HoBi-like Pestivirus Is Highly Prevalent in Cattle Herds in the Amazon Region (Northern Brazil).

Pestiviruses are globally distributed and cause substantial economic losses to the cattle industry. In Brazil, the country with the world's largest cattle population, pestivirus infections are well described in some regions, such as in the south, where a high frequency of BVDV-2 is described and contrasts with the high prevalence of HoBi-like pestivirus (HoBiPeV) in the northeast. However, there is a lack of information about pestiviruses in the Amazon Region, in northern Brazil, with a cattle population estimated at 55.7 million head, which has a significant impact on the international livestock market. Therefore, this study investigated the seroprevalence and genetic variability of ruminant pestiviruses in 944 bovine serum samples from four states in northern Brazil: Pará (PA), Amapá (AP), Roraima (RR), and Amazonas (AM). Our results showed that 45.4% of the samples were seropositive (19.8% for BVDV-1, 14.1% for BVDV-2, and 20.9% for HoBiPeV). All samples were tested by RT-qPCR, and three were positive and classified as HoBiPeV in a phylogenetic analysis. These serological and molecular results contrast with those from other regions of the world, suggesting that the northern Brazilian states have a high prevalence of all bovine pestiviruses including HoBiPeV.

A Serosurvey for Ruminant Pestivirus Exposure Conducted Using Sera From Stray Mexico Origin Cattle Captured Crossing Into Southern Texas.

The US Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor a quarantine zone along the Texas border to prevent the introduction of stray livestock carrying cattle fever ticks entering the United States from Mexico. Stray cattle collected by CFTEP are checked for ticks and several infectious disease-causing pathogens, but not for bovine viral diarrhea virus (BVDV). BVDV is one of the most economically impactful viruses affecting US cattle producers. BVDV is present in all parts of the world, but it has been demonstrated that another distantly related pestivirus, HoBi-like pestivirus (HoBiPev), can also cause BVD. To date, HoBiPev has not been detected in the United States, but is commonly found in Brazil, and sporadically in Europe and Asia. The objective of the current study was to evaluate the seroprevalence of pestiviruses, with a specific focus on HoBiPev, in stray cattle. Virus neutralization (VN) assay was used to determine seroprevalence (or antibody titers) of BVDV-1, BVDV-2, and HoBiPev. Approximately 50% (67 of 134) of the samples were seropositive for pestiviruses; all 67 positive samples were positive (50%) for BVDV-1, 66 samples of the 67 were positive (49.3%) for BVDV-2, and the same 66 samples of the 67 were also positive (49.3%) for HoBiPev. Due to the antigenic cross-reactivity among Pestiviruses, the comparative antibody against each pestivirus was calculated from all VN-positive samples. Titers were clearly higher against BVDV-1, and only one sample had a titer clearly higher against BVDV-2. No sample had an antibody titer higher for HoBiPev, and while this does not prove the absence of HoBiPev, it does provide evidence that the prevalence of HoBiPev is less predominant than BVDV-1. Additionally, data from these samples provide evidence on the susceptibility of animals that may enter into the United States, with ~50% of the animals seronegative for bovine pestiviruses. This cattle population provides a unique opportunity to evaluate and monitor changes in seroprevalence of economically important cattle diseases affecting the cattle industry.

Epidemiology of Pestivirus H in Brazil and Its Control Implications.

