Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Respiratory illness in young and adult cattle caused by bovine viral diarrhea virus subgenotype 2b in singular and mixed bacterial infection in a BVDV-vaccinated dairy herd.

Bovine respiratory disease (BRD) is a common global health problem in dairy cattle. The definitive diagnosis of BRD is complex because its etiology involves several predisposing and determining factors. This report describes the etiology of a BRD outbreak in a dairy herd in the mesoregion of Central Eastern Paraná, which simultaneously affected young (calves and heifers) and adult (cows) Holstein-Friesian cattle. Nine biological samples, consisting of five lung samples from two cows and three suckling calves, and four nasal swab samples from heifers, were used for etiological diagnosis. The nucleic acids extracted from lung fragments and nasal swabs were subjected to PCR and RT-PCR assays for partial amplification of the genes of five viruses [bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoAHV1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (BPIV-3), and bovine coronavirus (BCoV)] and four bacteria (Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) involved in the etiology of BRD. All nine biological samples from the animals with BRD tested negative for BoAHV1, BRSV, BPIV-3, BCoV, and H. somni. Therefore, the involvement of these microorganisms in the etiology of BRD outbreak can be ruled out. It was possible to identify the presence of BVDV and M. bovis in singular and mixed infections of the lower respiratory tract in cattle. BVDV was also identified in two nasal swabs: one as a single etiological agent and the other in association with two bacteria (P. multocida and M. haemolytica). The phylogenetic analysis conducted in the nucleotide sequence of the 5'UTR region and Npro gene of the BVDV amplicons demonstrated that the BVDV field strains of this BRD outbreak belong to subgenotype 2b. To the best of our knowledge, this is the first report of BVDV-2b involvement in the etiology of BRD in Brazil. Finally, it is necessary to highlight that the cattle were obtained from an open dairy herd with biannual vaccinations for BVDV-1a and - 2a.

Hepatitis B Virus Genotype D: An Overview of Molecular Epidemiology, Evolutionary History, and Clinical Characteristics.

The hepatitis B virus (HBV) genotype D (HBV/D) is the most extensively distributed genotype worldwide with distinct molecular and epidemiological features. This report provides an up-to-date review on the history of HBV/D subgenotyping and misclassifications, along with large-scale analysis of over 1000 HBV/D complete genome sequences, with the aim of gaining a thorough understanding of the global prevalence and geographic distribution of HBV/D subgenotypes. We have additionally explored recent paleogenomic findings, which facilitated the detection of HBV/D genomes dating back to the late Iron Age and provided new perspectives on the origins of modern HBV/D strains. Finally, reports on distinct disease outcomes and responses to antiviral therapy among HBV/D subgenotypes are discussed, further highlighting the complexity of this genotype and the importance of HBV subgenotyping in the management and treatment of hepatitis B.

Bovine Viral Diarrhea Virus in Cattle From Mexico: Current Status.

Bovine viral diarrhea (BVD) is an infectious disease, globally-distributed, caused by bovine Pestiviruses, endemic of cattle and other ruminant populations. BVD leads to significant economic losses to the cattle industry due to the wide range of clinical manifestations, including respiratory and gastrointestinal diseases and reproductive disorders. Within the Pestivirus genus of the family Flaviviridae three viral species are associated with BVD; Pestivirus A (Bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (Bovine viral diarrhea virus 2, BVDV-2), and Pestivirus H (HoBi-like pestivirus, atypical ruminant pestivirus). These species are subdivided into subgenotypes based on phylogenetic analysis. The extensive genetic diversity of BVDV has been reported for several countries, where the incidence and genetic variation are more developed in Europe than in the Americas. The first report of BVDV in Mexico was in 1975; this study revealed seropositivity of 75% in cows with a clinical history of infertility, abortions, and respiratory disease. Other studies have demonstrated the presence of antibodies against BVDV with a seroprevalence ranging from 7.4 to 100%. Recently, endemic BVDV strains affecting cattle populations started to be analyzed, providing evidence of the BVDV diversity in several states of the country, revealing that at least four subgenotypes (BVDV-1a, 1b, 1c, and 2a) are circulating in animal populations in Mexico. Little information regarding BVD epidemiological current status in Mexico is available. This review summarizes available information regarding the prevalence and genetic diversity viruses associated with BVD in cattle from Mexico.

