Celso Ramos

Dengue virus in the brain of a fatal case of hemorrhagic dengue fever

Neurologic complications associated with dengue fever are in general unusual. However, recent reports evidence more frequent neurologic alterations. In Mexico, neurologic involvement has not been reported in dengue cases. This report demonstrates the detection of dengue virus in the brain of a fatal case of dengue hemorrhagic fever. Serotype 4 was detected by immunohistochemistry and by RT-PCR in the inferior olivary nucleus of medulla and in the granular layer of cerebellum. Immunoreactivity was observed in neurons, astrocytes, microglia and endothelial cells. Our results emphasize the importance of neurologic manifestations in patients with dengue fever.

HLA-DR antigen frequencies in Mexican patients with dengue virus infection: HLA-DR4 as a possible genetic resistance factor for dengue hemorrhagic fever.

The human leukocyte antigen DRB1 locus (HLA-DRB1) was typed in genomic DNA extracted from whole blood samples of 34 Mexican dengue hemorrhagic fever (DHF) patients and 47 dengue fever (DF) patients, by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot. HLA-DRB1*04 was negatively associated with risk of DHF (OR 0.31, 95% CI 0.11-0.85). HLA-DR4 homozygous individuals were 11.6 times less likely to develop DHF in comparison to DR4 negative persons (OR 0.08, 95% CI 0.01-0.75). After adjusting for gender and infection type by logistic regression, DR4 positive individuals were 3.6 times less likely to develop DHF than DR4 negative persons (OR 0.28, 95% CI 0.12-0.66). A secondary dengue virus infection was also positively linked with DHF risk (OR 2.89, 95% CI 0.92-9.07). This data suggests that genes of the major histocompatibility complex play a major role in the susceptibility and/or resistance to develop DHF. In Mexicans, HLA-DR4 may be a genetic factor that is protective against DHF. Because HLA-DR4 has been positively selected in Latin American populations, these results may apply also to other similar ethnic groups, particularly those with high percentages of admixture with indigenous Amerindian genes.

A 65-kDa trypsin-sensible membrane cell protein as a possible receptor for dengue virus in cultured neuroblastoma cells

Dengue virus infects primary neurons in mouse experimental model and tissue culture cells of the central nervous system (CNS). In the present work, a mouse neuroblastoma cell line (N1E-115) and a human neuroblastoma cell line (SK-N-SH), susceptible to dengue virus infection were used to study the presence of cell membrane receptor for dengue-2. By day 5 postinfection (pi), viral antigen was detected by immunofluorescence in the cytoplasm and surrounding the nucleus of N1E-115 cells, while on day 7 pi, it was also present along neural extensions. Infection of N1E-115 cells was diminished with trypsin treatment but not with neuraminidase or endoglycosidase H. Partially purified cell membrane proteins from neuroblastoma cells were analyzed by the Virus Overlay Protein Blot Assay (VOPBA), and a single band migrating at 65 kDa was detected in mouse and human neuroblastoma cells but not in C6, a non-susceptible rat glial cell line which was included as a negative control. The 65 kDa protein was eliminated only when nitrocellulose membranes were treated with trypsin. Analysis of neuronal cell infection by dengue virus provides a useful tool to understand the nature of cellular receptors and mechanisms involved in the infection of the nervous system by dengue viruses.

