Detection of Vaccinia Virus in Urban Domestic Cats, Brazil.

We investigated possible vaccinia virus (VACV) in urban house cats in Brazil. Serum samples from 6 cats were positive for VACV by PCR, indicating likely VACV circulation among house cats in urban areas of Brazil. This finding highlights the importance of epidemiologic surveillance to avoid outbreaks among urban human populations.

Detection of Vaccinia Virus in Dairy Cattle Serum Samples from 2009, Uruguay.

We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out.

Seroprevalence of Orthopoxvirus in rural Brazil: insights into anti-OPV immunity status and its implications for emergent zoonotic OPV.

BACKGROUND: Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus, a virus from Orthopoxvirus genus (OPV) that affects mainly cattle herds and humans in rural areas in Brazil. Because most studies have focused on outbreaks situations, data on BV epidemiology is limited. A cross sectional study in Brazilian rural areas during 2012-2013 was conducted to determine the neutralizing antibodies seroprevalence and risk factors for BV. METHODS: A structured questionnaire was applied to elicit demographics data and farming practices considered risk factors for BV exposure. Neutralizing anti-OPV antibodies were investigated using plaque reduction neutralization test. The neutralizing antibodies prevalence rates were calculated and the risk factor analysis was performed using multivariate logistic regression. RESULTS: Two hundred and forty participants were enrolled in this study with a prevalence of neutralizing antibodies of 30.8 % (95 % confidence interval [CI], 25.3-36.9). In multivariate analysis, age > 35 years (Odds Ratio [OR] = 18.2; CI 95 % = 7.7 - 43.2) and previous outbreak in property (OR = 3.9; C I95 % = 1.2 - 12.6) were independently associated with anti-OPV neutralizing antibodies. CONCLUSIONS: In this study, anti-OPV protective immunity (neutralizing antibody titers) was assessed in an endemic BV Brazilian rural area. Our findings indicate that epidemiological surveillance is required and should be applied by public health authorities to create interventions and/or prevention strategies to avoid viral spread causing future outbreaks among individuals who are under risk of infection.

The detection of Vaccinia virus confirms the high circulation of Orthopoxvirus in buffaloes living in geographical isolation, Marajó Island, Brazilian Amazon.

In Brazil, serologic evidence of Orthopoxvirus (OPV) circulation showed positivity around 20% in cattle, humans, monkeys and rodents. Although OPV seropositivity has been described in buffalo herds in southeastern Brazil, no Vaccinia virus (VACV) (member of genus OPV) outbreaks in buffalo herds have been described in this country. This study aimed to investigate the detection of anti-OPV antibodies and to study the OPV genome in Brazilian buffalo herds. Our results demonstrated a high OPV seropositivity in buffalo herds on Marajó Island and molecular data confirmed the circulation of VACV. The geographical isolation conditionmight be a sine qua non condition to explain our results.

Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil.

A case of dengue virus 3 (DENV-3) genotype I infection with neurological manifestations occurred in Belo Horizonte, Minas Gerais in October 2012. The serotype was detected by PCR, and the genotype was assessed by sequencing and phylogenetic analysis of the C-prM region. The virus causing neurological manifestations clustered with other sequences of DENV-3 genotype I. Because neurological manifestations of DENV are possibly misdiagnosed in Brazil, this study serves as an alert of the importance of DENV diagnoses in CNS infections.

Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil

Abstract: A case of dengue virus 3 (DENV-3) genotype I infection with neurological manifestations occurred in Belo Horizonte, Minas Gerais in October 2012. The serotype was detected by PCR, and the genotype was assessed by sequencing and phylogenetic analysis of the C-prM region. The virus causing neurological manifestations clustered with other sequences of DENV-3 genotype I. Because neurological manifestations of DENV are possibly misdiagnosed in Brazil, this study serves as an alert of the importance of DENV diagnoses in CNS infections.

Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era.

Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks.