Drug-resistant and immune-escape hepatitis B virus mutants, occult hepatitis B infection and coinfections in public hospital patients from Argentina.

Argentina exhibits low serological prevalence for Hepatitis B virus (HBV); however, occult hepatitis B infection (OBI) has been reported in blood donors, Amerindians and individuals coinfected with hepatitis C virus (HCV), and/or human immunodeficiency virus (HIV). The aim of this study was to analyze the genetic diversity of HBV and to evaluate serological marker associations and coinfections with HCV and HIV in patients attending and treated in a public hospital in the province of Buenos Aires, Argentina. A total of 189 HBV reactive samples (HBsAg and/or anti-HBc) were analyzed for HBV DNA characterization. All reactive samples were tested for anti-HCV and HIV-antigen/antibody using CMIA assays. Thirty-six samples exhibited detectable HBV DNA, 7 of which were OBI. HBV sequences were classified as subgenotypes A1, A2, B2, D3, F1b, F3 and F4. Mutations related to the ability to escape the host's immune response, resistance to antiviral therapy and progression to disease were found in patients, partly due to the variable sensitivity of HBsAg, the reverse transcriptase, the basal core promoter and the preCore. HCV and HIV prevalence was 10% and most of the genotypes found in the sequences were genotype 1 and B/F recombinant subtype, respectively. Of the total samples analyzed, 7 exhibited coinfections. This study shows the frequency of OBI, subgenotype distribution, HBV mutations and coinfections, which may have important clinical implications in public hospital patients. Planned prevention, detection and treatment adherence are needed to reduce transmission and morbidity in vulnerable populations.

Detection of Pestivirus A (bovine viral diarrhea virus 1) in free-living wild boars in Brazil.

Bovine viral diarrhea virus (BVDV) is a major pathogen in cattle herds. Considering the epidemiological importance of pestiviruses and the process of wild boar invasion in Brazil, this study aimed to investigate the presence of BVDV in free-living boars. Forty-nine free-living wild boars were collected by exotic wildlife controller agents in 2017 and 2018. The presence of BVDV antibodies was evaluated in 42 serum samples using the virus neutralization test, and the detection of BVDV RNA was performed from the 5'UTR genomic region by RT-PCR assay in 49 lung tissue samples followed by sequencing of amplicons. BVDV neutralizing antibodies in serum were not identified in any of the evaluated samples. However, 3/49 (6.12%) lung samples were positive for BVDV RNA and classified one as BVDV-1a and two as 1d subgenotype. This report identified BVDV RNA in free-living wild boars and these results should be considered in BVDV control programs, especially in extensive beef cattle rearing systems.

Hepatitis C virus genotypes and associated risk factors in the state of Pará, Northern Brazil.

BACKGROUND: Despite the emergence of more effective therapies, hepatitis C virus (HCV) infection remains a serious public health problem at the global level. Currently, this virus is classified into seven genotypes and 67 subgenotypes, which in turn are distributed heterogeneously in Brazil and worldwide. Studies have shown that this genetic divergence results in differences in the progression of chronic disease associated with HCV infection and its treatment. OBJECTIVE: The aim of this study was to report the frequency of HCV genotypes in the state of Pará, Northern Brazil, and to assess the association between genotype and different clinical and laboratory characteristics, as well as risk factors for infection. METHOD: Data from 85 medical records of untreated patients who had chronic hepatitis C infection were analyzed; the patients were evaluated at two hospitals in Belem, Pará, Brazil. RESULTS: Circulation of genotypes 1 and 3 was detected, with a higher prevalence of genotype 1 (75.3%) than genotype 3 (24.7%). In addition, there was a predominance of subgenotype 1b (60.34%) compared to 1a (20.69%) and 3a (18.97%). Reuse of needles and/or glass syringes was significantly associated with infection by HCV genotype 1 than genotype 3; however, the small number of patients infected with genotype 3 may have biased the results. No associations between genotype and the evaluated clinical and laboratory characteristics were observed. CONCLUSION: This study reinforces the differences in the distribution of HCV genotypes in Brazil and showed no association between HCV genotype and progression of chronic hepatitis C in the studied group.

