Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somniwith sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.

PREVALENCE OF HERPES SIMPLEX VIRUS TYPE 2 AND RISK FACTORS ASSOCIATED WITH THIS INFECTION IN WOMEN IN SOUTHERN BRAZIL

SUMMARY The herpes simplex virus type 2 (HVS-2) is the most prevalent infection worldwide. It is a cofactor in the acquisition of human immunodeficiency virus (HIV) and the persistence of human papillomavirus (HPV). This study evaluated the prevalence of HSV-2, using the polymerase chain reaction (PCR), and associated factors in patients treated at the Federal University of Rio Grande (FURG) and Basic Health Units (BHU) in Rio Grande, Brazil. The observed prevalence of HSV-2 was 15.6%. Among the 302 women studied, 158 had received assistance in BHU and 144 were treated at FURG. The prevalence of HSV-2 in these groups was 10.8% and 20.8%, respectively, RR 1.9 and p = 0.012. Knowledge about the Pap smear, and the presence of lesions showed no association with HSV-2 infection. Multivariate analysis showed that the variable that most influenced the risk of HSV-2 infection was the presence of HIV infection, with a relative risk of 1.9 and p = 0.04. Discussion: Genital ulcers are an important entry point for HIV, and condom use is an important strategy to reduce transmission of HIV and HSV-2.

A expressão da proteína p16 e herpes simples vírus tipo 2 em lesões pré-neoplásicas e neoplásicas do colo do útero

OBJETIVO: demonstrar a expressão de biomarcadores, detectados por técnicas de imunohistoquímica, em tecidos sadios, lesões pré-neoplásicas e neoplásicas do colo do útero. MÉTODOS: para avaliação da reatividade imunohistoquímica de tecidos do colo do útero ao p16 e ao herpes simples vírus tipo 2 (HSV-2), foram avaliadas 187 amostras de lesões intra-epiteliais de baixo grau (LIE-BG) e lesões intra-epiteliais de alto grau (LIE-AG) e carcinoma do colo do útero, e comparadas com grupo de pacientes sem lesões no colo uterino. A análise estatística foi realizada pelo teste do chi2 para tendências. O nível de significância foi de alfa=0,05. RESULTADOS: foi avaliada a reatividade ao p16 com a seguinte distribuição: grupo sem lesão no colo do útero: 56% (24/43), LIE-BG: 92% (43/47), LIE-AG: 94% (43/46) e câncer: 98% (46/47) (p<0,001, tendência linear). Com relação ao HSV-2: grupo sem lesão no colo do útero: 27% (12/45), LIE-BG: 58% (22/38), LIE-AG: 78% (35/45) e câncer: 59% (29/49) (p<0,001, tendência linear). Foi observado aumento na proporção de reatividade para os dois marcadores entre os grupos controle, LIE-BG, LIE-AG e câncer do colo do útero (p<0,001). Não houve diferença significativa, quando comparamos apenas os grupos LIE-BG e LIE-AG entre si. CONCLUSÕES: FOI VErificado um aumento progressivo nas taxas de reatividade aos marcadores de imunohistoquímica estudados, com a severidade das lesões.

Antibodies against Bovine herpesvirus 1 in dairy herds in the state of Espirito Santo, Brasil

Bovine herpesvirus 1 (BoHV-1) causes major losses in worldwide livestock, affecting the respiratory and reproductive tracts of bovine. In the past decades, the number of cases in Brazil has been gradually increasing. Therefore, it is important to assess the distribution of infection in different regions of the country. In the state of Espírito Santo (ES) the BoHV 1 infection rate in dairy cattle herds is unknown. Thus, the aim of this study was to detect neutralizing antibodies against BoHV-1 in serum samples from 1,161 non-vaccinated cows from 59 dairy cattle herds in 23 municipalities of the Metropolitan, North, Northwest and South macro-regions. The identification of seropositive cows was evaluated by the virus neutralization test. The results showed that of all serum samples evaluated 775 (66.75%) had neutralizing antibodies against BoHV-1. Moreover, all herds were found positive; however, the percentage of positive cows varied among regions; 49.06%, 62.15%, 67.21% and 80.04% for the Metropolitan, South, North and Northwest macro-regions, respectively. In this study, the results clearly indicate the dissemination of the viral agent in dairy cattle in the ES state, requiring the monitoring and control of diseases related to BoHV-1 infection.

