Made available in DSpace on 2016-04-28T20:15:37Z (GMT). No. of bitstreams: 1 CELSO EDUARDO DE SOUZA1.pdf: 1961117 bytes, checksum: 9c58964339128b2a9f91e38568ada01c (MD5) Previous issue date: 2010-04-22 / Brazilian spotted fever (BSF) is an acute infectious disease and its etiologic agent Rickettsia rickettsii is a gram negative intracellular binding and transmitted through the bite of infected ticks. In the epidemiology of spotted fever, the amplifying hosts in general are wild animals. They play an important role for developing temporary rickettsemia, thus contributing to the possibility of infection of new generations of ticks. This study aims to verify the occurrence of anti rickettsiae IgG antibodies of the spotted fever group in the serum of wild rodents. The presence of rickettsiae of the brazilian spotted fever group in fragments of the spleen of wild rodents was studied by PCR and clarify the involvement of small rodents as amplifying hosts of R. rickettsii in endemic areas in the region of Campinas in S?o Paulo state. Thus, we selected the municipalities of Jaguari?na, Pedreira and Piracicaba, where human been cases were detected. Through the use of one hundred traps distributed in Sharm lines, 222 rodents were captured the following species: Akodon sp 35 (15.6%), Necromys lasiurus 61 (27,1%), Calomys tener 35 (15,6%), Mus muscullus 19 (8,4%), Nectomys squamipes 1 (0,4%), Oligoryzomys nigripes 63 (28,0%), Oxymycterus nigripes 1 (0,4%), Rattus rattus 7 (3,1%). All rodents were examined, but no tick was found. We analyzed a total of 186 serum samples for antibodies of the IgG class anti-Rickettsia rickettsii, R. parkeri, R. felis, R. amblyommii, R. rhipicephali and R. bellii. It was found that 39 (20.97%) sera were reactive to at least one Rickettsia spp. with titers ranging from 1:64 to 1:512 and 147 (79.03%) were negative. The prevalence of anti R. rickettsii spp antibodies IgG found in these mice was 9.34%. Of the 186 animals examined were positive only 17 of these, 15 animals showed evidence of 1/64 and only one animal reacted to the title of 1/512.One hundred and fifty six spleens were processed by PCR and was not detected in Rickettsia spp in any of them. Considering the results presented we can conclude that small rodents are not amplifying hosts of Rickettsia of Brazilian spotted fever group in the study area. / A febre maculosa brasileira (FMB) ? uma doen?a infecciosa aguda e tem como agente etiol?gico a Rickettsia rickettsii que ? uma bact?ria gram negativa intracelular obrigat?ria e transmitida atrav?s da picada de carrapatos infectados. Na epidemiologia da febre maculosa, os hospedeiros amplificadores em geral s?o animais silvestres, os quais exercem fun??o importante por desenvolverem rickettsemia tempor?ria. Este estudo teve como objetivos estudar a ocorr?ncia de anticorpos da classe IgG anti Rickettsia spp. do grupo da febre maculosa no soro dos pequenos roedores silvestres, detectar atrav?s da PCR a presen?a de esp?cies de Rickettsia do grupo da febre maculosa em fragmentos de ba?o dos roedores e esclarecer a participa??o destes animais como hospedeiros amplificadores de R. rickettsii em ?reas end?micas na regi?o de Campinas estado de S?o Paulo. Foram selecionados os munic?pios de Jaguari?na, Pedreira e Piracicaba por situarem-se em ?rea end?mica e com registro de casos em seres humanos. Foram utilizadas um total de cem armadilhas tipo Sharm distribu?das em linhas. Um total de 222 roedores das seguintes esp?cies foram capturadas: 35 (15,6%) Akodon sp, 61 (27,1%) Necromys lasiurus, 35 (15,6%) Calomys tener, 19 (8,4%)Mus muscullus, 1 (0,4%) Nectomys squamipes, 63 (28,0%) Oligoryzomys nigripes, 1 (0,4%) Oxymycterus nigripes e 7 (3,1%) Rattus rattus. Todos os roedores foram examinados, mas nenhum carrapato foi encontrado. Foram analisados um total de 186 soros para detec??o de anticorpos da classe IgG anti- Rickettsia rickettsii, R. parkeri, R. felis, R. amblyommii, R. rhipicephalus e R. bellii. Verificou-se que 39 (20,97%) dos soros foram reativos a pelo menos para uma das esp?cies Rickettsia com t?tulos variando de 1:64 a 1:512 e 147 (79,03%) foram negativos. A preval?ncia de anticorpos da classe IgG anti R. rickettsii encontrada nestes roedores foi de 9,34%. Dos 186 animais examinados apenas 17 foram positivos, destes, 15 animais apresentaram t?tulos de 1/64 e apenas um animal reagiu at? o t?tulo de 1/512. Foram processados 182 ba?os pela t?cnica da PCR e n?o foi detectado Rickettsia spp. em nenhum deles. Diante dos resultados apresentados podemos concluir que os pequenos roedores n?o s?o os hospedeiros amplificadores de Rickettsia do grupo da Febre Maculos na ?rea estudada.

