Tiempo de permanencia de anticuerpos y reinfección en pacientes con antecedente de infección por SARS-CoV-2 / Length of stay of antibodies and reinfection in patients with a history of SARS-CoV-2 infection

ANTECEDENTES: La enfermedad causada por el coronavirus de tipo 2 causante del síndrome respiratorio agudo severo (SARS-CoV-2) denominada como COVID-19 fue reportada inicialmente en la ciudad de Wuhan en China en diciembre de 2019 (1,3). Al poco tiempo, el 11 de marzo de 2020, fue caracterizada como pandemia por la Organización Mundial de la Salud (OMS). El espectro de la enfermedad es amplio e incluye desde cuadros leves y autolimitados hasta neumonía atípica severa y progresiva, falla multiorgánica y muerte (4,5). De acuerdo a la OMS, en el 2020 se subregistraron más de 1.8 millones de muertes a causa de esta enfermedad a nivel mundial. El Perú ha sido uno de los países más afectado en Latinoamérica con, al 30 de julio de 2021, más de 2 millones de casos y 196 214 fallecidos (letalidad de 9.31%) (6). Por lo tanto, conocer el tiempo de duración de esta inmunidad adquirida por la infección causada por SARS-CoV-2 es importante para poder guiar estrategias de vigilancia epidemiológica y de inmunizaciones. OBJETIVO: El objetivo de esta revisión fue identificar y sistematizar la evidencia disponible sobre el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac contra el SARS-CoV-2, la incidencia de reinfección y el tiempo hasta el evento en personas con antecedente de infección por SARS-CoV-2. METODOLOGÍA: Se realizó una revisión rápida basada en dos preguntas: En pacientes con antecedente de infección por SARS-CoV-2 ¿Cuál es el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac contra el SARS-CoV-2? ¿Cuál es la incidencia de reinfección y el tiempo hasta este evento? Se incluyeron estudios de cohorte. Para ello, se realizó una búsqueda sistemática en las bases de datos MEDLINE/PubMed, EMBASE/Ovid y LOVE/Epistemonikos. Luego de eliminar duplicados, los autores seleccionaron los ítems que cumplieran con las preguntas establecidas. RESULTADOS: Se incluyeron 32 estudios: 20 evaluaron el tiempo de permanencia de seropositividad de Ac anti SARS-CoV-2: 11 estudios evaluaron únicamente el tiempo de permanencia de seropositividad de los Ac, 9 estudiaron el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac, y 5 estudiaron únicamente el tiempo de permanencia de la capacidad de neutralización de los Ac 7 estudios evaluaron la incidencia de reinfección y el tiempo hasta este evento. La población incluida correspondió a adultos con antecedente de infección por SARS-CoV-2, 23 estudios incluyeron cohortes en población general, 6 incluyeron a personal de salud y 3 a donantes de plasma convaleciente. Para los estudios que evaluaron el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac, los periodos de observación fluctuaron entre 6 meses a 1 año. La mayoría (85%, 17/20) de los estudios coinciden en reportar la persistencia de Ac a los 6 meses de seguimiento, con variaciones en las frecuencias de las inmunoglobulinas (Ig) específicas (IgA, IgM, IgG) para las proteínas Spike (S), el complejo de unión al receptor (RBD) o a la proteína de nucleocápside (N). Los estudios reportaron que, hasta al menos 6 meses, la persistencia de la capacidad de neutralización de los Ac varió entre el 30.7 y 100% en los participantes. La incidencia de reinfección varió desde 0 a 4.85% en un tiempo aproximado de seguimiento de 180 días. De los 4 (57.14%) estudios que consideraron como intervalo de tiempo mínimo de 90 días en su definición de reinfección: la menor media de tiempo hasta la reinfección reportada fue de 116 ± 21 días y la mayor fue de 212 ± 25 días. Las personas que tuvieron una infección inicial por SARS-CoV-2 en comparación a aquellos sin infección previa (grupo control) tuvieron un menor riesgo de infección (reinfección), observándose una reducción de la incidencia de 84 a 89% en pacientes con infección previa en relación al grupo control; esta evidencia procede de 2 estudios. Hall et al., (41) realizó un sub-análisis de acuerdo a la presencia o no de síntomas de COVID-19 durante la infección inicial, reportando que el riesgo de reinfección en aquellos que fueron asintomáticos durante la infección inicial fue menor en comparación al grupo control (RTIa: 0.48, IC 95%: 0.37 ­ 0.63; reducción de riesgo relativo (RRR): 52%), aunque la reducción del riesgo fue mayor para el subgrupo con presencia de síntomas de COVID-19 en la infección inicial en comparación al grupo control (RTIa: 0.074, IC 95%: 0.06 ­ 0.10; RRR: 92.6%). La valoración de riesgos de sesgos indicó que los estudios que presentaron serias preocupaciones de riesgo de sesgos fueron el 65.75%: en los estudios sobre el tiempo de permanencia de seropositividad y capacidad de neutralización de los Ac anti-SARS-CoV-2, esta seria preocupación se presentó principalmente en el dominio 2 (sesgo de deserción) debido la pérdida de más del 60% de los participantes al final del seguimiento lo cual podría afectar la validez de los resultados. En los estudios sobre incidencia de reinfección, fue más frecuente (71.4%) la valoración de alguna preocupación de riesgo de sesgo debido a que el 71.4% fueron estudios retrospectivos y, en el 85.7% de los estudios las definiciones de reinfección no pudieron ser respaldadas por la realización de un análisis de secuenciación genómica. Las limitaciones de esta revisión son: que, debido a la heterogeneidad de las pruebas utilizadas para medir Ac, las definiciones de reinfección y la manera de reportar los desenlaces entre los diferentes estudios no fue posible realizar un meta-análisis para ninguno de los desenlaces. Adicionalmente, se incluyeron 8 (25%) estudios en pre-print y estos podrían modificar sus resultados y/o conclusiones en sucesivas versiones hasta su publicación, no siendo posible garantizar que respondan satisfactoriamente la revisión por pares y sean finalmente publicados. CONCLUSIONES: La evidencia disponible mostró que la mayoría (85%) de los estudios coinciden en reportar la persistencia de Ac a los 6 meses de seguimiento, con variaciones en las frecuencias de (Ig) específicas (IgA, IgM, IgG) para las proteínas S, RBD o a la proteína N. Hasta al menos 6 meses, la persistencia de la capacidad de neutralización de los Ac varió entre el 30.7 y 100% en los participantes. La incidencia de reinfección varió entre 0 a 4.85%, en un tiempo aproximado de seguimiento de 180 días. De los 4 (57.14%) estudios que consideraron como intervalo de tiempo mínimo de 90 días en su definición de reinfección: la menor media de tiempo hasta la reinfección reportada fue de 116 ± 21 días y la mayor fue de 212 ± 25 días. El antecedente de infección por SARS-CoV-2 redujo la incidencia de reinfección entre 84 y 89% en comparación a aquellos individuos sin infección previa (grupo control). La reducción de riesgo se mantuvo inclusive para el subgrupo de personas con infección inicial de tipo asintomática (reducción de incidencia de 52% en relación control); aunque, en menor magnitud que en aquellos que desarrollaron síntomas de COVID-19 (reducción incidencia de 93% en relación al control).