Along with viruses in the Pestivirus A (Bovine Viral Diarrhea Virus 1, BVDV1) and B species (Bovine Viral Diarrhea Virus 2, BVDV2), members of the Pestivirus H are mainly cattle pathogens. Viruses belonging to the Pestivirus H group are known as HoBi-like pestiviruses (HoBiPev). Genetic and antigenic characterization suggest that HoBiPev are the most divergent pestiviruses identified in cattle to date. The phylogenetic analysis of HoBiPev results in at least five subgroups (a-e). Under natural or experimental conditions, calves infected with HoBiPev strains typically display mild upper respiratory signs, including nasal discharge and cough. Although BVDV1 and BVDV2 are widely distributed and reported in many South American countries, reports of HoBiPev in South America are mostly restricted to Brazil. Despite the endemicity and high prevalence of HoBiPev in Brazil, only HoBiPev-a was identified to date in Brazil. Unquestionably, HoBiPev strains in BVDV vaccine formulations are required to help curb HoBiPev spread in endemic regions. The current situation in Brazil, where at this point only HoBiPev-a seems present, provides a more significant opportunity to control these viruses with the use of a vaccine with a single HoBiPev subtype. Despite the lack of differentiation among bovine pestiviruses by current BVDV tests, the reduced genetic variability of HoBiPev in Brazil may allow reliable identification of cases within the region. On the other hand, introducing foreign ruminants, biologicals, and genetic material to South America, especially if it originated from other HoBiPev-endemic countries, should consider the risk of introducing divergent HoBiPev subtypes.

Virome characterization in serum of healthy show pigs raised in Oklahoma demonstrated great diversity of ssDNA viruses.

In the United States, show pigs are raised to compete in agricultural events. These animals are usually raised in small herds with extensive human, domestic, and wild animal contact. Therefore, pathogen monitoring in this animal category is critical for improved disease surveillance and preparedness. This study characterized the virome of healthy show pigs using high-throughput sequencing using pooled serum samples from 2018 or 2019 (200 samples each pool). Results demonstrated the presence of DNA viral families (Parvoviridae, Circoviridae, and Herpesviridae) and RNA families (Arteriviridae, Flaviviridae, and Retroviridae). Twenty-three viral species were identified, including the first detection of porcine bufavirus in the US. Moreover, important swine pathogens identified included porcine reproductive and respiratory syndrome virus, atypical porcine pestivirus, and porcine circovirus (PCV). Additionally, complete coding genomes of 17 viruses from the Parvoviridae, Anelloviridae, and Circoviridae families were retrieved and included the first near full-length genomes of US Ungulate bocaparvovirus 3 species.

Analysis of tRNA halves (tsRNAs) in serum from cattle challenged with bovine viral diarrhea virus.

Acute infections of bovine viral diarrhea virus (BVDV) lead to a range of clinical presentations. Laboratory tests for detection depend on collection of samples during a short viremia. Acutely infected animals remain largely undiagnosed. Transfer RNA halves (tsRNAs) are hypothesized to function like microRNAs to regulate gene expression during an immune response. The objective of this study was to identify tsRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Colostrum-deprived neonatal Holstein calves were either challenged with BVDV (n=5) or mock challenged (n=4). Sera was collected prior to challenge and days 4, 9, and 16 post challenge. RNA was extracted and read counts of small non-coding RNAs were assessed using next-generation sequencing. A total of 87,838,207 reads identified 41 different tsRNAs. Two 5' tsRNAs, tsRNAProAGG and tsRNAValAAC, differed across time. Two 5' tsRNAs, tsRNAGlyCCC and tsRNAGlyGCC, differed between treatment groups across time. Four days post challenge, 5' tsRNAGlyCCC and tsRNAGlyGCC were significantly lower in the challenged group than the control group. Further studies are needed to identify the importance and function of 5' tsRNAGlyCCC and tsRNAGlyGCC in serum samples of cattle challenged with BVDV.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV) 1-7-4-type strains in Peru.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. While PRRSV has been endemic in North America since 1989, it was not until 1999 that the virus was first described in South America. Notably, recently an increased number of PRRSV outbreaks have been reported in South American countries. However, epidemiological information related to these outbreaks is limited and the genetic characteristics of the PRRSV strains circulating in the region are poorly understood. In this study, we describe the genetic analyses of PRRSV strains associated with severe PRRS outbreaks in Peru. Samples originating from 14 farms located in two Departments in Peru (Lima and Arequipa), were subjected to RT-PCR amplification of the PRRSV ORF5 gene and sequencing followed by restriction fragment length polymorphism (RFLP) analysis. Results demonstrated the circulation of PRRSV-2 in Peru. Notably ORF5 RFLP typing revealed that 15 (75%) of the PRRSV strains detected in this study belong to the RFLP 1-7-4 type. Phylogenetic analysis showed that the Peruvian strains are closely related to the highly virulent PRRSV 1-7-4 strains that emerged in the US in 2013-2014. Results here indicate the presence of highly virulent PRRSV 1-7-4 strains in Peru and provide important information on the geographical distribution of PRRSV, confirming the recent geographical expansion of this important swine pathogen towards South America.

