SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Molecular evolution of coxsackievirus A24v in Cuba over 23-years, 1986-2009.

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10-3 and 5.80 × 10-3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.

Induction of neutralizing antibodies and partial protection from viral challenge in Macaca fascicularis immunized with recombinant dengue 4 virus envelope glycoprotein expressed in Pichia pastoris.

A recombinant vaccine that expresses the envelope (E) gene of dengue virus type 4 was tested for immunogenicity and protection in Macaca fascicularis. One hundred micrograms of semipurified recombinant E protein (E4rec) expressed in Pichia pastoris was used to immunize three animals. Neutralizing antibodies to dengue 4 virus with a titer of 1:30 were detected in all immunized monkeys prior to challenge. Animals were challenged with 10(5) plaque-forming units of dengue 4 virus. One vaccine-immunized monkey was protected from viremia, while the other two were partially protected. Monkeys immunized with E4rec elicited the highest neutralizing antibody titers (P < 0.05) ranging from 1:85 to 1:640 at day 30. In both immunized and control animals, the longest duration of viremia correlated with earliest and highest level of IgM antibody to dengue virus. The vaccinated animals showed anamnestic antibody responses upon virus challenge, indicating successful priming by the recombinant vaccine. Our results suggest that E4rec expressed in P. pastoris can provide partial protection against viremia. However, the results were not effective enough to use it as a vaccine candidate. Further work is required to improve the quality of the immunogen.

Prevalence of Chlamydia trachomatis in human immunodeficiency virus-infected women in Cuba

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10 percent in group I and the estimated prevalence was 6.6 percent for group II; 83.3 percent of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50 percent). The control group C. trachomatis-infected women referred a history of cervicitis in 75 percent of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50 percent. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups

Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively

Aplicacion de la técnica de reacción en cadenade la polimerasa para la detección de secuencias de Papillomavirus humano / Aplication of polymerase chain reaction to detect sequences of human Papillomavirus

Se aplicó la técnica dereacción en cadena de la polimerasa para la detección de secuencias de papillomavirus humano (PVH) mediante controles de líneas celulares de cáncer cervical y tejidos obtenidos por biopsia con diagnóstico clínico positivo a PVH. Se utilizóun juego de oligonucleótidos consenso, que son complementarios a una región altamente conservada dentro del marco de lectura abierta. El del genoma viral de los PVH que afectan la mucosa cervical. Con este juego de cebadores fue posible amplificar secuencias de ácido desoxirribonucleico (ADN) correspondientes a los PVH 6 y 11, considerados dentro del grupo de bajo riesgo y de los PVH 16, 18, 31 y33 comprendidos en el grupo de alto riesgo. El estudio de la sensibilidad de latécnica de amplificación arrojó como resultado un nivel de detección de 3,5 partículas virales por cada genoma diploide celular

Estudio por Western Blot de sueros de pacientes con neuropatía epidémica / Study by Western Blot of sera from patients with epidemical neuropathy

Se estudió la respuesta inmune de un grupo de pacientes con neuropatía epidémica y de individuos controles mediante la tècnica de inmunoblotting frente a las proteinas del virus Coxsackie y a las proteínas de la cepa de efecto lento aislada en nuestro laboratorio. Se estudiaron 13 sueros de pacientes con neuropatía epidémica y 9 sueros controles. De los 13 sueros estudiados, 8 (61,5 por ciento) reconocieron a la proteína VPI y 2 sueros (15,3 por ciento) a la proteína VPO de la cepa 47/93. De los 9 controles estudiados, 4 (44,4 por ciento) reconocieron la proteína VPI y 3 sueros (33,3 por ciento) la proteína VPO solamente. Con el antígeno preparado a partir de la cepa de efecto lento, en 5(38,5 por ciento) sueros de pacientes con 2 sueros (22,5 por ciento) de controles se obtuvo una señal específica. Es de destacar en este último caso que la proteína observada tenía un peso molecular de 41 300 D, era de menor talla que la proteína precursora detectada frente a la cepa 47/93, que fue de 45 000 D

Neuropatia epidemica cubana. Parte II. Algunas caracteristicas antigenicas de los aislamientos virales / Cuban epidemic neuropathy. Part II. Certain antigenic characteristics of viral isolations

Con el objetivo de caracterizar antigenicamente los aislamientos productores de efectos citopatico ligero (ECP-L) obtenidos del liquido cefalorraquideo (LCR) de pacientes con neuropatia epidemica se realizaron prueba de Western Blot y neutralizacion utilizando sueros hiperinmunes de pacientes y sueros de conejos hiperinmunes. Se comprobo que los aislamientos de ECP-L estudiados tienen iguales caracteristicas antigenicas y estan relaciondos con los virus Coxsackie A9 y B4. No se pudo detectar en las cepas de ECP-L proteinas estructurales y solamente se comprobaron antigenos que por su alto peso molecular fueron considerados proteinas precursoras del ciclo de replicacion viral

Dengue 2 virus enhancement in asthmatic and non asthmatic individual

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorragic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X* p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data

Diagnóstico de la infección por Citomegalovirus en pacientes infectados por el VIH. Comparación de las técnicas de inmunofluorescencia indirecta y aislamiento viral / Diagnosis of cytomegalovirus infection in patients presenting with HIV infection. Comparison of indirect immunofluorescence technique and viral isolation

Se comparó la técnica para la detección de antígenos precoces fluorescentes (DAPF) con el método clásico de aislamiento viral en células de pulmón humano (PH). El estudio fue a partir de 85 muestras de orina provenientes de 64 pacientes infectados con el virus de la inmunodeficiencia hunam (VIH). La técnica de DAPF presentó una sensibilidad del 91 por ciento , especificidad del 97 por ciento y coincidencia del 94 por ciento con respecto al aislamiento viral. La principal ventaja que presenta la DAPF con respecto al aislamiento viral es que brinda un diagnóstico acelerado de infección por Citomegalovirus en 48 a 72 h, además de ser muy fácil de ejecutar

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CVM) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CVM from urine and the technical advantage of each is discussed.

Identificación mediante la técnica de inmunofluorescencia indirecta de varias cepas de dengue aisladas durante la epidemia de Nicaragua en 1985 / Identification by indirect immunofluorescence technique of several strains of dengue isolated during the epidemic of Nicaragua in 1985

Se describe la metodología seguida para la identificación, mediante la técnica de inmunofluorescencia indirecta, de varias cepas de dengue aisladas durante la epidemia de Nicaragua en 1985. De las 24 cepas identificadas en este estudio, 16 correspondieron al serotipo 1, y 8 al serotipo 2. Las 16 cepas de dengue 1, aisladas en las células de mosquito C6/36 fueron identificadas mediante diluciones seriadas de los líquidos ascíticos hiperinmunes, preparados contra los 4 serotipos de dengue. Las 8 cepas de dengue 2, aisladas en ratón lactante fueron identificadas con anticuerpos monoclonales. Se discuten algunos aspectos relacionados con las dificultades técnicas confrontadas al utilizar los anticuerpos monoclonales en la identificación