Development of a novel plaque reduction neutralisation test for hantavirus infection

In the Americas, hantaviruses cause severe cardiopulmonary syndrome (HCPS) with a high fatality rate. Hantavirus infection is commonly diagnosed using serologic techniques and reverse transcription-polymerase chain reaction. This paper presents a novel plaque reduction neutralisation test (PRNT) for detecting antibodies to Brazilian hantavirus. Using PRNT, plaque detection was enhanced by adding 0.6% of dimethyl sulfoxide into the overlay culture medium of the infected cells. This procedure facilitated clear visualisation of small plaques under the microscope and provided for easy and accurate plaque counting. The sera from 37 HCPS patients from the city of Ribeirão Preto, Brazil was evaluated for the Rio Mamoré virus (RIOMV) using PRNT. Six samples exhibited neutralising antibodies; these antibodies exhibited a low titre. The low level of seropositive samples may be due to fewer cross-reactions between two different hantavirus species; the patients were likely infected by Araraquara virus (a virus that has not been isolated) and RIOMV was used for the test. This assay offers a new approach to evaluating and measuring neutralising antibodies produced during hantavirus infections and it can be adapted to other hantaviruses, including viruses that will be isolated in the future.

Leite de mulheres brasileiras apresenta anticorpos IgA secretores (SIgA) que neutralizam o rotavírus G9P[5] / Milk from Brazilian women presents secretory IgA antibodies and neutralizes rotavirus G9P[5]

OBJETIVO: Verificar a presença de SIgA anti-rotavírus sorotipo G9P[5] e a capacidade de neutralização do vírus de amostras de leite de mulheres brasileiras. MÉTODOS: Foram determinados os níveis de anticorpos SIgA reativos contra rotavírus G9 em 30 amostras de leite materno por ELISA usando suspensões purificadas do vírus. A capacidade das amostras de neutralizarem o rotavírus G9P[5] foi analisada em ensaio de de Neutralização utilizando células MA-104. RESULTADOS: Foram observadas grandes variações individuais referentes aos níveis de SIgA e títulos de neutralização, mas todas as amostras mostraram certa capacidade de neutralizar o G9P[5]. Verificamos uma correlação positiva altamente significativa entre os níveis de anticorpos e os títulos de neutralização. CONCLUSÕES: A alta correlação entre níveis de anticorpos anti-rotavírus e a capacidade neutralizante das amostras de leite sugere um possível papel protetor desses anticorpos contra a infecção. Esses resultados também apoiam o incentivo à prática do aleitamento materno.

OBJECTIVE: To verify the presence of anti-rotavirus serotype G9P[5] SIgA and the virus neutralization capacity of milk samples from Brazilian women. METHODS: SIgA antibody levels reactive to rotavirus G9 were determined in 30 maternal milk samples by enzyme-linked immunosorbent assay (ELISA) using purified virus suspensions. The samples' capacity to neutralize rotavirus G9P[5] was analyzed using the MA-104 cells neutralization assay. RESULTS: Great individual variations were observed regarding the SIgA levels and neutralization titers, but all samples showed some G9P[5] neutralizing ability. A highly significant positive correlation was observed between antibody levels and neutralization titers. CONCLUSIONS: The high correlation between anti-rotavirus antibody levels and neutralizing capacity of the milk samples suggests a possible protective role of these antibodies against infection. These results also support the encouragement of the breast-feeding practice.

Teste imunoenzimático com base em anticorpo monoclonal para a detecção de anticorpos contra os herpesvírus bovino tipos 1 e 5 / A monoclonal antibody-based enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus types 1 and 5

Os herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5) são agentes virais genética e antigenicamente relacionados, associados com diversas manifestações clínicas em bovinos, incluindo doença respiratória, genital, neurológica e abortos. Estudos epidemiológicos indicam que esses vírus estão amplamente disseminados no rebanho bovino brasileiro. O diagnóstico sorológico, que permite identificar animais portadores da infecção latente, se constitui em importante ferramenta para monitoramento individual e de rebanho. O presente artigo relata a padronização de um teste imunoenzimático do tipo ELISA, com base em anticorpo monoclonal (AcM), para a detecção de anticorpos séricos que reagem contra BoHV-1 e/ou BoHV-5. Inicialmente, determinou-se o AcM mais adequado para a sensibilização das placas, as diluições apropriadas do antígeno e dos soros-teste e o ponto de corte do ensaio. Após a padronização, o ensaio foi validado testando-se 506 amostras de soro bovino, previamente testadas para anticorpos neutralizantes contra BoHV-1 e/ou BoHV-5 pela técnica de soroneutralização (SN). Comparando-se com os resultados da SN frente a BoHV-1, o teste de ELISA apresentou sensibilidade e especificidade de 96,6 por cento e 98,3 por cento, respectivamente. Os valores preditivos positivo e negativo foram de 97,6 por cento, a concordância foi de 97,6 por cento e o índice de correlação kappa entre os testes foi de 0,95, o que indica uma excelente concordância. Comparando-se com os resultados da SN frente o BoHV-5, o ELISA apresentou 94,3 por cento de sensibilidade; 97,9 por cento de especificidade; 97,1 por cento de valor preditivo positivo e 95,9 por cento de valor preditivo negativo. Para BoHV-5, a concordância entre os testes foi de 96,4 por cento e o índice de correlação foi de 0,92, também excelente. Esses resultados demonstram que o teste padronizado apresenta sensibilidade e especificidade adequados para o diagnóstico sorológico das infecções por BoHV-1 e BoHV-5...

