In vitro susceptibility of eighteen clinical isolates of human monkeypox virus to tecovirimat

BACKGROUND In 2022, an outbreak of mpox that started in European countries spread worldwide through human-to-human transmission. Cases have been mostly mild, but severe clinical presentations have been reported. In these cases, tecovirimat has been the drug of choice to treat patients with aggravated disease. OBJECTIVES Here we investigated the tecovirimat susceptibility of 18 clinical isolates of monkeypox virus (MPXV) obtained from different regions of Brazil. METHODS Different concentrations of tecovirimat were added to cell monolayers infected with each MPXV isolate. After 72 hours, cells were fixed and stained for plaque visualization, counting, and measurement. The ortholog of F13L gene from each MPXV isolate was polymerase chain reaction (PCR)-amplified, sequenced, and the predicted protein sequences were analyzed. FINDINGS The eighteen MPXV isolates generated plaques of different sizes. Although all isolates were highly sensitive to the drug, two showed different response curves and IC50 values. However, the target protein of tecovirimat, F13 (VP37), was 100% conserved in all MPXV isolates and therefore does not explain the difference in sensitivity. MAIN CONCLUSIONS Our results support screening different MPXV isolates for tecovirimat susceptibility as an important tool to better use of the restricted number of tecovirimat doses available in low-income countries to treat patients with mpox.

Comentario en torno al artículo "A propósito del bicentenario de la independencia de Colombia: las prácticas de lectura de Antonio Nariño y el desarrollo de una vacuna presuntamente efectiva contra la viruela" / Comment on the article "With regard to the bicentennial of the independence of Colombia: Reading practices of Antonio Nariño and the development of a presumably effective vaccine against smallpox"

Resumen | La viruela significó para las colonias americanas un proceso que desestabilizaba de forma dramática las dinámicas sociodemográficas de las colonias, lo que incentivó el desarrollo de estudios científicos sobre el virus causante. Cada libro acerca de la viruela en la biblioteca de Nariño constituyó una herramienta en la lucha contra el virus emprendida por el prócer. Tras la revisión del artículo "A propósito del bicentenario de la independencia de Colombia: las prácticas de lectura de Antonio Nariño y el desarrollo de una vacuna presuntamente efectiva contra la viruela" quise comentar y profundizar en torno al saber médico de Nariño mediante el acercamiento a las obras a las que recurrió para instruirse sobre la enfermedad. A partir de la semblanza de cada una de ellas, analicé el proceso de variolización en el Reino de la Nueva Granada y la necesidad de fabricar una vacuna propia.

Abstract | For the American colonies, smallpox implied a process that dramatically destabilized their sociodemographic dynamics, which explains why scientific development took place around the causative virus. Each book about smallpox in Nariño's library was a tool in the fight against smallpox undertaken by the founding father. After reviewing the article "About the bicentennial of the independence of Colombia: The reading practices of Antonio Nariño and the development of a vaccine that is presumably effective against smallpox", I set myself to study Antonio Nariño's medical knowledge further. Through the approach to the works that Nariño used to educate himself on smallpox and the development of a biographical sketch of each of them, I analyzed the process of variolization in the Kingdom of Nueva Granada and the need to manufacture a vaccine locally.

Vaccinia zoonótica en Colombia: evidencia acumulativa de la emergencia de los poxvirus en el mundo / Zoonotic vaccinia in Colombia: Cumulative evidence of the emergence of poxviruses in the world

RESUMEN La reciente ocurrencia de infecciones por el virus vaccinia en animales y humanos en distintos lugares de la geografía colombiana, sumadas a otras por éste y por otros virus pertenecientes al género Orthopoxvirus (familia Poxviridae), ocurridas en algunos países de Suramérica, África, Asia y Europa se convierten en evidencia de la inminente emergencia y re-emergencia de este género, con características biológicas y epidemiológicas que le confieren gran interés para la salud pública del mundo, como lo fue en el pasado una de sus especies representativas: el virus de la viruela. Esta emergencia y re-emergencia parecen estar relacionadas con la suspensión en las décadas de los 70s y 80s de las campañas de vacunación contra la viruela, las cuales; insospechadamente estuvieron protegiendo a la población, no únicamente contra este virus, sino contra otros del mismo género. En el presente artículo se hace una revisión de la biología y epidemiología de los principales miembros del género Orthopoxvirus, su presentación clínica, antecedentes históricos, contexto social, e impacto en la salud pública mundial en el pasado, presente y a futuro.(AU)