Hepatitis C virus genotypes and associated risk factors in the state of Pará, Northern Brazil

Abstract Background Despite the emergence of more effective therapies, hepatitis C virus (HCV) infection remains a serious public health problem at the global level. Currently, this virus is classified into seven genotypes and 67 subgenotypes, which in turn are distributed heterogeneously in Brazil and worldwide. Studies have shown that this genetic divergence results in differences in the progression of chronic disease associated with HCV infection and its treatment. Objective The aim of this study was to report the frequency of HCV genotypes in the state of Pará, Northern Brazil, and to assess the association between genotype and different clinical and laboratory characteristics, as well as risk factors for infection. Method Data from 85 medical records of untreated patients who had chronic hepatitis C infection were analyzed; the patients were evaluated at two hospitals in Belem, Pará, Brazil. Results Circulation of genotypes 1 and 3 was detected, with a higher prevalence of genotype 1 (75.3%) than genotype 3 (24.7%). In addition, there was a predominance of subgenotype 1b (60.34%) compared to 1a (20.69%) and 3a (18.97%). Reuse of needles and/or glass syringes was significantly associated with infection by HCV genotype 1 than genotype 3; however, the small number of patients infected with genotype 3 may have biased the results. No associations between genotype and the evaluated clinical and laboratory characteristics were observed. Conclusion This study reinforces the differences in the distribution of HCV genotypes in Brazil and showed no association between HCV genotype and progression of chronic hepatitis C in the studied group.

Toward unified molecular surveillance of RSV: A proposal for genotype definition.

BACKGROUND: Human respiratory syncytial virus (RSV) is classified into antigenic subgroups A and B. Thirteen genotypes have been defined for RSV-A and 20 for RSV-B, without any consensus on genotype definition. METHODS: We evaluated clustering of RSV sequences published in GenBank until February 2018 to define genotypes by using maximum likelihood and Bayesian phylogenetic analyses and average p-distances. RESULTS: We compared the patterns of sequence clustering of complete genomes; the three surface glycoproteins genes (SH, G, and F, single and concatenated); the ectodomain and the 2nd hypervariable region of G gene. Although complete genome analysis achieved the best resolution, the F, G, and G-ectodomain phylogenies showed similar topologies with statistical support comparable to complete genome. Based on the widespread geographic representation and large number of available G-ectodomain sequences, this region was chosen as the minimum region suitable for RSV genotyping. A genotype was defined as a monophyletic cluster of sequences with high statistical support (≥80% bootstrap and ≥0.8 posterior probability), with an intragenotype p-distance ≤0.03 for both subgroups and an intergenotype p-distance ≥0.09 for RSV-A and ≥0.05 for RSV-B. In this work, the number of genotypes was reduced from 13 to three for RSV-A (GA1-GA3) and from 20 to seven for RSV-B (GB1-GB7). Within these, two additional levels of classification were defined: subgenotypes and lineages. Signature amino acid substitutions to complement this classification were also identified. CONCLUSIONS: We propose an objective protocol for RSV genotyping suitable for adoption as an international standard to support the global expansion of RSV molecular surveillance.

Genetic Diversity of the Hepatitis B Virus Subgenotypes in Brazil.

Hepatitis B virus (HBV) subgenotypes may be related to clinical outcomes and response to antiviral therapy. Most Brazilian studies on HBV subgenotypes are restricted to some regions and to specific population groups. Here, we provide an insight about genetic diversity of HBV subgenotypes in 321 serum samples from all five geographical regions, providing a representative overview of their circulation among chronic carriers. Overall, HBV/A1 was the most prevalent subgenotype, being found as the major one in all regions except in South Brazil. Among HBV/D samples, subgenotype D3 was the most prevalent, found in 51.5%, followed by D2 (27.3%) and D4 (21.2%). D2 and D3 were the most prevalent subgenotypes in South region, with high similarity with European strains. D4 was found in North and Northeast region and clustered with strains from Cape Verde and India. For HBV/F, the most frequent subgenotype was F2 (84.1%), followed by F4 (10.1%) and F1 (5.8%), closely related with strains from Venezuela, Argentina and Chile, respectively. Phylogeographic analyses were performed using an HBV full-length genome obtained from samples infected with genotypes rarely found in Brazil (B, C, and E). According to Bayesian inference, HBV/B2 and HBV/C2 were probably introduced in Brazil through China, and HBV/E from Guinea, all of them mostly linked to recent events of human migration. In conclusion, this study provided a comprehensive overview of the current circulation of HBV subgenotypes in Brazil. Our findings might contribute to a better understand of the dynamics of viral variants, to establish a permanent molecular surveillance on the introduction and dispersion patterns of new strains and, thus, to support public policies to control HBV dissemination in Brazil.