Association of human herpesvirus 6 infection with exanthem subitum in Belem, Brazil

Recent human herpesvirus 6 (HHV-6) infection was detected in cases of exanthem subitum (ES) involving four children, aged 10 to 24 months, between April and August 1994, in Belém, Brazil. By using the indirect immunofluorescence antibody assay (IFA), significant increases (at least eight times) in antibody concentrations were noted from the acute to the convalescent serum samples, with titers ranging from <1:10/1:80 to <1:10/1:640 (patients 3 and 2, respectively). All children had high fever (over 39ºC) for three days, followed by generalized, maculo-papular skin rash. A physical examination of the children also revealed concomitant, cervical lymph node swelling and tonsillar pharyngitis in two of them.

Human herpesvirus-7 as a cause of exanthematous ilnesses in Belém, Pará, Brazil

We screened sera from 370 patients suffering from exanthematous illnesses in Belém, North Brazil, for the presence of human herpesvirus-7 (HHV-7) IgM and IgG antibodies. Samples were obtained from January 1996 to December 2002 and were processed by a HHV-7-specific indirect immunofluorescence assay (IFA). HHV-7-specific IgM and/or IgG antibodies were found in 190 (51.4%) of these patients, with similar prevalence rates (IgM+ and IgG+ subgroups taken together) for female and male subjects: 52.5% and 50.3%, respectively. Serological status as defined by IgG was identified in 135 (36.5%) patients. In 55 (14.9%) of the patients HHV-7 IgM antibodies were detected. HHV-7 IgM- and- IgG antibody rates were similar (p > 0.05) when male and female subjects are compared: 14.4% versus 15.3% and 38.1% versus 35.0%, respectively. Statistically significant difference (p = 0.003) was noted when HHV-7-IgM-positive female and male patients aged 5-8 months are compared. Prevalence rates ranging from 4.6% (female, 5-8 months of age) to 93.3% (female, > 10 years of age) and 12.2% (male, 5-8 months) to 80.0% (male, 8-10 years of age) were noted in the IgG- positive subgroups. A subgroup (n = 131) of patients with IgM or IgG HHV-7 antibodies were examined for the presence of DNA using a polymerase chain reaction/nested PCR. Recent/active HHV-7 infection occurred at a rate of 11.0% (6/55) among patients whose samples presented IgM+ specific antibodies. In a subgroup (n = 76) of patients with high HHV-7-IgG antibody levels (titre > 1:160) DNA could not be detected in sera examined by PCR/nested PCR. Of the six recent/active infections, four subjects with less than 1 year and two with 3 and 6 years of age, presented typical exanthem subitum (E.S), as defined by higher fever (> 38.0 ºC) with duration of 24 to 72 hours, followed by a maculopapular skin rash. Our results underscore the need for searching HHV-7 infection in patients with exanthematous diseases, particularly those presenting with typical E.S. HHV-7 appears therefore to emerge as a newly recognized pathogen of exanthem in our region.