Serologic evidence of HoBi-like virus circulation in Argentinean water buffalo.

HoBi-like pestiviruses (also known as bovine viral diarrhea virus 3) have been sporadically reported from naturally infected cattle in Brazil, Asia, and Europe. Although HoBi-like viruses seem to be endemic in Brazilian cattle and buffalo, they have not been studied in the other countries of South America to our knowledge. Herein we report serologic results of buffalo from 12 large farms in Argentina located near the Brazilian border. These buffalo were not vaccinated against pestiviruses. Our results indicate that HoBi-like virus may be circulating in the northeastern region of Argentina given that half of the analyzed animals showed high levels of neutralizing antibodies against the pestivirus. The HoBi-like seropositive animals were also checked for neutralizing antibodies against BVDV-1a, BVDV-1b, and BVDV-2, and in most cases these animals had low levels or no detectable antibodies against these other pestiviruses. Our study suggests a need for continued pestivirus surveillance in Argentinean cattle and buffalo.

Genetic diversity of bovine viral diarrhea virus in cattle from Mexico.

Bovine viral diarrhea virus (BVDV) infects cattle populations worldwide, causing significant economic losses though its impact on animal health. Previous studies have reported the prevalence of BVDV species and subgenotypes in cattle from the United States and Canada. We investigated the genetic diversity of BVDV strains detected in bovine serum samples from 6 different Mexican regions. Sixty-two BVDV isolates from Mexico were genetically typed based on comparison of sequences from the 5' untranslated region (5'-UTR) of the viral genome. Phylogenetic reconstruction indicated that 60 of the samples belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of partial 5'-UTR sequences clustered 49 samples within BVDV-1c, 8 samples within BVDV-1a, 3 samples within BVDV-1b, and 2 samples clustered with the BVDV-2a subgenotypes. Our study, combined with information previously published on BVDV field strain diversity in the United States and Canada, benefits the development of effective detection assays, vaccines, and control programs for North America.

Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed.

The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and 'HoBi'-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and 'HoBi'-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey's Multiple Comparison Test (P˂0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.

Temporal dynamics of 'HoBi'-like pestivirus quasispecies in persistently infected calves generated under experimental conditions.

'HoBi'-like virus is an atypical group within the Pestivirus genus that is implicated in economic losses for cattle producers due to both acute and persistent infections. Pestivirus strains exist as quasispecies (swarms of individual viruses) in infected animals and the viral populations making up the quasispecies differ widely in size and diversity in each animal. In the present study the viral quasispecies circulating in persistently infected (PI) calves, generated and maintained under experimental conditions using two different 'HoBi'-like strains, was observed over time. An increase in genetic variability and the development of certain mutations was observed over time. Mutations observed included the loss of a putative N-linked glycosylation site in the E2 region and the change of specific residues in E1/E2. It is hypothesized that these changes may be the results on continued adaption of the pestivirus to individual hosts. This is the first study characterizing variation in the viral swarms of animals persistently infected with HoBi-like viruses over time. Studies of the shifts in PI viral swarms will contribute to our understanding of the host and viral mechanisms that function in the maintenance of pestivirus persistent infections.

Emerging animal viruses: real threats or simple bystanders? / Vírus emergentes de animais: verdadeiras ameaças ou meros espectadores?

