. 23 isolations (53.4%) were obtained and identified as Coxsackievirus B5. Besides, in 43 matched sera investigated by the neutralization test against some Enteroviruses, 21 proved to be positive (48.8%) to the isolated agent. This allowed us to affirm, supported by the clinical picture and by epidemiology, that we are in the presence of an VME outbreak produced by Coxsackievirus B5.