ABSTRACT The recent occurrence of vaccinia virus infections in humans and animals in Colombia, together with that reported for this and other species of the genus Orthopoxvirus in some South American, African, Asian and European countries, is supporting evidence of the emergence and re-emergence of the genus. This fact has become of great interest for public health around the world due to its biological and an epidemiological features, as was in the past the variola virus, one of its representatives. The emergence and re-emergence of the genus Orthopoxvirus may be a consequence of stopping vaccination against the variola virus in the 1970s and 1980s. This vaccination unsuspectedly induced cross-protective immunity to other species of that genus. This is a review of the history, biology and epidemiology of the main species of the genus Orthopoxvirus, together with its clinical presentation, social context and public health impact in the past, present and future.(AU)

Preliminary exploration of replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan / 中华实验和临床病毒学杂志

Objective@#To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan.@*Methods@#We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected.@*Results@#We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot.@*Conclusions@#The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Advance in the development of modified vaccinia virus Ankara (MVA)vectors for novel influenza vaccines / 中国人兽共患病学报

Influenza,caused by influenza virus,is a respiratory infectious disease with a serious hazard to human health.Prevention of influenza through vaccine development is the most effective strategy.It is important to build a rapid response platform for research and production of influenza vaccine.As virus vectors,live vaccine provides a new prevention and treatment way for infectious disease.Modified vaccinia virus Ankara(MVA) is a replication-deficient viral vector that is safe and can encode one or more foreign antigens and induce humoral and cellular immune response.MVA holds great promise as a vaccine platform.In this review,we discuss the use of MVA for vaccine development against influenza virus.

Zoonosis associated to Rattus rattus and the impacts of the public actions to control the species / Zoonoses associadas à Rattus rattus e os impactos das ações públicas de controle sobre a espécie

The occurrence of the black rat, Rattus rattus , in major Brazilian cities has increased in the recent years. To evaluate if the efforts of public rodent control have been reaching the species in the city of São Paulo, a survey was conducted in flooding areas using live trapping before and after implementation of the control program. Captures were carried out from 2009 to 2011, and animals were evaluated for their risk of zoonosis transmission. The number of R. rattus captured after control did not differ from the number of animals captured before the control program in the Spring/Summer season, but, in the Autumn/Winter season, effective control was observed. Low infestations of Ctenocephalides felis (Siphonaptera) and Echinolaelaps echidninus (Acarina) were observed, and antibodies against Leptospira spp. were detected in just 1 of 65 serum samples. Ten out of 18 serum samples were sororeactive for Vaccinia virus, suggesting contact between R. rattus and the pathogen Calodium hepaticum (= Capillaria hepaticum ) (Nematoda) was found in the liver of 73.4% of captured R. rattus. In conclusion, R. rattus may not be effectively controlled by the rodent Control Program in the city of São Paulo, and represents a threat to human health, justifying permanent environmental management and educational programs.(AU)

A ocorrência do rato de telhado, Rattus rattus , nas grandes cidades brasileiras aumentou nos últimos anos. A fim de investigar se os esforços do controle público de roedores têm atingido essa espécie, foi conduzido um levantamento em áreas com risco de inundação na cidade de São Paulo, com armadilhas para captura viva dos roedores, antes e depois do controle público. O estudo foi realizado entre 2009 e 2011, e os animais capturados foram estudados para identificar seu risco como transmissores de zoonoses. O número de R. rattus capturados após o controle não diferiu entre o número de animais capturados antes do programa de controle, no período de primavera/verão, mas, no período de outono/inverno, foi observada efetividade do controle. Foram registradas infestações baixas de Ctenocephalides felis (Siphonaptera) e Echinolaelaps echidninus (Acarina). Anticorpos contra Leptospira spp. foram detectados em apenas 1 das 65 amostras de soro. Dez entre 18 amostras de soro foram sororeativas para o vírus Vaccinia , sugerindo o contato entre R. rattus e o patógeno Calodium hepaticum (Nematoda) foi encontrado no fígado de 73,4% dos R. rattus capturados. Concluiu-se que R. rattus pode não ser controlado pelas ações propostas pelo Programa de Controle de Roedores na cidade de São Paulo e representa uma ameaça para a saúde humana, justificando permanentes programas de gestão ambiental e programas educacionais.(AU)