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. [...] Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators. (AU)

O número de vírus animais cresce continuamente, causando preocupação permanente a virologistas e veterinários. O potencial patogênico e associação com doença tem sido claramente demonstrado para alguns - mas não para todos - vírus emergentes. Esse artigo apresenta uma breve revisão das recentes descobertas de vírus animais e a sua potencial relevância para saúde animal. Cães eram considerados refratários aos vírus da influenza até 2004, quando um vírus influenza A subtipo H3N8 foi transmitido de equinos e causou doença respiratória severa em cães galgos na Flórida/EUA. O novo vírus, denominado vírus da influenza canina (CIV), agora considerado uma linhagem distinta do vírus da influenza equina, disseminou-se na população canina urbana dos EUA. Um novo Pestivirus (Flaviviridae) - provisoriamente denominado pestivírus Hobi-like - foi identificado em 2004 em soro fetal bovino importado do Brasil. Os vírus Hobi-like são genética e antigenicamente relacionados com o vírus da diarreia viral bovina (BVDV) e induzem manifestações clínicas semelhantes. A sua origem e distribuição são desconhecidas, mas estão aparentemente disseminados no rebanho brasileiro e já foram identificados no sudeste asiático e na Europa. Em 2011, um novo buniavírus transmitido por mosquitos, denominado vírus Schmallemberg (SBV), foi associado com febre, redução da produção de leite, abortos e malformações fetais em bovinos e ovinos da Alemanha. [...] Finalmente, a longa e intensiva busca por vírus animais relacionados ao vírus da hepatite C humana (HCV) tem levado a identificação de "novos" pestivírus em cães (canine hepacivirus [CHV]), equinos (hepacivirus de não-primatas [NPHV] ou vírus associado à doença de Theiler [TDAV]) e em roedores. Para estes, uma associação clara e definitiva com doença ainda não foi demonstrada e apenas tempo e investigação irão dizer se são patógenos reais ou apenas espectadores. (AU)

Emerging animal viruses: real threats or simple bystanders? / Vírus emergentes de animais: verdadeiras ameaças ou meros espectadores?

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. [...] Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators.

O número de vírus animais cresce continuamente, causando preocupação permanente a virologistas e veterinários. O potencial patogênico e associação com doença tem sido claramente demonstrado para alguns - mas não para todos - vírus emergentes. Esse artigo apresenta uma breve revisão das recentes descobertas de vírus animais e a sua potencial relevância para saúde animal. Cães eram considerados refratários aos vírus da influenza até 2004, quando um vírus influenza A subtipo H3N8 foi transmitido de equinos e causou doença respiratória severa em cães galgos na Flórida/EUA. O novo vírus, denominado vírus da influenza canina (CIV), agora considerado uma linhagem distinta do vírus da influenza equina, disseminou-se na população canina urbana dos EUA. Um novo Pestivirus (Flaviviridae) - provisoriamente denominado pestivírus Hobi-like - foi identificado em 2004 em soro fetal bovino importado do Brasil. Os vírus Hobi-like são genética e antigenicamente relacionados com o vírus da diarreia viral bovina (BVDV) e induzem manifestações clínicas semelhantes. A sua origem e distribuição são desconhecidas, mas estão aparentemente disseminados no rebanho brasileiro e já foram identificados no sudeste asiático e na Europa. Em 2011, um novo buniavírus transmitido por mosquitos, denominado vírus Schmallemberg (SBV), foi associado com febre, redução da produção de leite, abortos e malformações fetais em bovinos e ovinos da Alemanha. [...] Finalmente, a longa e intensiva busca por vírus animais relacionados ao vírus da hepatite C humana (HCV) tem levado a identificação de "novos" pestivírus em cães (canine hepacivirus [CHV]), equinos (hepacivirus de não-primatas [NPHV] ou vírus associado à doença de Theiler [TDAV]) e em roedores. Para estes, uma associação clara e definitiva com doença ainda não foi demonstrada e apenas tempo e investigação irão dizer se são patógenos reais ou apenas espectadores.