LACK OF ASSOCIATION BETWEEN HERPESVIRUS DETECTION IN SALIVA AND GINGIVITIS IN HIV209;INFECTED CHILDREN

The aims of this study were to compare the detection of human herpesviruses (HHVs) in the saliva of HIV-infected and healthy control children, and to evaluate associations between viral infection and gingivitis and immunodeficiency. Saliva samples were collected from 48 HIV-infected and 48 healthy control children. Clinical and laboratory data were collected during dental visits and from medical records. A trained dentist determined gingival indices and extension of gingivitis. Saliva samples were tested for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by nested polymerase chain reaction assays. Thirty-five HIV-infected and 16 control children had gingivitis. Seventeen (35.4%) HIV-infected children and 13 (27%) control children were positive for HHVs. CMV was the most commonly detected HHV in both groups (HIV-infected, 25%; control, 12.5%), followed by HSV-1 (6.2% in both groups) and HSV-2 (HIV-infected, 4.2%; control, 8.3%). The presence of HHVs in saliva was not associated with the presence of gingivitis in HIV-1-infected children (p = 0.104) or healthy control children (p = 0.251), or with immunosuppression in HIV-infected individuals (p = 0.447). Gingivitis was correlated with HIV infection (p = 0.0001). These results suggest that asymptomatic salivary detection of HHVs is common in HIV-infected and healthy children, and that it is not associated with gingivitis.

Detection of human herpesvirus-7 by qualitative nested-PCR: comparison between healthy individuals and liver transplant recipients

Diagnosis of human herpesvirus-7 active infection in transplant patients has proved difficult, because this virus is ubiquitous and can cause persistent infections in the host. The significance of viral DNA detected in leukocytes by PCR is unclear and cross-reaction in serological tests may occur. This study aimed to evaluate nested-PCR to detect human herpesvirus-7 active infection in liver transplant recipients compared to healthy individuals. human herpesvirus-7 nested-PCR was performed on leukocytes and sera of 53 healthy volunteers and sera of 29 liver transplant recipients. In healthy volunteers, human herpesvirus-7 was detected in 28.3% of leukocytes and 0% of serum. human herpesvirus-7 was detected in sera of 48.2% of the liver transplant recipients. Nested-PCR on DNA extracted from leukocytes detected latent infection and the study suggests that nested-PCR performed on serum could be useful to detect human herpesvirus-7 active infection in liver transplant recipients.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8), some can also cause systemic disease (CMV and HHV-8). The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032) and EBV (OR: 3.44, p= 0.0081). No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028). CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.

Diagnosis of human herpesvirus 6B primary infection by polymerase chain reaction in young children with exanthematic disease

INTRODUCTION: Exanthem subitum is a classical rash disease of early childhood caused by human herpesvirus 6B (HHV-6B). However, the rash is frequently misdiagnosed as that of either measles or rubella. METHODS: In this study, a nested multiplex polymerase chain reaction (PCR) was used to diagnose HHV-6B primary infection, differentiate it from infections caused by HHV-6A and compare it to antibody avidity tests. The samples were separated into case group and control group according to the results of the indirect immunofluorescence assay (IFA) technique. RESULTS: From the saliva samples analyzed, HHV-6A DNA was detected in 3.2% of the case group and in 2.6% of the control group. Regarding HHV-6B, PCR detected viral DNA in 4.8% of the case group and in 1.3% of the control group. Among the serum samples studied, a frequency of 1.7% was determined for HHV-6A in the case group and 1.2% in the control group. PCR did not detect HHV-6B DNA in serum samples. The sensitivity and specificity of the PCR technique ranged from 0% to 4.8% and 97.5% to 100%, respectively, compared to IFA. CONCLUSIONS: The PCR technique was not suitable for diagnosing primary infection by HHV-6B in children with exanthematic disease and should not substitute the IFA.

Current status of herpesvirus identification in the oral cavity of HIV-infected children

INTRODUCTION: Some viruses of the Herpesviridae family are frequently the etiologic agents of oral lesions associated with HIV. The aim of this study was to identify the presence of herpes simplex virus types 1 and 2 (HSV-1, HSV-2), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus type 6, type 7 and type 8 (HHV-6, HHV-7 and HHV-8) in the oral cavity of HIV-infected children/adolescents and verify the association between viral subtypes and clinical factors. METHODS: The cells of oral mucosa were collected from 50 HIV infected children/adolescents, 3-13 years old (mean age 8.66). The majority (66%) of selected were girls, and they were all outpatients at the pediatric AIDS clinic of a public hospital in Rio de Janeiro. Nested-PCR was used to identify the viral types. RESULTS: Absence of immunosuppression was observed in 66% of the children. Highly active antiretroviral therapy (HAART) was used by 72.1% of selected and moderate viral load was observed in 56% of the children/adolescents. Viral types were found in 86% of the children and the subtypes were: HSV-1 (4%), HSV-2 (2%), VZV (4%), EBV (0%), HCMV (24%), HHV6 (18%), HHV-7 (68%), HHV8 (0%). CONCLUSIONS: The use of HAART has helped to reduce oral lesions, especially with herpes virus infections. The health professionals who work with these patients should be aware of such lesions because of their predictive value and the herpes virus can be found circulating in the oral cavity without causing lesions.