Effect of myxomavirus on the proliferation of ovarian cancer cells and its Mechanism / 肿瘤

Objective: To investigate the effect of myxomavirus (MV) on the proliferation of human ovarian cancer SKOV3 cells, and its molecular mechanism. Methods: The human ovarian cancer SKOV3 cells were cultured in vitro and infected with MV. At the same time, SKOV3 cells infected with inactivated virus or only cultured with RPMI 1640 medium were used as the negative control group or the blank group, respectively. The proliferation of SKOV3 cells in the three groups was determined by CCK-8 assay. The mRNA levels of Bcl-2 and survivin were detected by real-Time fluorescent quantitative PCR. The cell cycle distribution was analyzed by FCM. The expressions of total extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), Akt, p-Akt, Bcl-2 and survivin proteins were measured by Western blotting. The activities of caspase-3 and caspase-8 were also quantified by colorimetric method. Results: Compared with the negative control group and the blank group, MV significantly inhibited the proliferation and cell cycle progression of human ovarian cancer SKOV3 cells (all P < 0.05). After SKOV3 cells were infected with MV for 96 h, the mRNA and protein expressions of Bcl-2 and survivin were significantly down-regulated (both P < 0.05), while the phosphorylation levels of ERK1/2 and Akt were significantly decreased (both P < 0.05), but the activities of caspase-3 and caspase-8 were obviously enhanced (both P < 0.05). Conclusion: MV can inhibit the proliferation of ovarian cancer cells, and its mechanism may be related to blocking cell cycle progression, down-regulating the expressions of anti-Apoptotic proteins Bcl-2 and survivin, increasing the activation of caspase-3 and caspase-8, and inhibiting the phosphorylation of ERK and Akt in proliferation-related signal pathway.

Oncolytic vaccinia virus：a promising agent for cancer treatment / 中华微生物学和免疫学杂志

Vaccinia virus ( VACV) has been widely used in humans for the eradication of small-pox. Since its natural ability of selective infection and replication in tumor cells without harming the normal tissue, VACV becomes a promising candidate in cancer therapy. In recent years, a variety of strategies have been successfully applied to further enhance the tumor selectivity and anti-tumor efficacy of VACV. These engineered VACVs, such as JX-594, have shown promising results in cancer treatment and have made re-markable progress in clinical trials. This review first briefly introduces the oncolytic VACV, and then focuses on the strategies applied in VACV engineering. We also discuss the main challenges and the future directions in the development of oncolytic VACV.

The humoral immune response in guinea pigs primed with recombinant vaccinia virus strains ex-pressing Transmitted/Founder virus HIV-1 membrane proteins and boosted with gp140 protein / 中华微生物学和免疫学杂志

Objective To analyze the antibody responses in guinea pigs vaccinated with recombi-nant vaccinia virus( rTT) strains expressing transmitted/founder ( T/F) HIV-1 membrane proteins in combi-nation with gp140 protein.Methods Guinea pigs were primed with rTT strains and boosted twice with gp140 protein in every four weeks.Serum samples were collected from guinea pigs before immunization and in 2, 6 and 10 weeks after the last immunization for the detection of HIV-1-specific binding antibodies, neu-tralizingantibodiesandtherelativeavidityofantibodies.Results (1)Thebindingantibodiesspecificto HIV-1 B′/C, B, AE subtypes were efficiently induced by the immunization of rTT-B, rTT-C and rTT-CON vaccinia strains in combination with gp140 protein.The antibody titers ranged from 111 430 to 1 024 000. More antibodies against HIV-1 B′/C and AE subtypes were induced in guinea pigs by the immunization of rTT-C and rTT-CON strains in combination with gp140 protein than those by using rTT-B strain prime-protein boost strategy (P<0.05).No significant differences with the titers of HIV-1 B subtype specific antibody were observed among the guinea pigs immunized with the three strategies.( 2 ) High titers of SF162 and ZM109 neutralizing antibodies were induced in guinea pigs immunized with rTT-B, rTT-C and rTT-CON vac-cinia strains in combination with gp140 protein, ranging from 83.76 to 649.30.No significant differences were found among the three groups.(3) The HIV-1 V1V2-gp70 specific antibodies associated with protec-tive immunity were induced by immunization of the three virus prime-protein boost strategies.No significant differences were observed among them.(4) Antibodies induced in guinea pigs by immunization of the three strategies showed strong affinity to membrane proteins of HIV-1 B′/C, B, AE subtype strains.No significant differences were found among the three immunization strategies.Conclusion A strong humoral immune re-sponse was induced in guinea pigs primed with recombinant vaccinia virus strains expressing T/F virus HIV-1 membrane proteins and boosted with gp140 protein.