In vitro neutralization of HoBi-like viruses by antibodies in serum of cattle immunized with inactivated or modified live vaccines of bovine viral diarrhea viruses 1 and 2.

HoBi-like viruses are an emerging species of pestiviruses with genetic and antigenic similarities to bovine viral diarrhea viruses 1 and 2 (BVDV1 and BVDV2). Vaccines for HoBi-like viruses are not yet available. However, both modified live virus (MLV) and killed virus (KV) vaccines against BVDV are widely used worldwide. This study evaluated the cross reactive antibody response against HoBi-like pestiviruses in sera of cattle immunized with BVDV1 and BVDV2 vaccines. Groups "KV" and "MLV", with 25 calves each, received killed or modified live vaccines, respectively, containing both BVDV1 and BVDV2 antigens. The antibody response was evaluated by virus neutralization test. The average of geometric mean titers (GMTs) of neutralizing antibodies in serum against HoBi-like viruses in the MLV group was 12.9, whereas GMTs to BVDV1, BVDV2 and border disease virus (BDV) were 51.1, 23.5, and 12.4, respectively. In this group, neutralizing antibodies against BVDV1, BVDV2, HoBi-like viruses and BDV were detected in 100%, 94%, 68% and 68% of calves, respectively. The GMT of neutralizing antibodies in serum against BVDV1, BVDV2, HoBi-like viruses and BDV in the KV group were 24.7, 14.5, 10.4 and 11, respectively. Similarly, the percentage of animals with neutralizing antibodies against BVDV1, BVDV2, HoBi-like viruses and BDV were 84%, 56%, 34% and 44%, respectively. These results indicate that MLV or killed BVDV1 and BVDV2 vaccines induce a cross reactive antibody response comparatively weak to HoBi-like viruses, and this response would likely not suffice to confer protection.

HoBi-like viruses: an emerging group of pestiviruses.

The genus Pestivirus is composed of 4 important pathogens of livestock: Bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), Classical swine fever virus (CSFV), and Border disease virus of sheep (BDV). BVDV are major pathogens of cattle, and infection results in significant economic loss worldwide. A new putative pestivirus species, tentatively called "HoBi-like," "BVDV-3," or "atypical pestiviruses," was first identified in Europe in fetal bovine serum (FBS) imported from Brazil. HoBi-like viruses are related to BVDV at the genetic and antigenic levels. Further, the disease caused by these new viruses resembles clinical presentations historically associated with BVDV infection, including growth retardation, reduced milk production, respiratory disease, reduced reproductive performance, and increased mortality among young stock. Current BVDV diagnostic tests may fail to detect HoBi-like viruses or to differentiate between BVDV and HoBi-like viruses. Further, commercial tests for BVDV exposure, based on serological response, do not reliably detect HoBi-like virus exposure, and cross protection against HoBi-like viruses conferred by current BVDV vaccines is likely limited. As many HoBi-like viruses, characterized to date, were isolated from FBS originating from Brazil, it is assumed that the agent is probably widespread in Brazilian herds. Nevertheless, reports of natural infection in Southeast Asia and Europe demonstrate that these viruses are not restricted to South America. Increased demand for FBS has led to widespread distribution of FBS originating in HoBi-like virus endemic regions. The contamination of such FBS with HoBi-like viruses may lead to spread of this virus to other regions.

Antigenic relationships between Bovine viral diarrhea virus 1 and 2 and HoBi virus: possible impacts on diagnosis and control.