RAP1 GTPase overexpression is associated with cervical intraepithelial neoplasia.

RAP1 (RAS proximate 1), a small GTP-binding protein of the RAS superfamily, is a putative oncogene that is highly expressed in several malignant cell lines and types of cancers, including some types of squamous cell carcinoma. However, the participation of RAP1 in cervical carcinogenesis is unknown. We conducted a cross-sectional study of paraffin-embedded cervical biopsies to determine the association of RAP1 with cervical intraepithelial neoplasia (CIN). Standard and quantitative immunohistochemistry assessment of RAP1 expression in fixed tissue was performed on 183 paraffin-embedded cervical biopsies that were classified as normal or non-dysplastic mucosa (NDM) (n = 33); CIN grade 1 (n = 84) and CIN grade 2/3 (n = 66). A gradual increase in RAP1 expression in NDM < CIN 1 < CIN 2/3 (p<0.001) specimens was observed and was in agreement with the histopathologic diagnosis. A progressive increase in the RAP1 expression levels increased the risk of CIN 1 [odds ratio (OR) = 3.50; 95% confidence interval (CI) 1.30-10.64] 3.5 fold and the risk of CIN 2/3 (OR = 19.86, 95% CI 6.40-70.79) nearly 20 fold when compared to NDM. In addition, stereotype ordinal regression analysis showed that this progressive increase in RAP1 expression more strongly impacted CIN 2/3 than CIN 1. Our findings suggest that RAP1 may be a useful biomarker for the diagnosis of CIN.

Outbreak of severe zoonotic vaccinia virus infection, Southeastern Brazil.

In 2010, a vaccinia virus isolate caused an atypically severe outbreak that affected humans and cattle in Brazil. Of 26 rural workers affected, 12 were hospitalized. Our data raise questions about the risk factors related to the increasing number and severity of vaccinia virus infections.

Caraparu virus induces damage and alterations in antioxidant defenses in the liver of BALB/c mice after subcutaneous infection.

Oxidative stress is a disturbance in the oxidant-antioxidant balance leading to potential cellular damage. Most cells can tolerate a mild degree of oxidative stress because they have a system that counteracts oxidation that includes antioxidant molecules such as glutathione (GSH) and superoxide dismutase (SOD). Disruption of the host antioxidant status has been recognized as an important contributor to the pathogenesis of many viruses. Caraparu virus (CARV) is a member of group C of the Bunyaviridae family of viruses. In South American countries, group C bunyaviruses are among the common agents of human febrile illness and have caused multiple notable outbreaks of human disease in recent decades; nevertheless, little is known about the pathogenic characteristics of these viruses. The purpose of this study was to examine the hepatic pathogenesis of CARV in mice and the involvement of oxidative stress and antioxidant defenses on this pathology. Following subcutaneous infection of BALB/c mice, CARV was detected in the liver, and histopathology revealed acute hepatitis. Increased serum levels of aspartate and alanine aminotransferases (AST/ALT) and greater hepatic expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) were found in infected animals. CARV infection did not alter the biomarkers of oxidative stress but caused an increase in GSH content and altered the expression and activity of SOD. This is the first report of an alteration of oxidative homeostasis upon CARV infection, which may, in part, explain the hepatic pathogenesis of this virus, as well as the pathogenesis of other Bunyaviridae members.

Differential upregulation of human 2'5'OAS genes on systemic sclerosis: Detection of increased basal levels of OASL and OAS2 genes through a qPCR based assay.