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are antigenic and genetically related viruses associated with different clinical syndromes in cattle, including respiratory, reproductive, neurological disease and abortion. Epidemiological studies indicate the widespread distribution of both viruses among Brazilian cattle. Serological diagnosis, that allows the identification of latently infected animals, represents an important tool for individual and herd monitoring. The present article describes the standardization of a monoclonal antibody (MAb)-based immunoenzymatic test (ELISA) for detection of antibodies to BoHV-1 and/or BoHV-5. The initial steps involved the determination of the most suitable MAb, the appropriate dilutions of viral antigen and serum samples, and the cut-off value of the assay. After standardization, the ELISA was validated by testing 506 cattle serum samples previously tested for neutralizing antibodies to BoHV-1 and BoHV-5 by virus neutralizing assay (VN). Comparing to the VN for BoHV-1 antibodies, the ELISA presented sensitivity and specificity of 96.6 percent and 98.3 percent, respectively. Positive and negative predictive values were 97.6 percent, the concordance between the tests was 97.6 percent and the coefficient of correlation k (kappa) was 0.95, demonstrating an excellent correlation. Comparing to the VN for BoHV-5 antibodies, the ELISA presented 94.3 percent of sensitivity, 97.9 percent of specificity, 97.1 percent of positive predictive value, 95.9 percent negative predictive value, concordance of 96.4 percent and kappa coefficient of 0.92. These results demonstrate that the ELISA presents suitable specificity and sensitivity to be used for individual and herd serological diagnosis of BoHV-1 and BoHV-5, thus, representing an alternative for VN assays and imported ELISA kits.

Animal infections by vaccinia-like viruses in the state of Rio de Janeiro: an expanding disease / Infecções animais por vírus semelhantes ao vaccínia no estado do Rio de Janeiro: uma doença em expansão

In the present study we investigated the presence of infections by vaccinia-like viruses in dairy cattle from 12 counties in the state of Rio de Janeiro in the last 9 years. Clinical specimens were collected from adult animals with vesicular/pustular lesions mainly in the udder and teats, and from calves with lesions around the nose and mouth. A plaque reduction neutralization test (PRNT) was applied to search for antibodies to Orthopoxvirus; the vesicular/pustular fluids and scabs were examined by PCR, electron microscopy (EM) and by inoculation in VERO cells for virus isolation. Antibodies to Orthopoxvirus were detected in most cases. The PCR test indicated a high nucleotide homology among the isolates and the vaccinia viruses (VACV) used as controls. By EM, typical orthopoxvirus particles were observed in some specimens. The agents isolated in tissue culture were confirmed as vaccinia-like viruses by EM and PCR. The HA gene of the vaccinia-like Cantagalo/IOC virus isolated in our laboratory was sequenced and compared with other vaccinia-like isolates, showing high homology with the original Cantagalo strain, both strains isolated in 1999 from dairy cattle. Antibodies to Orthopoxvirus were detected in one wild rodent (genus Akodon sp.) collected in the northwestern region of the state, indicating the circulation of poxvirus in this area. Nonetheless, PCR applied to tissue samples collected from the wild rodents were negative. Vesicular/pustular lesions in people in close contact with animals have been also recorded. Thus, the vaccinia-like virus infections in cattle and humans in the state seem to be an expanding condition, resulting in economic losses to dairy herds and leading to transient incapacitating human disease. Therefore, a possible immunization of the dairy cattle in the state should be carefully evaluated.

Neste estudo avaliou-se a presença de infecções por vírus semelhantes ao vírus vaccínia (VACV) em gado leiteiro em 12 municípios no estado do Rio de Janeiro, ao longo dos últimos nove anos. Amostras clínicas foram coletadas de animais com vesículas, pústulas e crostas no úbere e tetas, e da região do nariz e da cavidade oral de bezerros. Um teste de neutralização viral por redução de placas foi desenvolvido para investigar a presença de anticorpos contra Orthopoxvirus. Os fluidos de vesículas / pústulas e as crostas foram testadas por PCR, microscopia eletrônica (ME) e por inoculação em células VERO para isolamento viral. Anticorpos contra Orthopoxvirus foram detectados na grande maioria dos animais. O teste de PCR demonstrou homologia entre os vírus isolados e amostras de vírus vaccínia usados como controles. Na ME, partículas típicas de Orthopoxvirus foram observadas em vários espécimes analisados. Os vírus isolados em cultivo celular foram confirmados como Orthopoxvirus por PCR e ME. O gene HA da amostra Cantagalo/IOC isolada em nosso laboratório foi seqüenciado e comparado com outras amostras semelhantes ao vaccínia, mostrando uma alta homologia com a amostra original Cantagalo, tendo sido as duas amostras isoladas em 1999 de gado leiteiro. Anticorpos para Orthopoxvirus foram detectados em um roedor silvestre do gênero Akodon sp. coletado na região noroeste do estado, sugerindo uma circulação de poxvírus na natureza. No entanto, os testes de PCR aplicados a tecidos de roedores silvestres foram negativos. Infecções vesiculares / pustulares em humanos que mantinham contato com os animais afetados também foram relatadas. Assim, infecções por amostras semelhantes ao vírus VACV em bovinos e em humanos parecem em expansão no estado, gerando perdas econômicas em animais e quadros de doença incapacitante temporária em pacientes humanos. Dessa forma, a possibilidade da imunização do gado leiteiro no estado deve ser devidamente avaliada.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Características biológicas e inmunológicas del veneno de Bothrops cotiara (Serpentes: Viperidae) / Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

