Outbreak of persistently infected heifer calves with bovine viral diarrhea virus subgenotypes 1b and 1d in a BVDV-vaccinated open dairy herd.

Bovine viral diarrhea virus (BVDV) infection has a significant economic impact on beef and dairy industries worldwide. Fetal infection with a non-cytopathic strain may lead to the birth of persistently infected (PI) offspring, which is the main event in the epidemiological chain of BVDV infection. This report describes the birth of 99 BVDV-PI heifer calves within 52 days of birth in a regular BVDV-vaccinated Brazilian dairy cattle herd and the subgenotypes of the infecting field strains. This study was conducted in a high-yielding open dairy cattle herd that frequently acquired heifers from neighboring areas for replacement. The farm monitors the birth of PI calves by screening all calves born using an ELISA (IDEXX) for BVDV antigen detection. All calves aged 1-7 days were evaluated. For positive and suspected results, the ELISA was repeated when the calves were close to one month old. A total of 294 heifer calves were evaluated between February and March 2021. Of these, 99 (33.7 %) had positive ELISA results and were considered PI calves. To evaluate the predominant BVDV species and subgenotypes in this outbreak, whole blood samples were collected from 31 calves born during the study period. All samples were submitted to the RT-PCR assay for the partial amplification of the BVDV 5'-UTR region, and these amplicons were subjected to nucleotide sequencing. Phylogenetic analysis identified BVDV-1b and BVDV-1d in 16 and 13 heifer calves, respectively. In two calves, it was not possible to determine the BVDV-1 subgenotype. Detection of PI animals and monitoring of circulating BVDV subgenotype strains are central to disease control. This study shows that regular BVDV vaccination alone may be insufficient to prevent BVDV infection in high-yielding open dairy cattle herds. Other biosecurity measures must be adopted to avoid the purchase of cattle with acute infections by BVDV or BVDV-PI, which can cause a break in the health profile of the herd and economic losses.

Subtyping bovine viral diarrhea virus (BVDV): Which viral gene to choose?

Bovine viral diarrhea virus-1 (BVDV-1, Pestivirus A) and BVDV-2 (Pestivirus B) have been clustered into 21 and 4 subtypes, respectively. This genetic diversity, in addition to the lack of consensus on which genomic region to use for BVDV subtyping, has resulted in conflicting classifications depending on the target analyzed. Here, we investigated which genes or UTRs would reproduce the phylogeny obtained by complete genome (CG) analyses. The study was carried out with 91 (BVDV-1) and 85 (BVDV-2) CG available on GenBank database. The viruses were subtyped by analyzing their CG, as well as their individual genes and UTRs (complete 3' and 5'UTRs, and partial 5'UTR); and the phylogeny results were compared to each other. The sequences were aligned using the ClustalW multiple method (BioEdit Alignment Editor software, v.7.0.5.3) and the phylogenetic analyses were performed by the Maximum Likelihood method (MEGA-X software, v.10.2.4), with 1000 bootstrap replicates. The best analysis model for each gene/UTR was defined using the jModelTest software. The geodesic distance between the CG (reference) and individual genes/UTRs trees was also calculated (TreeCmp software, v.2.0). In general, 3'UTR-based analyses, followed by 5'UTR, presented the least reliable subtyping results. Regarding BVDV-1, phylogeny based on C, Erns, E1, E2, p7, NS2, NS3, NS4B, NS5A and NS5B was consistent with that of CG. In contrast, analyses performed with individual BVDV-2 genes showed at least one different clustering from the phylogeny based on the CG. After analyzing the geodesic distance between the CG and genes/UTRs trees, we observed that NS4B (for BVDV-1) and NS5A (BVDV-2) presented the closest topology and edge length to the CG analyses. Finally, comparing the phylogeny performed with the CG and the genes/UTRs, as well as the geodesic distance between them, we understand that NS4B and NS5A represent the most suitable targets for BVDV-1 and -2 subtyping, respectively, and may be considered in future phylogenetic studies.

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.

Molecular detection and genotyping of bovine viral diarrhea virus in Western China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is an important global viral pathogen of cattle and other ruminants. To survey the infection rate and genetic diversity of BVDV in western China, a total of 1234 serum samples from 17 herds of dairy cattle, beef cattle and yak in 4 provinces were collected in 2019. RESULTS: All the 1234 serum samples were screened individually for BVDV by RT-PCR. Our results demonstrated that the average positive rate of BVDV was 7.2% (89/1234) in animals and 82.4% (14/17) in herds. Thirteen BVDV strains were isolated from RT-PCR positive clinical samples and they were all NCP biotype. BVDV-1a and 1c subgenotypes were identified from 22 selected virus isolates in 14 BVDV-positive herds. These results confirmed that BVDV-1a and BVDV-1c were circulating in western China, similar to the BVDV epidemics in cattle in other regions of China. CONCLUSIONS: This study provides data for monitoring and vaccination strategies of BVDV in western China.

