Influenza A virus infection of healthy piglets in an abattoir in Brazil: animal-human interface and risk for interspecies transmission

Asymptomatic influenza virus infections in pigs are frequent and the lack of measures for controlling viral spread facilitates the circulation of different virus strains between pigs. The goal of this study was to demonstrate the circulation of influenza A virus strains among asymptomatic piglets in an abattoir in Brazil and discuss the potential public health impacts. Tracheal samples (n = 330) were collected from asymptomatic animals by a veterinarian that also performed visual lung tissue examinations. No slaughtered animals presented with any noticeable macroscopic signs of influenza infection following examination of lung tissues. Samples were then analysed by reverse transcription-polymerase chain reaction that resulted in the identification of 30 (9%) influenza A positive samples. The presence of asymptomatic pig infections suggested that these animals could facilitate virus dissemination and act as a source of infection for the herd, thereby enabling the emergence of influenza outbreaks associated with significant economic losses. Furthermore, the continuous exposure of the farm and abattoir workers to the virus increases the risk for interspecies transmission. Monitoring measures of swine influenza virus infections and vaccination and monitoring of employees for influenza infection should also be considered. In addition regulatory agencies should consider the public health ramifications regarding the potential zoonotic viral transmission between humans and pigs.

Etude du respect des mesures de prévention de la transmission verticale du virus de l'hépatite B chez les enfants nés dans les maternités des hôpitaux publics du canton de Vaud

Actuellement, en Suisse, environ 1% des femmes en âge de procréer ont une hépatite B chronique(8). En l'absence de mesures de prévention, le risque de transmission du virus de l'hépatite B, de la mère à son enfant, est estimé à 40%(3,4,5) lors de l'accouchement. Ce risque s'étend bien au-delà de la période péri¬natale. Les enfants infectés dans ces circonstances ont une probabilité de 90% de développer une infection chronique(5,7) et un quart meurent prématurément de cirrhose ou d'hépatocarcinome(2). L'Office fédéral de la santé publique recommande d'effectuer un dépistage anténatal de l'antigène HBsAg lors de toute grossesse(1) et d'effectuer une vaccination passive et active chez tous les enfants naissant d'une mère avec une hépatite B chronique. Cette prophylaxie doit être effectuée comme suit : immunoglobuline spécifique et lere dose de vaccin dans les 12 heures suivant la naissance (en maternité) ; 2eme dose de vaccin à 1 mois, 3eme dose de vaccin à 6 mois et contrôle de la réponse immune entre le 7eme et le 12eme mois (par le médecin traitant). Cette étude vise à évaluer la compliance du système de soins envers ces recommandations qui exigent l'intervention des maternités et des médecins traitants et qui s'étalent dans le temps. Pour ce faire, un recensement rétrospectif des enfants nés de mère avec une hépatite B chronique, en 2005 et 2006 dans 4 maternités vaudoises, a été effectué. Les mesures appliquées par les maternités, les informations transmises aux médecins traitants et les mesures appliquées par ces derniers ont été évaluées. Sur un total de 10'412 parturientes testées, 70 présentent une infection chronique et 51 acceptent le recrutement dans l'étude (représentant un collectif de 54 enfants). En maternité, l'immunisation active et passive est effectuée chez tous les enfants. L'évidence qu'elle est effectuée dans les 12 heures suivant la naissance est fournie dans 61% des cas (mais dans 100% des dossiers dans lequel ce renseignement est consigné). La nécessité de poursuivre la vaccination n'est mentionné au médecin traitant que dans 15% des cas, et dans seulement 11% des cas les modalités du calendrier vaccinal sont précisées. La recommandation d'effectuer un contrôle sérologique n'apparaît dans aucun document de transmission. Chez les médecins traitants, la 2eme dose de vaccin est administrée à 100% des enfants, mais seulement dans 15% des cas dans les délais recommandés. La 3eme dose de vaccination est administrée à 98% des enfants, mais seulement dans 43% des cas dans les délais recommandés. La sérologie de contrôle n'est effectuée que chez 24% des enfants, et seulement dans 7% des cas dans les délais recommandés. Les maternités appliquent les mesures de prophylaxie dans le délai imparti, tout au moins quand l'heure d'intervention est indiquée. Les médecins traitants sont rarement informés de la nécessité de compléter la vaccination et jamais des modalités ni de la nécessité d'effectuer un contrôle sérologique. L'application des mesures de prévention par les médecins traitants est non conforme aux recommandations. Nous émettons l'hypothèse que cet état reflète la carence d'information de la part des maternités et nous proposons que celles-ci utilisent un document de transmission standardisé qui indique précisément aux médecins traitants ce qui reste à faire, et quand, en matière de prévention de l'hépatite B chez le nouveau-né/nourrisson.