La influenza, un problema vigente de salud pública

La influenza estacional es una enfermedad respiratoria aguda, recurrente y comun que se conoce desde la antiguedad y se presenta sobre todo durante los meses de invierno con un elevado impacto para la salud publica mundial. La enfermedad se manifiesta con altas tasas de morbilidad en individuos de todas las edades y elevadas tasas de mortalidad en ninos, individuos mayores de 60 anos, pacientes con enfermedades cronicas y mujeres en gestacion. Las estrategias de prevencion incluyen el uso de vacunas: inactivadas, subunitarias o vacuna con virus geneticamente modificados. Dos subtipos de virus de influenza tipo A y un virus de influenza tipo B causan la enfermedad en humanos. Los virus de influenza A que afectan a los humanos mutan con facilidad, por lo que con frecuencia aparecen nuevas variantes antigenicas de cada subtipo, lo que obliga a incluir dichas variantes en las vacunas anuales para brindar una adecuada proteccion a la poblacion. La influenza pandemica se refiere a la introduccion y posterior diseminacion mundial de un nuevo virus de influenza en la poblacion humana, lo que ocurre de manera esporadica, y que debido a que los humanos carecen de inmunidad para el nuevo virus pueden suscitarse epidemias graves con elevadas tasas de morbilidad y mortalidad. Historicamente el origen de las pandemias de influenza se debe a la transmision de virus de aves al hombre o la transferencia de genes de estos a los virus de la influenza estacional. En las aves acuaticas silvestres, tanto migratorias como costeras, se mantiene una gran diversidad de subtipos de virus de influenza, los cuales se introducen eventualmente en aves domesticas, donde algunos virus adquieren la capacidad de infectar a mamiferos, incluido el hombre. El proceso de adaptacion de los virus aviarios a hospederos mamiferos requiere tiempo, por lo que la presentacion de estos casos puede tardar varios anos. Desde diciembre de 2003, en varios paises del sureste asiatico, las aves domesticas han sido afectadas por una epidemia de influenza aviaria (subtipo H5N1) de grandes proporciones. A febrero de 2006 la epidemia ya afecto a paises de Europa y Africa, con un fuerte impacto economico para la avicultura comercial por el sacrificio de mas de 180 millones de aves. Algunos linajes de este virus adquirieron la capacidad de cruzar la barrera de especie e infectaron de manera directa pero incipiente a la poblacion humana. El virus todavia no adquiere del todo la eficiencia de infeccion y transmision de persona a persona, lo que ha limitado su diseminacion en humanos. Debido a que la tasa de mortalidad en los individuos afectados con este virus es mayor de 50%, la Organizacion Mundial de la Salud (OMS) convoco a los paises miembros para que establezcan planes de preparacion y emergencia ante la amenaza de una posible pandemia causada por el virus H5N1 o por algun virus reasociante con el de la influenza comun; estas acciones han de evitar o disminuir el impacto de la amenaza y asi impedir catastrofes previas como la de 1918, en la que murieron alrededor de 40 millones de personas en todo el mundo. Los planes de prevencion y control previstos consideran, entre otras actividades, la vacunacion y el uso de medicamentos antiviricos. Sin embargo, a la fecha no hay vacunas aplicables en caso de emergencia por un virus pandemico de influenza y tal vez los paises que fabrican la vacuna anual del tipo endemico carezcan de capacidad para producir la del virus pandemico. Ademas, se han detectado cepas de los virus resistentes a los agentes antiviricos de uso comun. La presente revision pretende actualizar los conceptos basicos sobre la influenza con el fin de fortalecer la vigilancia epidemiologica de la enfermedad y resaltar las acciones de prevencion y control en caso de que acontezca una pandemia.

HLA class I and II polymorphisms in Mexican Mestizo patients with dengue fever

Host genetics in dengue hemorrhagic fever (DHF) pathophysiology has not been extensively investigated. Most studies have focused on HLA in different populations; however these reported associations have not been replicated. We performed a case-control study to analyze possible associations of HLA-A, HLA-B, HLA-Cw, HLA-DRB1 and HLA-DQB1 alleles with clinical disease severity caused by dengue virus infection. Our population consisted of 39 individuals (DF: 23, DHF: 16) and 34 healthy controls from the State of Morelos, Mexico. HLA loci were genotyped by nucleotide sequencing method. Statistical analyses revealed associations in three alleles: HLA-B*35 was negatively associated with symptomatic disease (p<1x10(-4), p(c)=0.01, OR=0.12, 95%CI=0.037-0.39), and DF (p=0.0007, p(c)=0.03, OR=0.13, 95%CI=0.031-0.51). HLA-DQB1*0302 was positively associated with DHF (p=0.018, p(c)=NS, OR=5.02, 95%CI=1.05-25.34), and negatively with DF (p=0.011, p(c)=NS, OR=0.23, 95%CI=0.06-0.84). HLA-DQB1*0202 was positively associated with DF only (p=0.012, p(c)=NS, OR=7.0, 95%CI=1.11-73.8). We identified possible associations of HLA-B and HLA-DQB1 alleles with the risk of developing symptomatic disease, DF and DHF in a Mexican Mestizo population.

Genetic diversity of hantaviruses in Mexico: identification of three novel hantaviruses from Neotominae rodents.