Natural infection by Cryptosporidium SPP. in production animals: first description of subtype IIaa15G2R1 in goat kids and piglets in Brazil

Cryptosporidiosis is a severe enteric disease, with varied clinical manifestations. In young animals the infection is more common and may be more severe. In this study the polymerase chain reaction (PCR) was used to detect Cryptosporidium parasites in goat kids, calves, lambs, piglets and colts sharing the same environment. Fecal samples were collected directly from the rectum of 192 goat kids, 184 calves, 44 lambs, 47 piglets and 26 colts aged up to twelve months, males and females, of different breeds, from the Brazilian states of Goiás, Mato Grosso do Sul, Minas Gerais and São Paulo. PCR was used for amplifying a fragment of 18S rRNA gene and the gene encoding the surface glycoprotein GP60. Positive PCR amplification was observed in 16.7% (32/192) goat kids, 6.5% (12/184) calves and 2.1% (1/47) piglets. Based on the sequencing of 18S rRNA PCR products, all samples from goat kids were identified as C. parvum. Among calves, C. parvum was identified in 41.7% (5/12), C. andersoni in 16.7% (2/12), C. ryanae in 16.7% (2/12) and C. bovis in 25% (3/12) of the animals. All GP60 sequences were classified as genotype IIaA15G2R1 and were found in goat kids, calves and piglets sharing the same environment. This is the first description of the molecular identification and genotyping of Cryptosporidium in goat kids and piglets in Brazil. We conclude that Cryptosporidium species and C. parvum GP60 subtypes that infect livestock in Brazil, may act as sources of zoonotic infection for other animals and humans.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher ...

Molecular and clinical evaluation of the acute human parvovirus B19 infection: comparison of two cases in children with sickle cell disease and discussion of the literature

Human parvovirus B19 is a well-known cause of severe conditions in patients with sickle cell disease, but the molecular mechanisms of the infection are insufficiently understood. The different clinical outcome of the acute parvovirus B19 infection in two pediatric patients with sickle cell disease has been examined. One of them developed life-threatening condition requiring emergency transfusions, while the other had asymptomatic infection, diagnosed occasionally. Both cases had high viral load and identical subgenotype, indicating that the viral molecular characteristics play a minimal role in the infection outcome.

Hepatitis A virus subgenotypes dissemination during a community outbreak in a surrounding region of Rio de Janeiro

From December 1999 to December 2001, many cases of hepatitis A were notified in the county of Belford Roxo involving individuals aged 0 to 79 years. Serum samples were collected to evaluate the prevalence of anti-hepatitis A virus (HAV) antibodies, to detect HAV-RNA and to correlate with possible risk factors of HAV infection. Serum samples were screened by commercial IgM and total anti-HAV antibody ELISA and HAV-RNA was isolated and subsequently amplified by reverse transcription-polymerase chain reaction (RT-PCR) at VP1/2A region, sequenced and analyzed. Total anti-HAV prevalence was 87.9 percent (203/231) and IgM anti-HAV prevalence was 38.7 percent (89/231). Multivariate analysis showed that individuals under 20 years old are risks groups to acquire the infection suggesting that hygienic habits of young subjects are the principal factor of transmission and so they could be the target for vaccine programs. HAV-RNA was amplified from 29 (32.5 percent) IgM anti-HAV positive patients and 26 samples were sequenced and classified into subgenotypes IB (8 isolates) and IA (18 isolates). Isolates classified into subgenotype IB were identical representing one distinct strain. We could observe both subgenotypes circulating during the study which suggests different sources of infection. Prophylactic measures as vaccination strategies added to improvements in hygienic and sanitary conditions would be highly effective to reduction of infection.