Introdução: Neutropenia febril é uma emergência médica que demanda um diagnóstico precoce e administração de antibióticos o mais breve possível. A procalcitonina (PCT) é um marcador inflamatório que vem sendo utilizado como um indicador de infecção bacteriana grave. A detecção precoce do quadro séptico é difícil, principalmente numa população heterogênea como no caso dos neutropênicos febris. A possibilidade de um único exame laboratorial poder identificar precocemente os quadros de sepse contribuiria de forma significativa para melhorar o prognóstico destes pacientes. Objetivo: Avaliar os níveis de PCT como marcador de infecção sistêmica comparados aos níveis de proteína C-reativa (PCR) em pacientes neutropênicos febris. Métodos: Foram estudadas amostras de 65 pacientes com a finalidade de determinar as concentrações séricas de PCT, PCR e outros parâmetros hematológicos em três momentos diferentes: antes da febre, no momento da febre e 72 após o término da febre. Os pacientes foram divididos inicialmente em quatro grupos: com infecção sistêmica comprovada laboratorial ou clinicamente (I), com febre de origem indeterminada - FOI- (II), com infecção localizada (III) e com infecção fúngica confirmada (IV). Posteriormente, os grupos I e IV foram denominados de 1 (com infecção sistêmica) e os grupos II e III de 2 (sem infecção sistêmica). Treze pacientes não apresentaram febre durante a internação sendo excluídos da comparação PCT/PCR. Resultados: A concentração de PCT mostrou estar associada com o diagnóstico de infecção grave e neutropenia febril. Não houve correlação entre os níveis de PCT e PCR. Conclusão: Fica evidente que a PCT demonstrou ser um marcador útil para o diagnóstico de infecção sistêmica em neutropenia febril, sendo provavelmente, superior à PCR. Pode-se caracterizar a PCT como um auxiliar de indicador de infecção sistêmica já no primeiro dia de apresentação da febre. A PCT, ao contrário da PCR, foi capaz de distinguir entre infecção sistêmica e infecção localizada ou febre de origem indeterminada, tendo boa capacidade diagnóstica. Entretanto, a PCT não se correlacionou com o prognóstico, possivelmente pelo pequeno tamanho da amostra, apesar da curva ROC da PCT do grupo com infecção sistêmica com evolução para óbito ter delimitado uma área estatisticamente diferente da esperada pelo acaso. / Introduction: Febrile neutropenia is a medical emergency that calls for a precocious diagnosis and the administration of antibiotics as soon as possible. The procalcitonin (PCT) is an inflammatory marker that has been used as an indicator of severe bacterial infection. Considering that neutropenic population is heterogeneous, an early and only reliable laboratory test that could identify septic patients would be of great value to improve its outcome. Objective: Assess the diagnostic value of PCT as a marker of systemic infection, comparing with C-reactive protein (CRP) levels in febrile neutropenia. Methods: Sixty-five adults patients were enrolled in the study. Blood sample was collected in order to determine the serum concentrations of PCT, CRP and other hematological parameters at three different moments: before the beginning of fever, at the onset of fever and 72 hours after cessation of it. Firstly, the patients were divided into four groups: with clinical or laboratorial proven systemic infection (I), with fever of undetermined origin (FUO) (II), with localized infection (III) and with proven fungal infection (IV). After that, the groups I and IV were named as 1:- with systemic infection. The groups II and III were named 2:- without systemic infection. Thirteen patients did not present