Bothrops cotiara es una serpiente que se encuentra en la provincia de Misiones (Argentina), el Sur de Brasil y Paraguay. La información sobre las características clínicas de los accidentes por esta serpiente es muy escasa y existen pocos datos sobre su veneno y la capacidad neutralizante de las actividades tóxicas del mismo por antivenenos terapéuticos. En este trabajo se estudiaron características bioquímicas, actividades tóxicas y la reactividad inmunoquímica del veneno de B. cotiara. Seis antivenenos anti Viperinos Sudamericanos fueron estudiados frente a este veneno por el método ELISA y se probó la capacidad neutralizante de tres de estos frente a las actividades hemorrágica, necrotizante, procoagulante, trombina-símil, hemolítica indirecta y la potencia letal de veneno de ejemplares de B. cotiara de la provincia de Misiones. Los patrones cromatográficos y electroforéticos mostraron características similares a los de otros venenos de Bothrops. Las actividades tóxicas estuvieron dentro de los ámbitos descritos para los venenos botrópicos. Los seis antivenenos mostraron gran reactividad inmunoquímica por ELISA y las potencias neutralizantes de los tres estudiados fueron muy próximas para las actividades letal, hemorrágica, necrotizante, hemolítica indirecta, coagulante y trombina-símil. Los resultados de los estudios de neutralización indicarían que ante la mordedura de esta poco común especie de Bothrops, pueden usarse los diferentes tipos de antivenenos botrópicos o botrópico-crotálicos para uso terapéutico disponibles en esa región.

Antibodies against pseudorabies virus in feral swine in southeast Brazil / Anticorpos contra o vírus da doença de Aujeszky em javalis no estado de São Paulo

Serum samples collected from 358 wild boars (Sus scrofa) in breeding farms in São Paulo, southeast Brazil, from 1998 to 2001, were tested for antibodies against pseudorabies virus (PRV) by means of serum neutralization (SN) and enzyme-linked immunobsorbent assay (ELISA). Seropositive animals were detected in three of seven herds analyzed. Overall seroprevalence as assessed by SN was 30.7 percent, ranging from 25.2 percent to 100 percent for the herds that presented seropositive animals. Indirect ELISA detected lower seroprevalence (19.3 percent). Sensitivity and specificity of ELISA were equal to 57.3 percent and 97.6 percent, respectively. Agreement was equal to 85.2 percent (P<0.0001). These results showed that PRV infections occurred in farmed feral swine in southeast Brazil, and affect pseudorabies eradication program.

Soros de 358 javalis (Sus scrofa), criados em sistema de semiconfinamento em propriedades do estado de São Paulo, foram coletados entre 1998 e 2000 e testados para anticorpos contra o vírus da doença de Aujeszky (VDA), pela técnica de soroneutralização (SN) e ensaio imunoenzimático (ELISA). Foram detectados animais soropositivos em três das sete propriedades analisadas. Do total de javalis testados, 30,7 por cento apresentaram anticorpos neutralizantes contra o VDA, com variação de 25,2 por cento a 100 por cento nas propriedades com animais sororreagentes. O ELISA detectou menor número de sororeagentes (19,3 por cento), sendo a sensibilidade e a especificidade 57,3 por cento e 97,6 por cento, respectivamente, e a correlação observada de 85,2 por cento (P<0,0001). Os resultados mostram que a infecção pelo vírus da doença de Aujeszky ocorre em criações de javalis no estado de São Paulo, e compromete o sucesso de um futuro programa de erradicação da doença na região.