The emergence of a newly recognized group of pestiviruses in cattle, the HoBi-like viruses, requires an evaluation of the available diagnostic tools and vaccines. The present study compared antigenic characteristics of Bovine viral diarrhea virus 1 and 2 (BVDV-1, -2) strains and HoBi virus. This comparison was based on detection of HoBi virus and antibodies against it by commercial enzyme-linked immunosorbent assays (ELISAs) and the level of cross-neutralizing antibodies present in sera from animals vaccinated with BVDV. Reactivity with a panel of monoclonal antibodies (mAbs) revealed greater cross-reactivity between BVDV species (BVDV-1, -2) and HoBi epitopes within E(rns) and NS2/3 proteins than between epitopes located in the E2 glycoprotein. The results suggest that a diagnostic test designed to detect both BVDV species and HoBi could be based on E(rns) or NS2/3 epitopes, while variation among E2 epitopes could be exploited in tests for differentiation of pestivirus species. The threshold of detection of HoBi virus by an antigen-capture ELISA kit based on detection of E(rns) was statistically similar to that for BVDV. In contrast, 2 commercial ELISA kits designed to detect antibodies against BVDV missed 22.2% and 77.7%, respectively, of serum samples harboring HoBi virus-neutralizing antibodies. In addition, sera of calves vaccinated with BVDV-1 and -2 presented low neutralizing activity against HoBi virus. The results demonstrate that in spite of antigenic similarities, HoBi virus is antigenically distinct from both BVDV species. Detection and control of HoBi virus infections in cattle would thus require the development of new diagnostic reagents and reformulation of current vaccines.

Bacterial biodiversity from an anaerobic up flow bioreactor with ANAMMOX activity inoculated with swine sludge

The present study aimed to describe the bacterial community present at an anaerobic up flow bioreactor with ANAMMOX activity, inoculated with the sludge from the anaerobic pond of a swine slurry treatment system. The description was based on the molecular DNA techniques using primers for amplification of complete 16S rRNA gene and also new primers to amplify smaller fragments from 16S rRNA. During the bioreactor operation time, the bacterial community changed significantly, increasing the nitrogen removal efficiency, reaching after 500 days a removal rate of 94 percent. The complete PCR amplification of 16S rRNA gene generated 17 clones, where three presented similarity with Candidatus Jettenia asiatica (97 percent), twelve with Janthinobacterium (99 percent) and two with uncultured clones. The PCR amplification of 436 base pairs had generated 12 clones, of which eight presented 96-100 percent similarity with Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 and one with Pseudomonas sp. (99 percent) and three with uncultured clones.

Cepa recombinante do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E é atenuada e imunogênica para bezerros / A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK􀀀Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 107.5TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (107.5TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 107.3TCID50)...

O herpesvírus bovino tipo 5 (BoHV-5) é um importante patógeno de bovinos na América do Sul e tem suscitado esforços para o desenvolvimento de vacinas mais seguras e eficazes. Além de conferirem proteção, vacinas contra os herpesvírus animais devem permitir a diferenciação sorológica entre animais vacinados e infectados naturalmente. Foi recentemente relatada a construção e caracterização in vitro de um recombinante do BoHV-5 defectivo na enzima timidina quinase (tk) para atenuação e, na glicoproteína E (gE) como marcador antigênico, como potencial cepa vacinal (Brum et al. 2010a). O presente artigo relata uma investigação sobre da atenuação e imunogenicidade do recombinante BoHV-5gE/TKΔ em bezerros. Em um primeiro experimento, bezerros soronegativos, com 80 a 90 dias de idade (n=6), inoculados pela via intranasal (IN) com o vírus recombinante (título de 107,5TCID50) excretaram baixos títulos de vírus nas secreções nasais por até seis dias, mas não desenvolveram sinais sistêmicos, respiratórios ou neurológicos. No dia 30 pós-infecção (pi), todos os animais possuíam anticorpos neutralizantes contra o BoHV-5, em títulos entre 4 e 8, mas permaneceram negativos para anticorpos contra a gE. Administração de dexametasona (Dx) a quatro desses bezerros no dia 42 pi (0.1mg/kg/dia durante 5 dias) não resultou em excreção viral ou em aumento dos títulos de anticorpos, indicando ausência de reativação viral. Em um segundo experimento, vacinação intramuscular (IM) de bezerros com 8 meses de idade (n=9) com o recombinante (107,5TCID50/animal) não resultou em excreção viral ou em manifestações clínicas. Os animais vacinados desenvolveram anticorpos neutralizantes em títulos de 2 a 8 no dia 42 pós-vacinação (PV) e permaneceram negativos para anticorpos anti-gE. Finalmente, 21 bezerros (aproximadamente 10 meses de idade) mantidos a campo foram vacinados com o recombinante (107,3TCID50) pela via IM...

Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates / Evidências de infecção persistente mista em bezerros nascidos de vacas inoculadas com um pool de isolados do vírus da diarréia viral bovina

Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.(AU)

Vacas prenhes infectadas com isolados não-citopáticos (NCP) do vírus da diarréia viral bovina (BVDV) entre os dias 40 e 120 de gestação frequentemente produzem bezerros imunotolerantes, persistentemente infectados (PI). Este artigo relata a caracterização de bezerros PI produzidos experimentalmente, pela inoculação de vacas prenhes com um pool de isolados brasileiros de BVDV-1 (n=2) e BVDV-2 (n=2) entre os dias 60 e 90 de gestação. Dois bezerros nasceram positivos para vírus, negativos para anticorpos, mas morreram aos dias 7 e 15 pós-nascimento, respectivamente. Outros seis bezerros nasceram saudáveis, soronegativos para o BVDV, replicaram e excretaram o vírus em secreções por até 210 dias. Análise antigênica dos vírus presentes nos bezerros PI com anticorpos monoclonais, ao nascimento e 30 dias após, revelou dois padrões de infecção. Três bezerros apresentavam apenas um dos vírus utilizados no desafio (BVDV-1 ou BVDV-2), enquanto outros três bezerros apresentavam co-infecção com mais de um vírus. Além disso, testando-se o sangue dos animais PI com um PCR capaz de diferenciar entre BVDV-1 e BVDV-2 observou-se a presença concomitante de vírus dos dois genótipos (BVDV-1 e BVDV-2) nestes três animais. Esses resultados indicam que infecção persistente - uma consequência bem conhecida da infecção fetal pelo BVDV - pode ser estabelecida concomitantemente por mais de um vírus, após infecção experimental. Nesse sentido, infecções persistentes mistas com isolados antigenicamente distintos podem ser úteis para o estudo das bases imunológicas e moleculares da imunotolerância e infecção persistente pelo BVDV.(AU)

Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates / Evidências de infecção persistente mista em bezerros nascidos de vacas inoculadas com um pool de isolados do vírus da diarréia viral bovina

Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.

Vacas prenhes infectadas com isolados não-citopáticos (NCP) do vírus da diarréia viral bovina (BVDV) entre os dias 40 e 120 de gestação frequentemente produzem bezerros imunotolerantes, persistentemente infectados (PI). Este artigo relata a caracterização de bezerros PI produzidos experimentalmente, pela inoculação de vacas prenhes com um pool de isolados brasileiros de BVDV-1 (n=2) e BVDV-2 (n=2) entre os dias 60 e 90 de gestação. Dois bezerros nasceram positivos para vírus, negativos para anticorpos, mas morreram aos dias 7 e 15 pós-nascimento, respectivamente. Outros seis bezerros nasceram saudáveis, soronegativos para o BVDV, replicaram e excretaram o vírus em secreções por até 210 dias. Análise antigênica dos vírus presentes nos bezerros PI com anticorpos monoclonais, ao nascimento e 30 dias após, revelou dois padrões de infecção. Três bezerros apresentavam apenas um dos vírus utilizados no desafio (BVDV-1 ou BVDV-2), enquanto outros três bezerros apresentavam co-infecção com mais de um vírus. Além disso, testando-se o sangue dos animais PI com um PCR capaz de diferenciar entre BVDV-1 e BVDV-2 observou-se a presença concomitante de vírus dos dois genótipos (BVDV-1 e BVDV-2) nestes três animais. Esses resultados indicam que infecção persistente - uma consequência bem conhecida da infecção fetal pelo BVDV - pode ser estabelecida concomitantemente por mais de um vírus, após infecção experimental. Nesse sentido, infecções persistentes mistas com isolados antigenicamente distintos podem ser úteis para o estudo das bases imunológicas e moleculares da imunotolerância e infecção persistente pelo BVDV.