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Detection of human herpesvirus 6 and 7 DNA in saliva from healthy adults from Rio de Janeiro, Brazil

In this study, we aimed to evaluate virus shedding in the saliva of healthy adults from the metropolitan region of the city of Rio de Janeiro, Brazil, in order to verify the prevalence of both human herpesviruses 6 and 7 (HHV-6, HHV-7). The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Saliva specimens were subjected to a multiplex polymerase chain reaction (PCR) to detect the presence of HHV-6A, HHV-6B and HHV-7. The total Roseolovirus DNA prevalence was 22.4%. The PCR detected a HHV-6 prevalence of 9.8%, with HHV-6A detected in 7.1% of the samples and HHV-6B in 2.7%. HHV-7 DNA was revealed in 12.6% of the studied cases. Multiple infections caused by HHV-6A and 7 were found in 2.1% of the samples. No statistical differences were observed regarding age, but for HHV-7 infection, an upward trend was observed in female patients. Compared to studies from other countries, low prevalence rates of herpesvirus DNA were detected in saliva from the healthy individuals in our sample. PCR methodology thus proved to be a useful tool for Roseolovirus detection and it is important to consider possible geographic and populations differences that could explain the comparatively low prevalence rates described here.

Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes infectadas experimentalmente com o herpesvírus bovino 2

A mamilite causada pelo herpesvírus bovino tipo 2 (BoHV-2) possui importante repercussão sanitária em gado leiteiro de alguns países, mas a sua patogenia permanece pouco conhecida. Este trabalho descreve a reprodução e caracterização da mamilite aguda em ovelhas lactantes inoculadas com o BoHV-2 na pele do úbere e dos tetos. Cinco de oito ovelhas inoculadas desenvolveram extensas placas com necrose focal, pequenas vesículas e formação de crostas nos locais de inoculação. As lesões foram inicialmente observadas no 4º dia pós-inoculação (pi), progrediram em extensão e gravidade até os dias 7-8pi e posteriormente regrediram. O vírus foi isolado das lesões entre os dias 7 e 8 pi. Antígenos virais e partículas típicas de herpesvírus foram demonstrados por microscopia eletrônica em biópsias de lesões coletadas nos dias 5, 6 e 10pi. Os achados histológicos foram caracterizados por necrose epitelial, erosões e úlceras, com formação de células sinciciais e infiltrado inflamatório linfoplasmocitário na derme. Associadas a essas lesões observaram-se inclusões eosinofílicas intranucleares em células epiteliais, sinciciais e inflamatórias. Em um segundo experimento, 7 de 10 cordeiros inoculados na mucosa nasal desenvolveram hiperemia e descarga nasal, bem como vesículas e erosões no focinho. O vírus foi isolado das secreções nasais por até três dias e todos os cordeiros soroconverteram ao BoHV-2. Tentativas de reativar a infecção latente pela administração de dexametasona no 40º dia pi foram infrutíferas, pois não foram observados re-excreção viral, recrudescência clínica ou soroconversão. Esses resultados demonstram que ovelhas são susceptíveis à infecção experimental pelo BoHV-2 e desenvolvem lesões semelhantes às observadas em casos naturais de mamilite herpética. Além disso, esses resultados sugerem o uso dessa espécie animal como modelo para o estudo de vários aspectos da biologia da infecção pelo BoHV-2.