fever during evolution and were excluded from the PCT/PCR comparison among febrile patients. Results: The PCT concentration showed it was associated with the diagnosis of severe infection in febrile neutropenia. No correlation could be found between the levels of PCT and CRP. Conclusion: PCT seems to be an useful marker for the diagnosis of systemic infection in febrile neutropenia, probably better than CRP. We could assume that PCT could indicate systemic infection at the very first day of the outcome of fever. Only PCT (and not CRP) could be able to distinguish between systemic infection and localized infection or FUO, with excellent diagnostic capacity. However none of the markers (PCT and CRP) could be correlated to prognosis, possibly due to the small size of the sample. Nevertheless, PCT ROC curve for evolution to death as a result of systemic infection limit an area under the curve statistically different that expected by chance.

Introdução: A idade ao diagnóstico do lúpus eritematoso sistêmico juvenil (LESJ) pode influenciar a expressão da doença em termos de apresentação clínica inicial, padrão de envolvimento de órgãos e achados sorológicos. Objetivo: Avaliar dados demográficos, características clínicas e alterações laboratoriais no momento do diagnóstico da doença em três grupos etários diferentes de pacientes com LESJ: grupo A com início precoce (< 6 anos), grupo B com início em idade escolar ( >= 6 e < 12 anos) e grupo C com início em adolescentes ( >= 12 e < 18 anos). Métodos: Estudo multicêntrico brasileiro de coorte retrospectiva em 10 centros de reumatologia, incluindo 847 pacientes com o diagnóstico de LESJ. Resultados: Os pacientes foram divididos em três grupos: A com 39 (4%), B com 395 (47%) e C com 413 (49%). Dos 39 pacientes com LESJ do grupo A, 3 (8%) tinham < 2 anos, 4 (10%) >= 2 e < 3 anos e 32 (82%) >= 3 e < 6 anos. Setenta e quatro pacientes com LESJ foram analisados para os níveis séricos de C1q e a deficiência completa de C1q foi observada em 3/74 (4%), todos estes pertencentes ao grupo A. Os grupos foram semelhantes quanto às altas frequências de sexo feminino, nefrite, envolvimento neuropsiquiátrico, SLEDAI-2K ( >= 8), perfil de autoanticorpos, proteínas de fase aguda elevada e baixos níveis de complemento (p > 0,05). No entanto, as frequências de febre (78% vs. 61% vs. 47%, p < 0,0001), hepatomegalia (42% vs. 29% vs. 14%, p < 0,0001), esplenomegalia (28% vs. 12% vs. 4%, p < 0,0001) e lúpus discoide (13% vs. 4% vs. 4%, p=0,020) foram significantemente maiores no grupo A em comparação com os grupos B e C. As frequências de perda de peso > 2kg (19% vs. 28% vs. 36%, p < 0,017), fotossensibilidade (34% vs. 41% vs. 51% p < 0,006), leucopenia < 4.000/mm3 (14% vs. 25% vs. 30%, p=0,048) e linfopenia < 1.500/mm3 (22% vs. 41% vs. 47%, p=0,011) foram significantemente menores no grupo A. Conclusão: O presente estudo multicêntrico identificou que a apresentação inicial de LESJ foi caracterizada por alta frequência de envolvimento de órgãos internos nos três grupos estudados e algumas características clínicas e laboratoriais distintas nos grupos de início precoce e adolescentes / Introduction: Age at diagnosis of childhood-onset systemic lupus erythematosus (cSLE) may influence disease expression in terms of initial clinical presentation, pattern of organ involvement and serological findings. Objective: To evaluate demographic data, clinical and laboratory features at disease diagnosis in three different age groups of childhood systemic lupus erythematosus (cSLE): group A early-onset ( < 6 years), group B school age ( >= 6 and < 12 years) and group C adolescent ( >= 12 and < 18 