Levantamento soro-epidemiológico da infecção pelo vírus da Anemia Infecciosa Eqüina, da Influenza Eqüina-2 e do Herpesvírus Eqüino-1 em rebanhos do sul do Estado do Pará, Brasil / Seroepidemiological survey of infection of equine infectious anemia, equine influenza type 2 and equine herpesvirus type 1 in herds of south Pará State, Brazil

Os vírus da anemia infecciosa eqüina (EIAV), da influenza eqüina tipo 2 (EIV-2) e o herpesvírus eqüino tipo 1 (EHV-1) são agentes causadores de enfermidades que podem causar graves prejuízos econômicos. O objetivo deste presente estudo foi estimar a freqüência de anticorpos contra o EIAV, EIV-2 e o EHV-1 em rebanhos do sul do Estado do Pará, Brasil. Os anticorpos contra EIAV, EIV-2 e EHV- 1 foram detectados pelo teste de IDGA, pelo método de inibição da hemaglutinação e pela técnica de soroneutralização (TCID50 =100), respectivamente. Amostras de sangue de 672, 514 e de 506 equídeos saudáveis e sem histórico de vacinação contra nenhum dos três vírus foram testadas, respectivamente, para EIAV, EIV-2, EHV-1. A seguinte freqüência de soro reativos foi observada: 1,34% para o EIAV; 35,79% para o EIV-2; 45,45% para o EHV-1. Estes resultados indicam que estes agentes estão presentes no rebanho paraense e a adoção de medidas de manejo e profilaxia devem ser priorizadas, garantindo deste modo, a prosperidade da eqüideocultura brasileira.

Equine infectious anemia virus (EIA V), equine influenza virus type 2 (EIV-2) and equine herpesvirus type 1 (EHV-1) are the causal agents of diseases that may bring economical Iosses. The aim of this present study was to estimate the frequency of antibodies against EIAV, EIV-2 and EHV-1 in herds of south Pará State, Brazil. Antibodies against EIAV, EIV-2 and EHV-1 were detected by AGID, hemagglutination inhibition method and serum neutralization technique (TCID50 =100), respectively. Blood samples of 572, 514, and 506 healthy equine unvaccinated against any of the three viruses were tested, respectively, for EIAV, EIV-2 and EHV-1. The following frequencies of serum reactors animals were observed: EIAV,1,34%; EIV-2, 35,79%; EHV-1, 45,45%. These results show that the agents are present in herds from Pará herds and the adoption of measures of management and prophylaxis should be prioritized, ensuring, thereby, the prosperity of brazilian's breeding equine.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

HIV-1 binding and neutralizing antibodies of injecting drug users

Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU) compared to that of individuals sexually infected with HIV-1 (S), but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B), the primary HIV-1 isolate 95BRRJ021 (genotype F), and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47) and S (20/60) groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23) than from the IDU group (15/47, P = 0.0108). No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.

Avaliação da dinâmica da resposta imune humoral desencadeada pelo papilomavirus humano / Evaluation of the dinamics of the hummoral immune response triggered by human papillomavirus

Os anticorpos neutralizantes são cruciais na proteção contra doenças virais mediadas por vacinação. A elevada proteção adquirida contra infecções persistentes causadas pelo papilomavírus humano, por meio da imunização de mulheres com capsídios vazios de HPV-16, é conferida por este tipo de anticorpos. Os anticorpos neutralizantes detectados em amostras séricas de mulheres vacinadas foram capazes de neutralizar variantes divergentes de HPV-16... Neste trabalho detectaram-se anticorpos neutralizantes em amostras séricas obtidas de mulheres não vacinadas participantes de um estudo sobre os fatores de risco para o desenvolvimento de infecções pelo HPV e das neoplasias cervicais. O impacto da presença deste tipo de anticorpos foi analisado por meio das principais manifestações virais durante o curso natural da infecção pelo HPV... A eliminação de infecções HPV-16 induzidas não pareceu ter sido facilitada pela presença de altos níveis de nenhum tipo de anticorpos reativos a HPV-16 (anticorpos neutralizantes – OR: 0,64 [IC95%: 0,16-2,58] X ELISA – OR: 0,64 [IC 95%: 0,19-2,16]... Essas correlações não foram encontradas para os anticorpos detectados por meio de ELISA. Logo, títulos de anticorpos neutralizantes equivalentes à diluição 640 ou superiores atuam como marcadores extremamente específicos da infecção causada pelo HPV-16 durante o curso habitual natural da infecções por HPV (AU)