Prevalence of occult hepatitis B virus infection in kidney transplant recipients

In this cross-sectional study, 207 hepatitis B surface antigen (HBsAg)-negative kidney transplant recipients were evaluated based on demographic and epidemiological data and on the levels of serological markers of hepatitis B virus (HBV) and hepatitis C virus infection and liver enzymes. Patients with HBV or human immunodeficiency virus infection were excluded. Sera were analysed for the presence of HBV-DNA. HBV-DNA was detected in two patients (1%), indicating occult hepatitis B (OHB) infection (the HBV-DNA loads were 3.1 and 3.5 IU/mL in these patients). The results of the liver function tests were normal and no serological markers indicative of HBV infection were detected. The prevalence of OHB infection was low among kidney transplant recipients, most likely due to the low HBsAg endemicity in the general population of the study area.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

Performance of diagnostic biomarkers in predicting liver fibrosis among hepatitis C virus-infected Egyptian children

The aim of the present study was to identify specific markers that mirror liver fibrosis progression as an alternative to biopsy when biopsy is contraindicated, especially in children. After liver biopsies were performed, serum samples from 30 hepatitis C virus (HCV) paediatric patients (8-14 years) were analysed and compared with samples from 30 healthy subjects. All subjects were tested for the presence of serum anti-HCV antibodies. Direct biomarkers for liver fibrosis, including transforming growth factor-β1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hyaluronic acid (HA), procollagen type III amino-terminal peptide (PIIINP) and osteopontin (OPN), were measured. The indirect biomarkers aspartate and alanine aminotransferases, albumin and bilirubin were also tested. The results revealed a significant increase in the serum marker levels in HCV-infected children compared with the healthy group, whereas albumin levels exhibited a significant decrease. Significantly higher levels of PIIINP, TIMP-1, OPN and HA were detected in HCV-infected children with moderate to severe fibrosis compared with children with mild fibrosis (p < 0.05). The diagnostic accuracy of these direct biomarkers, represented by sensitivity, specificity and positive predictive value, emphasises the utility of PIIINP, TIMP-1, OPN and HA as indicators of liver fibrosis among HCV-infected children.

Serologic survey of West Nile virus in horses from Central-West, Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmaniaantigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.

Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus

In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.

A comparative study of the effect of multiple immersions on Aedini (Diptera: Culicidae) mosquito eggs with emphasis on sylvan vectors of yellow fever virus

The effect of multiple immersions on Haemagogus janthinomys , Haemagogus leucocelaenus , Aedes albopictus and Ochlerotatus terrens eggs was studied. Eggs were collected in April, June, October and December of 2011 in Minas Gerais, Brazil. Most of the Aedes and Ochlerotatus eggs hatched upon the first immersion, while Haemagogus eggs showed a varied instalment hatching response. The number of immersions required for hatching increased for eggs collected closer to the dry winter season.

Clinical features and laboratory findings of human parvovirus B19 in human immunodeficiency virus-infected patients

Immunocompromised patients may develop severe chronic anaemia when infected by human parvovirus B19 (B19V). However, this is not the case in human immunodeficiency virus (HIV)-infected patients with good adherence to highly active antiretroviral treatment (HAART). In this study, we investigated the clinical evolution of five HIV-infected patients receiving HAART who had B19V infections confirmed by serum polymerase chain reaction. Four of the patients were infected with genotype 1a strains and the remaining patient was infected with a genotype 3b strain. Anaemia was detected in three of the patients, but all patients recovered without requiring immunoglobulin and/or blood transfusions. In all cases, the attending physicians did not suspect the B19V infections. There was no apparent relationship between the infecting genotype and the clinical course. In the HAART era, B19V infections in HIV-positive patients may be limited, subtle or unapparent.

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.