years). Methods: Brazilian multicenter cohort retrospective study in 10 Pediatric Rheumatology centers, including 847 cSLE patients. Results: Patients were divided in three groups: A with 39 (4%), B 395 (47%) and C 413 (49%). Of 39 cSLE patients of group A, 3 (8%) were < 2 years, 4 (10%) >= 2 to < 3 years and 32 (82%) >= 3 and < 6 years. Seventy-four cSLE patients were analyzed for C1q levels and complete C1q deficiency was observed in 3/74 (4%), all of them of group A. Groups were similar regarding high frequencies of female gender, nephritis, neuropsychiatric involvement, SLEDAI-2K ( >= 8), autoantibody profile, elevated acute phase proteins and low complement levels (p > 0.05). However, the frequency of fever (78% vs. 61% vs. 47%, p < 0.0001), hepatomegaly (42% vs. 29% vs. 14%, p < 0.0001), splenomegaly (28% vs. 12% vs. 4%, p < 0.0001) and discoid lupus (13% vs. 4% vs. 4%, p=0.020) was significantly higher in the group A compared to groups B and C. The frequency of weight loss > 2kg (19% vs. 28% vs. 36%, p=0.017), photosensitivity (34% vs. 41% vs. 51%, p=0.006), leukopenia < 4,000/mm3 (14% vs. 25% vs. 30%, p=0.048) and lymphopenia < 1,500/mm3 (22% vs. 41% vs. 47%, p=0.011) was significantly lower in the group A. Conclusions: Our large multicenter study identified that the initial presentation of cSLE is characterized by comparable high frequency of internal organ involvement and some distinct clinical and laboratory features in early-onset and adolescent groups

Foi conduzido um estudo sobre a ecologia da Febre Maculosa Brasileira, causada pela bactéria Rickettsia rickettsii, em uma área endêmica, no distrito de Taiaçupeba, Município de Mogi das Cruzes, SP. Para o melhor entendimento de quais animais silvestres são os hospedeiros das formas imaturas do carrapato vetor, Amblyomma aureolatum, foram capturados entre janeiro e dezembro de 2005, 243 animais silvestres em dois fragmentos de mata. Foram utilizadas estações de pitfall para captura de roedores e pequenos didelfídeos e armadilhas tomahawk para captura de Didelphis aurita, além da colocação de redes de neblina (14m x 3m cada) para captura de aves. Os animais foram sacrificados e tiveram sangue, baço e fígado extraídos. O baço de cada animal foi submetido a testes moleculares e bioensaios para pesquisa de bactérias do gênero Rickettsia. Os carrapatos capturados dos animais foram submetidos à identificação taxonômica morfológica ou molecular e à pesquisa de bactérias do gênero Rickettsia. Foram colhidos carrapatos dos gêneros Amblyomma, Haemaphysalis e Ixodes. Imaturos do carrapato Amblyomma aureolatum foram colhidos parasitando três indivíduos da espécie de Passeriforme Pyriglena leucoptera. Não foram encontrados exemplares desta espécie de carrapato parasitando roedores ou didelfídeos. Nenhum animal foi identificado sendo infectado por riquétsias, enquanto que três espécies de riquétsias, sendo duas do grupo da febre maculosa, foram identificadas infectando carrapatos das espécies Amblyomma longirostre, Ixodes aragaoi e Ixodes loricatus. Nenhum carrapato foi encontrado naturalmente infectado com a bactéria R. rickettsii. O estudo mostrou detalhes do ciclo de vida do carrapato A. aureolatum que podem auxiliar o entendimento do ciclo enzoótico da febre maculosa brasileira. / Brazilian Spotted Fever (BSF) is a tick-borne-disease caused by the bacterium Rickettsia rickettsii. An ecological study was conducted in a BSF-endemic area in Taiaçupeba County, Mogi das Cruzes Municipality, State of São Paulo. With the purpose to determine natural hosts of the immature stages of the tick vector Amblyomma aureolatum, a total of 243 wild animals were