Study of the immunogenicity and virulence of replication-competent recombinant vaccinia virus in CD4+T cell-depleted mice / 中华微生物学和免疫学杂志

Objective To investigate the effects of CD4+T cell depletion in BALB/c mice on the immunogenicity and virulence of replication-competent recombinant vaccinia virus. Methods Twelve BALB/c mice were inoculated with recombinant Tiantan vaccinia ( rTV, n=8 ) or vaccinia virus Tiantan strain ( VTT, n=4) by tail scarification after the depletion of CD4+T cells with anti-CD4 monoclonal anti-body( McAb) injected intraperitoneally for three days before virus inoculation.A control group without anti-body treatment was set up accordingly.Several parameters including the body weight, pocks on tail, viral shedding and the percentage of CD4+T cells were monitored.In the fourth week after virus infection, ovaries were collected from mice and viral loads in the tissue were titrated by plaque forming assay on chick embryo fibroblast ( CEF) cells.The specific cellular immune responses against vaccinia virus and HIV induced by inoculation of VTT and rTV respectively were detected by intracellular cytokine staining ( ICS) assay.ELISA was used to detect antibodies against vaccinia virus and HIV-1 gp120.Results All mice with or without CD4 McAb treatment showed typical poxvirus pocks on tails after inoculation of vaccinia viruses, but none of them developed secondary or satellite lesions.It took a longer time for CD4+T cell-depleted mice to heal from lesions, to regain body weights and to release viruses than the mice in control group.No vaccinia virus was detected in the ovaries of CD4+T cell-depleted mice or mice in control group.The mean absorbance( A) values for the detection of HIV-specific and vaccinia virus-specific antibodies in CD4+T cell-depleted mice with ELISA were respectively 0.119 and 0.168, which were significantly lower than those in mice of control group.The titers of neutralizing antibodies against vaccinia virus in McAb/rTV treated mice (1 ∶321) were lower than those in rTV treated mice (1 ∶1286) (P<0.05).The percentages of CD4+T cells secreting IFN-γ(0.654%) in McAb/rTV treated mice were significantly lower than those in rTV treated mice in the fourth week after immunization (P <0.0004).No significant differences with the vaccinia virus-specificCD8+ T cell responses were observed among mice with or without CD4+T cells depletion.Conclusion Thereplication and dissemination of replication-competent recombinant vaccinia virus could be effectively controlledin the mice with CD4+ T cell-depletion.The depletion of CD4+ T cells significantly reduced the humoraland CD4+ T cell responses, but had no effect on CD8+ T cell responses.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012) / Surtos de doença vesicular causados pelo Vaccinia virus em bovinos de leite no Estado de Goiás, Brasil (2010-2012)

Cases of vesicular and exanthematic disease by Vaccinia virus (VACV) have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goiás state, Midwestern Brazil (2010-2012), involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old) were affected in all cases and calves (2 to 9 months old) were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm), painful and coalescent papules (3-8 mm), which resulted in ulcers (5-25mm) and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to vgf gene of Orthopoxvirus was positive in samples collected from all outbreaks, and in some cases, genomic VACV sequences were identified by nucleotide sequencing of the PCR amplicons. Infectious virus was isolated in cell culture from scabs from one outbreak. Antibodies to Orthopoxvirus were detected in at least 3 or 4 animals in most outbreaks, by ELISA (outbreaks 1, 2, 3, 4, 5 and 7) or virus-neutralization (outbreak 6). Neutralizing titers ranging from 8 to 64 in outbreak 6. In all outbreaks, VACV infection was suspected based on the clinical and pathological findings and it was confirmed by laboratory tests. Upon the etiological confirmation, other agents associated with vesicular disease were discarded. In all outbreaks, at least one milker who handled the affected cows developed malaise, headache, fever, painful vesico-pustular lesions mainly in the hands, but also in the neck and nose. These results confirm the circulation of VACV in the region and call attention for a correct diagnosis and the adoption of prophylactic and control measures.