Neutralizing antibodies (NAb) are crucial for vaccine-mediated protection against viral diseases and presumably play the key role in the high degree of protection from persistent papillomavirus infection seen after virus-like particles (VLP) vaccination in recent clinicai trials. Neutralizing antibodies detected in serum samples of HPV-16 VLP vaccinated women were able to neutralize divergent variants of HPV-16 and their detection was achieved for serum samples collected from both vaccinated and non-vaccinated women through a HPV pseudovirus neutralization assay. However, little is known about the induction of NAb after genital HPV infections among nonvaccinated women and the putative role that these antibodies could play during the natural course of HPV infections. This work looked for HPV-16 NAbs in serum samples obtained from non-vaccinated women participating of a study for HPV infection and neoplasia risk factors. The impact of NAbs presence was analyzed through viral major outcomes during the natural course of HPV infection. The efficiency of Nabs was compared to HPV VLP ELISA seroreactivity in a 150 serum samples casuistic obtained at the enrollment of the study. The clearence of HPV-16 infection did not appear to be improved by the presence of high titers of any kind of anti-HPV-16 antibodies (Nabs- OR: 0,64 [CI 95o/o: O, 16-2,58] X ELISA- )R: 0,64 [CI95°/o: O, 19-2, 16]). Women with neutralizing antibodies titers equal ar superior to 640 dilutions showed higher risks of harboring the following HPV outcomes: cumulative HPV-16 infection (OR: 6,24 [CI95°/o: 0,88-270,1 ]), persistence of HPV-16 infection (OR: 5,17 [CI95°/o: 1,50-17,83] to OR: 27,33 [CI95°/o: 2,02-1376,6], according to the reference category used - transient or negative results, respectively), acquisition of new HPV-16 infection (OR: 3,45 [CI95°/o: O, 17-207 ,2]) and higher HPV vira I load (OR: 4,05 [CT95°/o: 1 ,05-15,66]). The same was not observed for women with higher titers of HPV-16 VLP seroreactivity. Neutralizing antibodies titers equal or superior to the 640 dilution play an important role as highly specific markers for HPV-16 infection during HPV natural history course (AU)

Detección de anticuerpos antirrábicos en personal de riesgo con el empleo de la técnica de neutralización por reducción del número de placas / Detection of antirabic antibodies in personnel at risk through the use of the neutralization technique by reducing the number of plaques

Se empleó la técnica de neutralización por reducción del número de placas (NRNP) para la detección de anticuerpos antirrábicos en personal de riesgo. Se estudiaron muestras de suero de individuos de alto riesgo y personas con antecedentes de vacunación y sin estos. La técnica de neutralización fue comparada con la prueba biológica en ratón y como resultado del estudio se obtuvo una concordancia de 100 por ciento. Se encontró que el sexo no influye en la respuesta de anticuerpos a este virus, aunque el contacto directo y la cantidad de veces que el individuo ha recibido la vacuna actúan positivamente en la respuesta inmune. La introducción de esta técnica en el laboratorio permite contar con una herramienta útil, en el seguimiento del personal que recibe vacunación preexposición y posexposición al virus rábico

Detection of Coxsackievirus B 4 antibodies in cases of suspected encephalopathy by microneutralization test.

A retrospective study of serum and cerebrospinal fluid (CSF) samples collected from suspected viral encephalitis and encephalopathy cases was carried-out and it included 100 CSF and 89 serum samples from Goa, collected during 1990-1994. These samples which were negative for antibodies to Japanese encephalitis (JE), West Nile (WN), Dengue-2 (DN-2) and herpes viruses, were tested for Coxsackievirus B 4 specific antibodies by 'in vitro' microneutralization technique along with 80 negative control serum samples. Out of 189 specimens (100 CSF and 89 serum), 23 CSF and 41 serum samples were positive for Coxsackievirus B 4 neutralizing antibodies. Antibody profile seemed to be IgG as revealed by mercaptoethanol treatment. The presence of neutralizing antibodies to coxsackievirus B 4 with titres as high as 1:512 in 8 CSF and 19 serum samples seemed to be suggestive of viral meningitis due to Cox B-4 viruses.

Application of HIV-1-green fluorescent protein (GFP) reporter viruses in neutralizing antibody assays.