captured in two fragments of Atlantic Forests between January and December of 2005. Pitfall trap stations were used for capture of rodents and small didelphids while tomahawk traps were used for Didelphis aurita capture. Mist nets (14m x 3m each) were used for bird capture. Captured animals were scarified and blood, spleen and liver were extracted. Spleen samples were submitted to molecular and bioassay tests for rickettsia research. Captured ticks were submitted to morphological or molecular taxonomic identification and to rickettsia research. Ticks from the genera Amblyomma, Haemaphysalis and Ixodes were collected. Immature tick stages of A. Aureolatum were collected on three individuals of the bird species Pyriglena leucoptera, but no other A. Aureolatum tick was found infesting neither rodents or didelphids. No animal was found infected by rickettsiae whereas three rickettsiae, two of them belonging to Spotted Fever Group, were found infecting the ticks Amblyomma longirostre, Ixodes aragaoi and Ixodes loricatus. No tick was found infected by R. Rickettsii, the agent of BSF. The present study revealed details about the A. Aureolatum life cycle in natural conditions, contributing for a better understanding about the enzootic cycle of BSF.

Febrile neutropenia is a potentially life-threatening oncologic emergency characterized by a dangerously low neutrophil count that places the patient at great risk. In these patients, fever may be the only sign of infection, which requires prompt treatment. With the increasing focus in shifting health care from inpatient centers to outpatient arenas, home health care clinicians will likely have an increased role in the care of neutropenic fever patients in the future. The article describes both the pharmacologic treatment and nonpharmacologic support required of these patients with particular attention to treatment that may be required in the patient?s home.

Dengue fever is a debilitating, viral, mosquito-borne disease occurring in tropical and subtropical areas in the world. The majority of dengue cases in the United States were acquired in endemic areas by travelers or immigrants. However, in recent years, autochthonous (locally acquired) dengue cases have been diagnosed in Florida. The purpose of this study was to find an association between potential risk factors and the expansion of dengue fever in the United States. Guided by the eco-bio-social framework, which offers a broad assessment of risk factors for the illness, a retrospective design was used with archival data to correlate changes in climatic variables and imported dengue cases with autochthonous dengue cases in Southeast Florida from 1980 to 2013. A Spearman correlation indicated weak correlations between temperature and autochthonous dengue cases (rs = .999, p = 000) and imported dengue cases with autochthonous dengue cases (rs = .162, p = 000). A negative binomial multivariate regression was used to analyze the expansion of dengue to each monthly unit of temperature, rainfall, and imported dengue cases over 34 years. The results indicated that temperature (IRR = 2.198; 95% CI [1.903, 2.538]) and precipitation (IRR = .991; 95% CI [.988, .994]) were predictors for the geographic expansion of dengue fever in Southeast Florida. The positive social changes include the use of the results to develop an understanding of how climatic variables and migration may influence the expansion of dengue fever to nonendemic regions. The results can be used by public health authorities to address risk factors and to formulate evidence-based decisions in regard to prevention and education concerning dengue fever.