Casos de doença vesicular e exantemática associados ao Vaccinia virus (VACV) têm sido descritos em rebanhos leiteiros em diversas regiões do Brasil, ocasionalmente afetando também os ordenhadores. Este artigo descreve oito surtos de doença vesicular associados ao VACV ocorridos em rebanhos leiteiros de seis municípios do estado de Goiás (2010-2012), com o envolvimento de 122 vacas em lactação, de 12 bezerros e de 11 pessoas. Vacas em lactação (3-9 anos de idade) foram afetadas em todos os casos. Em cinco rebanhos, bezerros de 2-9 meses apresentaram lesões orais. A morbidade nos rebanhos variou entre oito e 100% (vacas) e entre 1,5 e 31% (bezerros). As lesões iniciavam como vesículas (2-7mm) ou pápulas doloridas e coalescentes (3-8mm), que progrediam para úlceras (5-25mm) e crostas, localizadas principalmente nas tetas e, eventualmente, no focinho das vacas. O curso clínico variou entre 16 e 26 dias. Histopatologia de amostras de pele coletadas de bovinos revelou dermatite perivascular superficial com infiltrado de linfócitos, neutrófilos, plasmócitos e macrófagos, além de áreas multifocais de acantose, espongiose, hipergranulose e hiperceratose ortoceratótica ou paraceratótica com úlceras focalmente extensas. No citoplasma dos ceratinócitos adjacentes às úlceras, observaram-se numerosos corpúsculos de inclusão eosinofílicos. Em todos os surtos, amostras de lesões cutâneas dos bovinos foram positivas para o gene vgf dos Orthopoxvirus por PCR, e em alguns casos, a identificação do VACV foi confirmada por sequenciamento de nucleotídeos dos amplicons. O vírus foi detectado por isolamento em cultivo celular em um dos surtos e, pelo menos 2 a 3 vacas por rebanho, apresentaram sorologia positiva para Orthopoxvirus pelos testes de ELISA (surtos 1, 2, 3, 4, 5 e 7) e soroneutralização (surto 6). No surto 6, os títulos de anticorpos neutralizantes variaram de 8 a 64. O diagnóstico da infecção pelo VACV, inicialmente suspeito com base nos achados clínicos e patológicos, foi confirmado em todos os surtos por exames laboratoriais. Em todos os surtos, pelo menos um ordenhador que teve contato com os bovinos afetados apresentou mal-estar geral, febre alta, dor de cabeça e lesões vesiculo-pustulosas doloridas, principalmente nas mãos, mas também no pescoço e nariz. Esses resultados confirmam a circulação do VACV no rebanho bovino da região centro-oeste, alertando para necessidade de diagnóstico correto e adoção de medidas profiláticas e de controle.

Requirement of CD4 Help for Induction of CD8 T Cell Response Specific for Virally Derived H60

CD40-CD40L-mediated help from CD4 T cells is essential to induce primary CD8 T cell responses specific to the non-inflammatory cell-based antigen H60. In this study, using H60 as a model antigen, we generated recombinant vaccinia viruses (rVVs) expressing the H60 CD8 epitope and investigated whether CD4 help was required to activate the CD8 T cell response specific to the virally expressed H60. The immune response after infection with rVVs expressing H60 was similar to that after immunization with H60 congenic splenocytes, with a peak frequency of H60-specific CD8 T cells detected in the blood on day 10 post-infection. A CD8 T cell response specific for virally derived H60 was not induced in CD4-depleted mice, but was in CD40-deficient mice. These results provide insights into the characterization of the CD8 T cell response specifically for antigens originating from cellular sources compared to viral sources.