Teste imunoenzimático com base em anticorpo monoclonal para a detecção de anticorpos contra os herpesvírus bovino tipos 1 e 5 / A monoclonal antibody-based enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus types 1 and 5

Os herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5) são agentes virais genética e antigenicamente relacionados, associados com diversas manifestações clínicas em bovinos, incluindo doença respiratória, genital, neurológica e abortos. Estudos epidemiológicos indicam que esses vírus estão amplamente disseminados no rebanho bovino brasileiro. O diagnóstico sorológico, que permite identificar animais portadores da infecção latente, se constitui em importante ferramenta para monitoramento individual e de rebanho. O presente artigo relata a padronização de um teste imunoenzimático do tipo ELISA, com base em anticorpo monoclonal (AcM), para a detecção de anticorpos séricos que reagem contra BoHV-1 e/ou BoHV-5. Inicialmente, determinou-se o AcM mais adequado para a sensibilização das placas, as diluições apropriadas do antígeno e dos soros-teste e o ponto de corte do ensaio. Após a padronização, o ensaio foi validado testando-se 506 amostras de soro bovino, previamente testadas para anticorpos neutralizantes contra BoHV-1 e/ou BoHV-5 pela técnica de soroneutralização (SN). Comparando-se com os resultados da SN frente a BoHV-1, o teste de ELISA apresentou sensibilidade e especificidade de 96,6 por cento e 98,3 por cento, respectivamente. Os valores preditivos positivo e negativo foram de 97,6 por cento, a concordância foi de 97,6 por cento e o índice de correlação kappa entre os testes foi de 0,95, o que indica uma excelente concordância. Comparando-se com os resultados da SN frente o BoHV-5, o ELISA apresentou 94,3 por cento de sensibilidade; 97,9 por cento de especificidade; 97,1 por cento de valor preditivo positivo e 95,9 por cento de valor preditivo negativo. Para BoHV-5, a concordância entre os testes foi de 96,4 por cento e o índice de correlação foi de 0,92, também excelente. Esses resultados demonstram que o teste padronizado apresenta sensibilidade e especificidade adequados para o diagnóstico sorológico das infecções por BoHV-1 e BoHV-5 ...(AU)

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are antigenic and genetically related viruses associated with different clinical syndromes in cattle, including respiratory, reproductive, neurological disease and abortion. Epidemiological studies indicate the widespread distribution of both viruses among Brazilian cattle. Serological diagnosis, that allows the identification of latently infected animals, represents an important tool for individual and herd monitoring. The present article describes the standardization of a monoclonal antibody (MAb)-based immunoenzymatic test (ELISA) for detection of antibodies to BoHV-1 and/or BoHV-5. The initial steps involved the determination of the most suitable MAb, the appropriate dilutions of viral antigen and serum samples, and the cut-off value of the assay. After standardization, the ELISA was validated by testing 506 cattle serum samples previously tested for neutralizing antibodies to BoHV-1 and BoHV-5 by virus neutralizing assay (VN). Comparing to the VN for BoHV-1 antibodies, the ELISA presented sensitivity and specificity of 96.6 percent and 98.3 percent, respectively. Positive and negative predictive values were 97.6 percent, the concordance between the tests was 97.6 percent and the coefficient of correlation k (kappa) was 0.95, demonstrating an excellent correlation. Comparing to the VN for BoHV-5 antibodies, the ELISA presented 94.3 percent of sensitivity, 97.9 percent of specificity, 97.1 percent of positive predictive value, 95.9 percent negative predictive value, concordance of 96.4 percent and kappa coefficient of 0.92. These results demonstrate that the ELISA presents suitable specificity and sensitivity to be used for individual and herd serological diagnosis of BoHV-1 and BoHV-5, thus, representing an alternative for VN assays and imported ELISA kits.(AU)