Malignant catarrhal fever (MCF) is a lymphoproliferative and inflammatory syndrome affecting primarily ruminant species. The disease, which is often fatal, is most often described as affecting bovids and cervids. No vaccines are available for prevention of MCFV infection. The primary method to control spread of disease is to prevent contact between carriers and clinically susceptible species. There is no known method to control infection of malignant catarrhal fever virus-white-tailed deer variant (MCFV-WTD), as the carrier animal of this virus is unknown. To determine the prevalence of malignant catarrhal fever viruses in Tennessee ruminant populations, blood and/or lymph node samples were collected from farms, animal processing and disposal facilities, and hunter check-in stations from 2006-2008 from several species of animals including cervids, cattle, and goats. Strain-specific real time PCR was developed to detect ovine herpesvirus-2 (OvHV-2), caprine herpesvirus-2 (CpHV-2), and MCFV-WTD DNA. MCFV DNA was detected in all species of ruminants sampled. Although disease related to infection with MCFV-WTD and CpHV-2 has not been reported in Tennessee cattle or cervid populations, MCFV-WTD DNA was detected in 3 percent of cervid samples, and MCFV-WTD and CpHV-2 DNA was detected in 27 and 3 percent respectively of cattle samples from animal disposal facilities that process dead or debilitated animals. One hunter harvested deer (n=781) and 25 cattle (n=165) tested from animal disposal facilities were positive for OvHV-2 DNA. This study demonstrated that healthy cattle and cervids can be infected with OvHV-2 and MCFV-WTD without apparent disease, and dead or debilitated cattle were infected with OvHV-2, MCFV-WTD and CpHV-2 at a higher percentage than healthy herd animals. Prevalence of CpHV-2 in Tennessee goat populations (7%) was significantly lower than reported in other goat populations (73%). Low prevalence of CpHV-2 in Tennessee goat populations likely explains why no evidence of infection was found in cervids tested, and the low prevalence of CpHV-2 infection in dead or debilitated cattle compared to prevalence of infection with OvHV-2 and MCFV-WTD. The discovery of infection in cattle with CpHV-2 and MCFV-WTD opens a new avenue of investigation into the pathology and virulence of MCFV’s in domestic cattle.

Infectious diseases such as malaria and relapsing fever borreliosis (RF), cause severe human mortality and morbidity in developing countries. Malaria, caused by Plasmodium spp. parasites, is estimated by the World Health Organization to cause 1.5-2.7 million deaths annually. RF, caused by Borrelia spirochetes, has the highest prevalence described for any bacterial disease in Africa, with infection outcomes ranging from asymptomatic to fatal. RF borreliosis manifests in humans as a recurring fever and with other symptoms very similar to those of malaria. RF borreliosis has been regarded as a transient infection of the blood. However, B. duttonii exploits the brain as an immunoprivileged site escaping the host immune response while spirochetes in the blood are cleared. To investigate whether residual bacteria are dormant or actively dividing, mice with residual brain infection were administered ceftriaxone, a β-lactam antibiotic interfering with cell wall synthesis. Hence, it only affects actively dividing bacteria. Ceftriaxone eradicated brain RF infection in all treated mice, demonstrating that the bacteria are actively multiplying rather than in a dormant state. The findings support the therapeutic use of ceftriaxone for RF neuroborreliosis since penetration into cerebrospinal fluid is greater for ceftriaxone than for the often recommended doxycycline. The clinical features of malaria and RF are similar and diagnosis is further complicated by the frequently occurring concomitant malaria-RF infections. Therefore, we established a mouse model to study the pathogenesis and immunological response to Plasmodium/Borrelia mixed infection. Interestingly, malaria was suppressed in the co-infected animals whereas spirochete numbers were elevated 21-fold. The immune response in the concomitantly infected mice was polarized towards malaria leaving the spirochetes unharmed. Mice with co-infections also exhibited severe anemia and internal damages, probably attributed to escalating spirochete numbers. A secondary malaria infection reactivated the residual brain RF infection in 60% of the mice. This highlights the importance of co-infections as diagnostic pitfalls as well as the need for novel treatment strategies. Currently there is no commercial malaria vaccine and increasing drug resistance presents an urgent need for new malaria chemotherapeutics. Blood-stage malaria parasites are rapidly growing with high metabolic and biosynthetic activity, making them highly sensitive to limitations in polyamine supply. Disrupting polyamine synthesis in vivo with trans-4-methylcyclohexylamine (4MCHA) eradicated the malaria infection gradually, resulting in protective immunity. This leads the way for further biochemical and pharmacological development of the polyamine inhibitor 4MCHA and similar compounds as antimalarial drugs

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.