Impact of B8R elimination on vaccinia virus induced immune responses / 中华微生物学和免疫学杂志

Objective To investigate the antigenicity of foreign antigen in recombinant vaccinia virus (VACV) after elimination of B8R,and provide help to improve the efficacy of VACV-based vaccines,and provide guidance for vaccine design.Methods Transfer vector pSC11-OVA was generated,and OVAgene was in,rted into VACV with B8R deletion.The biological characteristics of recombinant VACV was investigated in vitro,and the immune responses against OVA were tested in vivo.Results The plague phenotypes and growth of wcombinant VACV and its parental strains were essentially identical.Cellular immuneresponse against OVA was augmented in mice infected with B8R-deleted recombinant VACV when comparedwith those infected with B8R-intact recombinant VACV.Conclusion Deletion of B8R and insertion of OVAdoes not affect the biological characteristics of VACV.Immunogenicity of exogenous target antigens can beimproved in VACV with B8R deficiency.Deletion of dominant epitopes may provide a vector with more efficiency for vaccines and gene therapy.

Efficacy of epidural infusion of a mixture of bupivacaine-fentanyl-dexamethasone on top of intravenous extract from rabbit skin inflamed by vaccinia virus for severe herpetic neuralgia / 中华麻醉学杂志

ObjectiveTo evaluate the efficacy of epidural infusion of a mixture of bupivacaine-fentanyl-dexamethasone on top of intravenous extract from rabbit skin inflamed by vaccinia virus (ERSVV) for severe herpetic neuralgia.MethodsForty-eight patients of both sexes with severe herpetic neuralgia aged 45-92 yr were randomly divided into 2 groups (n =24 each):control group (group C) and test group( group T).Both groups received intravenous ERSVV 6 ml/d.Group C received oral amitriptyline and gabapentin,while the group T received epidural infusion of 100 ml of a mixture of 0.075％ bupivacaine,fentanyl 2 μg/ml and dexamethasone 50 μg/ml in normal saline at 2-5 ml/h,once a day for 10 days and VAS score was maintained≤4.Epidural puncture was performed at the spinal segments severely affected by herpes virus.Intensity of pain was assessed with VAS (0 =no pain,10 =worst pain).In group C when VAS ＞ 4 oral amitriptyline 12.5 mg (once/d) and gabapenti 0.1 g (3 times/d) were given as rescue analgesics.Adverse effects of epidural infusion and incidence of post-herpetic neuralgia were recorded.ResultsThe incidence of urinary retention and incidence of post-herpetic neuralgia were lower in group T than in group C.No other adverse effects were found in group T.ConclusionEpidural infusion of a mixture of bupivacaine-fentanyl-dexamethasone on top of ERSVV can effectively reduce severe herpetic neuralgia and prevent development of post-herpetic neuralgia safely.

Establishment of crystal violet plaque assay for virus titration of recombined Tiantan vaccinia AIDS vaccine / 中华微生物学和免疫学杂志

Objective To establish the crystal violet plaque assay for detection of virus titer of recombined Tiantan vaccinia AIDS vaccine,and provide more stable method of virus titration for rTV AIDS Vaccine.Methods Optimized the concentration of Vero cells,the time and temperature of virus adsorption,and the time of determination for CPE,then established the crystal violet plaque assay for virus titer of rTV.Counting and analysis the plaques by BioSpot Reader,then analyzed the relativity of plaques counted with BioSpot Reader and manual; Several lots rTV AIDS Vaccine and Tiantan vaccinia were titrated by the method of plaque formation-hemadsorption assay,neutral red and crystal violet plaque assay,then analyzed the relativity of the results of three methods ; meanwhile,the virus titer of samples were determine repeatedly by the crystal violet plaque assay,then calculated the coefficient of variation( CV),and verified the precision of the method; SPSS17.0 was used in statistical analysis of the experimental results.Results When the concentration of Vero cells was 5.0×105-9.O×105 cells/ml,virus been adsorbesd 2 h at 37℃,then cultivated 72 h after adding the culture medium containing methyl cellulose.Plaques counted by BioSpot Reader was highly related with counted by manual (r =0.985),so BioSpot Reader counting can objectively reflect the virus plaques with various size,and reduce the error by manual counting; compared the virus titration for different lots of rTV AIDS vaccine and Tiantan vaccinia with three methods,the crystal violet plaque assay was highly related with plaque formation-hemadsorption assay (r =0.997,P＜0.01 ) and neutral red plaque assay(r=0.980,P＜0.01 ).Conclusion Crystal violet plaque assay was established for virus titration of rTV AIDS Vaccine.