2'5'OAS are template-independent RNA polymerases with antiviral activity and important to homeostasis maintenance. Here we have developed quantitative PCR (qPCR) reactions for the detection of each individual 2'5'OAS human gene and used them to evaluate these gene levels in systemic sclerosis patients cells. The method was efficient for quantification of 2'5'OAS genes on human cells after interferon (IFN) treatment, and revealed that primary cells from patients with systemic sclerosis have increased basal levels of OASL and OAS2 genes. When treated, patients cells are able to induce all four 2'5'OAS genes. Our hypothesis is that abnormally circulating type I IFNs on the disease could be establishing a desensitized state on patients cells, making them refractory to subsequent IFN doses, and that OASL and OAS2 genes upregulation may be due to an IFN-independent stimulus. Further characterizing the biological activities of these genes, their induction pathways and their regulatory functions can lead to better understanding of systemic sclerosis molecular mechanisms and of their biological activities.

Group 1 Vaccinia virus zoonotic outbreak in Maranhao State, Brazil.

In Brazil, several exanthematic autochthone Vaccinia virus (VACV) outbreaks affecting dairy cattle and rural workers have been reported since 1999. Although outbreaks had been first described in the Brazilian Southeast, VACV outbreaks were notified in all Brazilian regions in < 10 years. However, in this context, VACV outbreaks had not been described in some Brazilian States, likely because of a lack of notification, or yet unknown epidemiological reasons. Here, we describe the first VACV outbreak in Maranhão State, northeastern Brazil. The virus isolated from this outbreak showed several biological and molecular features that resemble other Group 1 Brazilian VACV, including a deletion signature in the A56R gene. This study raises new questions about diversity and epidemiology of Brazilian VACV.

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.

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.

Recombinant envelope protein-based enzyme immunoassay for IgG antibodies is comparable to neutralization tests for epidemiological studies of dengue infection.

Dengue virus (DENV) is the most prevalent arbovirus in the world, found mainly in tropical regions. As clinical manifestations present frequently as nonspecific febrile illness, laboratory diagnosis is essential to confirm DENV infections and for epidemiological studies. Recombinant envelope (E) antigens of four serotypes of DENV were used to develop an immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA). To evaluate the IgG-ELISA, a panel of serum samples that had been tested previously by a plaque reduction neutralization test (PRNT) was investigated for the presence of anti-E antibodies against the four DENV serotypes. IgG-ELISA was found to have a sensitivity (91%) and specificity (98%) at a receiver-operating characteristic (ROC) optimized cutoff and demonstrated high performance as well as good indexes. A concordance of 97% was achieved between both assays, and only 21/704 (3%) samples were not concordant. The results of the present study demonstrate a moderate correlation between neutralizing antibody titers and IgG-ELISA values. These findings indicate that the recombinant protein-based IgG-ELISA is a suitable method for routine serodiagnosis, monitoring and seroepidemiological studies of DENV infections.

Group 2 vaccinia virus, Brazil.

In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil.

Virucidal activity of chemical biocides against mimivirus, a putative pneumonia agent.

BACKGROUND: Acanthamoeba polyphaga mimivirus (APMV), the largest known virus, has been studied as a putative pneumonia agent, especially in hospital environments. Despite the repercussions of the discovery of APMV, there has been no study related to the control of APMV and the susceptibility of this virus to disinfectants. OBJECTIVES: This work investigated the virucidal activity against mimivirus of chemical biocides commonly used in clinical practice for the disinfection of hospital equipment and rooms. STUDY DESIGN: APMV was dried on sterilized steel coupons, exposed to different concentrations of alcohols (ethanol, 1-propanol and 2-propanol) and commercial disinfectants (active chlorine, glutaraldehyde and benzalkonium chloride) and titrated in amoebas using the TCID50 value. The stability of APMV on an inanimate surface was also tested in the presence and absence of organic matter for 30 days. RESULTS: APMV showed a high level of resistance to chemical biocides, especially alcohols. Only active chlorine and glutaraldehyde were able to decrease the APMV titers to undetectable levels. Dried APMV showed long-lasting stability on an inanimate surface (30 days), even in the absence of organic matter. CONCLUSIONS: The data presented herein may help health and laboratory workers plan the best strategy to control this putative pneumonia agent from surfaces and devices.