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 Characterization of Coxsackievirus A24v from Feces and Conjunctiva Reveals Epidemiological Links.

Coxsackievirus A24 variant (CVA24v), the main causative agent of acute hemorrhagic conjunctivitis (AHC), can be isolated from both the eyes and lower alimentary tract. However, the molecular features of CVA24v in feces is not well-documented. In this study, we compared the VP1 and 3C sequences of CVA24v strains isolated from feces during AHC epidemics in Cuba in 1997, 2003, and 2008-2009 with those obtained from conjunctival swabs during the same epidemic period. The sequence analyses of the 3C and VP1 region of stool isolates from the three epidemics showed a high degree of nucleotide identity (ranging from 97.3-100%) to the corresponding conjunctival isolates. The phylogenetic analysis showed that fecal CVA24v isolates from the 1997 and 2003 Cuban outbreaks formed a clade with CVA24v strains isolated from conjunctival swabs in Cuba and other countries during the same period. There were three amino acid changes (3C region) and one amino acid change (VP1 region) in seven CVA24v strains isolated sequentially over 20 days from fecal samples of one patient, suggesting viral replication in the intestine. Despite these substitutions, the virus from the conjunctival swab and fecal samples were genetically very similar. Therefore, fecal samples should be considered as a reliable alternative sample type for the routine molecular diagnosis and molecular epidemiology of CVA24v, also during outbreaks of AHC.

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.

Vaccine-mismatched influenza B/Yamagata lineage viruses in Cuba, 2012-2013 season.

Annual trivalent influenza vaccines contain one of influenza B lineages; influenza B/Victoria-lineage or influenza B/Yamagata viruses. Theoretically, these vaccines should protect against viruses expected to circulate in the next influenza season. The National Influenza Centers, based on surveillance data from National Reference Laboratories, selects the strains composing each annual trivalent or tetravalent vaccine. Nevertheless, in some epidemics, vaccine strains do not match genetically with circulating strains. The aim of the present study is to compare the HA1-domain of 42 influenza B viruses circulating in Cuba during the 2012-2013 season with the vaccine strain B/Wisconsin/01/2010-like virus from the B/Yamagata lineage, included in the 2012-2013 Northern-Hemisphere Influenza vaccine. The efficacy of the influenza vaccine was also estimated. The analysis of the present study indicates that the B/Victoria and B/Yamagata lineages co-circulated in Cuba in the 2012-2013 season. In 2012-2013 season, according to the sequences analysis, trivalent vaccine did not match with the circulating strains. The present study also detected amino acid substitutions which could have altered the antigenic properties of HA gene. The results presented here suggest the need to consider a possible introduction of tetravalent influenza vaccine in Cuba, as has been recommended by the WHO to ensure higher levels of protection.

New genetic variants of influenza A(H1N1)pdm09 detected in Cuba during 2011-2013.

Influenza A(H1N1)pdm09 virus has evolved continually since its emergence in 2009. For influenza virus strains, genetic changes occurring in HA1 domain of the hemagglutinin cause the emergence of new variants. The aim of our study is to establish genetic associations between 35 A(H1N1)pdm09 viruses circulating in Cuba in 2011-2012 and 2012-2013 seasons, and A/California/07/2009 strain recommended by WHO as the H1N1 component of the influenza vaccine. The phylogenetic analysis revealed the circulation of clades 3, 6A, 6B, 6C and 7. Mutations were detected in the antigenic site or in the receptor-binding domains of HA1 segment, including S174P, S179N, K180Q, S202T, S220T and R222K. Substitutions S174P, S179N, K180Q and R222K were detected in Cuban strains for the first time.

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.

Dengue vaccine: priorities and progress.

Dengue transmission has increased considerably in the past 20 years. Currently, it can only be reduced by mosquito control; however, the application of vector-control methods are labor intensive, require discipline and diligence, and are hard to sustain. In this context, a safe dengue vaccine that confers long-lasting protection against infection with the four dengue viruses is urgently required. This review will discuss the requirements of a dengue vaccine, problems, and advances that have been made. Finally, new targets for research will be presented.

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

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% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. 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

Aplicación de la técnica de reacción en cadena de la polimerasa para la detección de secuencias de Papillomavirus humano / Application of polymerase chain reaction to detect sequences of human Papillomavirus

Se aplicó la técnica de reacción 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 utilizó un 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 y 33 comprendidos en el grupo de alto riesgo. El estudio de la sensibilidad de la técnica de amplificación arrojó como resultado un nivel de detección de 3,5 partículas virales por cada genoma diploide celular(AU)

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

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(AU)