Effect of anti-inflammation-analgesic injection combined with extract from rabbit skin inflamed by vaccinia virus on sciatic nerve injury in rats / 中华麻醉学杂志

Objective To investigate the effect of anti-inflammation-analgesic injection (AIAI) combined with extract from rabbit skin inflamed by vaccinia virus (ERSVV) on repair of the damaged sciatic nerve in rats.Methods Fifty adult SD rats of both sexes weighing 260-300 g were randomly divided into 5 gronps ( n = 10each): sham operation group (group S); sciatic nerve chronic constriction injury group (group CCI); CCI + AIAI group (group A); CCI + ERSVV group (group E) and CCI + AIAI + ERSVV group (group A + E). Right sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4/0 catgut in CCI,A,E and A + E groups. A catheter was placed around sciatic nerve, and fixed to the nearby muscle and kept unclogged by injecting 0.2 nl distilled water daily. AIAI and/or ERSVV 0.2 ml were injected via the catheter starting from the 14th day after operation. AIAI 0.2 ml contained dexamethasone palmitate (4 mg/ml) 0.05 ml, 2% lidocaine 0.05 ml and vitamin B12 (0.5 mg/ml) 0.05 ml in distilled water. Dexamethasone palmitate was omitted in AIAI starting from the 2nd of drug administration in group A and A + E. Paw withdrawal threshold to mechanical stimulation (MWT) was measured before (baseline) and at 1, 5, 7, 14, 21 and 28 days after operation. Sciatic nerves were exposed at 14 and 28 days after operation in 5 rats in each group. Conduction velocity of motor nerve (NCV) and action potential (AP) of gastrocnemius muscle were measured. Sciatic nerve at the site of CCI was examined for pathologic changes, the number of axons (NA) and thickness of myelin sheath (TMS) with light microscope. Results CCI significantly decreased MWT, AP, NA, TMS and NCV in group CCI as compared with group S (P ＜0.01). AIAI and/or ERSVV significantly attenuated CCI-induced decrease in MWT, AP, NA, TMS and NCV in A,E and A + E groups as compared with CCI group ( P ＜ 0.05). Their curative effects were potentiated by combined use. Conclusions Both AIAI and ERSVV have curative effects against CCI-induced sciatic nerve injury and their actions are potentiated by combined used.

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Effect of aldosterone on the amplification of oncolytic vaccinia virus in human cancer lines / 대한간학회지

BACKGROUND/AIMS: JX-594 is an oncolytic virus derived from the Wyeth vaccinia strain that causes replication-dependent cytolysis and antitumor immunity. Starting with a cross-examination of clinical-trial samples from advanced hepatocellular carcinoma patients having high levels of aldosterone and virus amplification in JX-594 treatment, we investigated the association between virus amplification and aldosterone in human cancer cell lines. METHODS: Cell proliferation was determined by a cell-counting-kit-based colorimetric assay, and vaccinia virus quantitation was performed by quantitative polymerase chain reaction (qPCR) and a viral plaque assay. Also, the intracellular pH was measured using a pH-sensitive dye. RESULTS: Simultaneous treatment with JX-594 and aldosterone significantly increased viral replication in A2780, PC-3, and HepG2 cell lines, but not in U2OS cell lines. Furthermore, the aldosterone treatment time altered the JX-594 replication according to the cell line. The JX-594 replication peaked after 48 and 24 hours of treatment in PC-3 and HepG2 cells, respectively. qPCR showed that JX-594 entry across the plasma membrane was increased, however, the changes are not significant by the treatment. This was inhibited by treatment with spironolactone (an aldosterone-receptor inhibitor). JX-594 entry was significantly decreased by treatment with EIPA [5-(N-ethyl-N-isopropyl)amiloride; a Na+/H+-exchange inhibitor], but aldosterone significantly restored JX-594 entry even in the presence of EIPA. Intracellular alkalization was observed after aldosterone treatment but was acidified by EIPA treatment. CONCLUSIONS: Aldosterone stimulates JX-594 amplification via increased virus entry by affecting the H+ gradient.