We made reporter HIV-1 DNA constructs carrying green fluorescent protein (GFP) gene and exchangeable env of subtype E. The recombinant constructs were used to produce infectious reporter viruses, which induced infected cells to emit green fluorescent light and rendered them easily detectable at single cell level. Because the env in this construct can be easily exchanged, viruses with different antigenic epitopes can be made. We used these reporter viruses to set up a neutralizing antibody assay based on fluorescence reduction by flow cytometric measurement. The result of this new assay correlated with the standard infectivity reduction assay using primary isolates. Because this new assay is faster and much less costly than the standard assay using a p24 endpoint and can be performed in peripheral blood mononuclear cells (PBMC), it provides a useful tool for analysis of HIV-1 immune responses.

Anticuerpos neutralizantes frente a 5 cepas de VIH-1 en macacos inmunizados con el polipéptido multiepitópico TAB9 / Neutralizing antibodies to 5 HIV-1 strains in macaques immunized with the multi-epitope polypeptide TAB9

Presence of neutralizing antibodies(Nab) to 5 laboratory strains of human immunodeficiency virus type-1 (HIV-1) was studied and compared in the sera of eight macaca (Macaca fascicularis) after the third and fourth immunizations with multi-epitope polypeptide TAB9, emulsified with adjuvant Montanide ISA 720. Four animals were inoculated 1 mg and the others 200 micrograms, 2 animals were used as controls and injected only with adjuvant. Although the presence of neutralizing antibodies to homologous strains of HIV-group group B was confirmed in most animals after immunization through antigen p24 capture ELISA (DAVIH-Agp24, Cuba), no statistically significant differences were found neither in titers caused by antigen concentrations nor in the responses after the third and fourth immunizing doses. Controls did not develop neutralizing antibodies.

Seroprevalencia de infeccion por el virus encefalitis San Luis en la Provincia de Formosa / Seroprevalence of infection by St. Louis encephalitis virus in the Province of Formosa

El objetivo del presente trabajo fue conocer la prevalencia de anticuerpos para el virus encefalitis San Luis (ESL) en sueros humanos del Laboratorio de Vigilancia Epidemiológica del departamento Formosa (provincia de Formosa, Argentina), extraídos en los años 1995 y 1997, se emplearon las pruebas de inhibición de la hemaglutinación (IH) y de neutralización (NT). El tamizaje realizado mediante la prueba de NT mostró una prevalencia del 21 por ciento (60/284) y 32 por ciento (50/157) en las muestras obtenidas en los años 1995 y 1997 respectivamente. El 14 por ciento de los sueros del año 1995 presentaron títulos de anticuerpos neutralizantes (NT) bajos (1/20 y 1/40) mientras que en el año 1997 el 19 por ciento de los sueros tenían títulos de anticuerpos NT iguales o mayores a 1/80. En la titulación simultánea de anticuerpos por NT e IH se observaron sueros con títulos bajos de anticuerpos NT (1/20-1/40) y negativos en IH, mientras que otros poseían títulos altos por ambas pruebas. Esta relación entre los títulos de anticuerpos indica la presencia de infecciones pasadas y recientes y la circulación continua de este virus. Por otra parte la prevalencia de anticuerpos NT en la población estudiada se incrementó significativamente en 2 años (p < 0.0075), lo que confirma la endemicidad de este agente en la zona y muestra la necesidad de realizar estudios de las enfermedades febriles de etiología viral no confirmadas, a fin de conocer su importancia como patógeno humano en nuestro país.

Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes

An indirect hemagglutination test for a seroepidemiological survery of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1 per cent glutaraldehyde at 56 degrees Celsius for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4 degrees Celsius. A total of 564 serum samples with high, moderate and low anti-streptolysin 0 antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (r(s)=0.726). Five reagent batches sucessively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.

Neutralizing antibodies in brazilian sera against three strains of human immunodeficiency virus type 1 (HIV-1)

A cepa MN do HIV-1 e conhecida por ser mais prevalente no Brasil, a cepa BRU e mais prevalente na Europa e a cepa NDK na Africa. Tem sido sugerido na literatura que diferentes cepas devam ser incluidas em uma mesma vacina contra o HIV-1. Com o intuito de se contribuir para o desenvolvimento de uma vacina universal, a ocorrencia de anticorpos (Ac) contra tres cepas do HIV-1 (MN, BRU, NDK) foram determinadas em amostras de soros de 85 pacientes voluntarios, adultos, soropositivos para o HIV-1, atendidos em um Hospital Universitario. Cem unidades infectantes para cultura de tecido (TCIU) dos virus reagiram com diluicoes seriadas de soro (2x) e celulas MT4 adicionadas a uma concentracao final de 0,3 x 10 elevado a sexta potencia celulas/ml...