A variety of hantaviruses are harbored by rodents in North and South America, some of which can cause hantavirus pulmonary syndrome. To obtain greater evolutionary insight into hantaviruses in the Americas, a total of 211 rodents were captured in the Mexican states of Guerrero and Morelos in 2006. Anti-hantavirus antibodies were detected in 27 of 211 serum samples (12.8%) by ELISA. The distribution of seropositive rodents was: 17 Peromyscus beatae, 1 Megadontomys thomasi, 1 Neotoma picta, 6 Reithrodontomys sumichrasti, and 2 Reithrodontomys megalotis. The hantavirus small (S), medium (M), and large (L) genome segments from P. beatae, R. sumichrasti, and R. megalotis were amplified and the sequences covering the open reading frames were determined. The hantaviruses from P. beatae, R. sumichrasti, and R. megalotis were provisionally designated Montano (MTN), Carrizal (CAR), and Huitzilac (HUI), respectively. The M segment amino acid identities among the Mexican hantaviruses were 80.8-93.0%. When these M segments were compared to those of known hantaviruses, MTN virus was most closely related to Limestone Canyon (LSC) virus (88.9% amino acid identity), while the CAR and HUI viruses were most closely related to El Moro Canyon (ELMC) virus (90-91% identity). Phylogenetic analysis revealed that the MTN, CAR, and HUI viruses occupy a monophyletic clade with the LSC, ELMC, and Rio Segundo viruses, which are harbored by Peromyscus boylii, R. megalotis, and Reithrodontomys mexicanus, respectively. The data obtained in this study provide important information for understanding the evolution of hantaviruses in the Americas.

Cytokine production in brain of mice experimentally infected with dengue virus.

Dengue virus encephalopathy was experimentally induced in newborn mice by i.p. inoculation of dengue 2 virus. At 6 and 9 days post-infection, motor incoordination and posterior limb paralysis were observed with focal necrosis, apoptotic cells, perivascular inflammatory cuffing and astrocytosis, mainly in the cortex and hippocampus. Expression of dengue virus genome and mRNA encoding for TNF-&agr;, IL-1&agr;, IL-2, IL-6, IL-1&bgr;, IL-12 p40, IFN&agr;, and IFN&bgr;; in addition host survival and inflammatory response genes MAC-1, EB22, GFAP, ICAM-1 and A20, were increased, suggesting that experimental dengue encephalitis could be associated with virus replication, inflammatory cytokine production or both. Similar findings have been observed in human dengue virus infection. Therefore, our results can be useful to elucidate and support the physiopathology of the disease.

Prevalencia de infección por el virus del Nilo Occidental en dos zoológicos del estado de Tabasco

Objective. To determine the prevalence of West Nile Virus (WNV) infection in animals, mosquitoes and employees from two zoos of Tabasco state, Mexico. Material and Methods. WNV antibodies were detected by blocking ELISA in serum samples from animals. Viral RNA was detected by RT-PCR from mosquitoes and serum samples from employees at “Yum-Ka” zoo. Results. Seroprevalence in birds was 25.65% (19/74) and 85% (6/7) in reptiles from “La Venta” zoo. Thirty-one percent of birds (50/160) and 34.48% mammals (16/29) at the “Yum-Ka” zoo, were seropositive. All human serum samples from Yum-ka zoo were negative by RT-PCR. A pool of mosquitoes (Culex quinquefasciatus) was positive for WNV. Conclusions. The presence of WNV antibodies in animals from both zoos and the detection of viral genome in mosquitoes demonstrate the presence of WNV in this region and indicates a potential risk of infection in animals and humans.

The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses

The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.

Los hantavirus causantes de la fiebre hemorrágica con síndrome renal y del síndrome pulmonar

Este trabajo de revision se enfoca en el analisis de la informacion basica sobre los hantavirus, agentes causales de dos enfermedades importantes en salud publica: la fiebre hemorragica con sindrome renal (FHSR) y el sindrome pulmonar por hantavirus (SPH), dos zoonosis distribuidas en Asia/Europa y el continente americano, respectivamente. Los hantavirus se transmiten al hombre a traves de la manipulacion y contacto directos de roedores infectados o tejidos y secreciones (orina, heces y saliva). La FHSR y el SPH comparten algunas caracteristicas clinicas, aunque las hemorragias y la afectacion renal son propias de la FHSR,y los problemas respiratorios del SPH. Se aportan algunos datos sobre estudios realizados en Mexico sobre hantavirus y se mencionan las condiciones ecologicas vinculadas con la distribucion de los virus y sus reservorios naturales, asi como algunas medidas para evitar o reducir el riesgo de infeccion.