Identification and genotyping of a new subtype of bovine viral diarrhea virus 1 isolated from cattle with diarrhea.

In 2019, diarrhea cases occurred on cattle farms in Qionglai and Guang'an, Sichuan Province. Two out of 20 (10%) serum and nasal swab samples were positive when tested using a bovine viral diarrhea virus (BVDV) antigen-capture ELISA kit. Two non-cytopathic strains of BVDV were isolated and named QL1903 and GA190608, respectively. The nucleotide sequences of the genomes of the two isolates were 89.52% identical. Phylogenetic analysis based on the 5'-UTR sequence revealed that the BVDV isolate QL1903 belonged to BVDV subtype 1b, whereas isolate GA190608 clustered with strains HN1814, EN-19, and BJ09_26 in a separate branch, which has tentatively been classified as a new genetic subtype, "1v".

The NADL strain of bovine viral diarrhea virus induces the secretion of IL-1ß through caspase 1 in bovine macrophages.

Bovine viral diarrhea virus (BVDV) infects different cell types including antigen-presenting cells such as macrophages. The infection induces pro-inflammatory cytokines like interleukin 1 beta (IL-1ß), which is necessary to trigger a successful inflammatory response against infections. Several authors have reported differences between IL-1ß gene expression and protein detection in BVDV-infected macrophages. These patterns may be related to inflammasome assembly, which promote the formation of active caspase 1 in order to produce mature IL-1ß molecules. Our goal was to assess BVDV ability to induce the release of IL-ß through a caspase 1 dependent pathway in bovine macrophages. We infected peripheral blood monocyte-derived macrophages using BVDV NADL strain at 0.001, 0.1, 2 and 10 multiplicities of infection (MOI) and we measured IL-1ß at different times 2, 6, 12, 24, 48, 72 h. We found an increase of 1140-2154 pg for a MOI of 10:1 and 2:1 respectively. To inhibit caspase 1, we used either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD) or carbobenzoxy-tyr-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Y-VAD). We found decreased IL-1ß secretion 2154 pg/ml to 854 pg/ml IL-1ß secretion using Y-VAD and we observed decrease from 2154 pg/ml to 22.33 pg/ml with Z-VAD, and this inhibition was followed by diminished viral replication from 2.25 × 107 to 2.1 × 105 CCID50, which suggests that caspase 1-dependent secretion of the IL-1ß active molecule is important for viral replication. This is the first report showing that BVDV infected-bovine macrophages trigger the caspase 1 dependent pathway for IL-1ß activation and that activation increases viral replication.

Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay.

BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

Origin and transmission of bovine viral diarrhea virus type 1 in China revealed by phylodynamic analysis.

Bovine viral diarrhea virus type 1 (BVDV-1) is prevalent worldwide and causes significant economic loss in animal husbandry. Since its first report in the 1980s in China, several genotypes of BVDV-1 had been reported, but an in-depth phylogenetic analysis on the BVDV isolates from China is lacking. To investigate the molecular evolution and phylodynamics of BVDV-1 genotypes circulating in China, comprehensive phylogenetic and phylodynamic analyses were performed to reconstruct the origin and spatial-temporal distribution, and to trace main viral flows among different areas. BVDV-1 5'-UTR sequences from China and Mongolia were collected from Genbank, and the phylogeny was built using the maximum likelihood method. The Bayesian Skygrid was used to estimate the evolution and population dynamics of BVDV-1. Eight BVDV-1 genotypes were identified, of which 1b and 1 m are the main genotypes. The results indicated that BVDV-1 might be introduced in China in the 1960s, and after a long period of population growth, it gradually leveled off after 2010. The phylodynamic inference clearly shows a more steady BVDV-1 population growth, and the transmission of BVDV-1 may be confined to specific regions. This study will help to understand the molecular epidemiology and long-term evolutionary dynamics of BVDV-1 in China, therefore providing a scientific basis for the prevention and controlof the virus.

In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Slaughterhouse survey for detection of bovine viral diarrhea infection among beef cattle in Kyushu, Japan.

Bovine viral diarrhea virus (BVDV) footprint has spread across the globe and is responsible for one of the most economically important diseases in cattle. In Japan, some regional surveillance and preventive measures to control bovine viral diarrhea (BVD) have been implemented. However, BVDV infection is poorly understood in cattle industries, and there is no systematic BVD surveillance system and control program. Kyushu is the center for raising beef cattle in Japan. Therefore, this study aimed to determine the BVDV infection using a slaughterhouse survey among beef cattle in Kyushu, Japan. A total of 1,075 blood samples were collected at two regional slaughterhouses in Miyazaki prefecture from December 2015 to June 2016. Antigen ELISA was used for detection of BVDV antigen in blood samples. Two samples showed positive results (2/1,075; 0.18%). BVDV RNA was extracted from positive blood samples; the sequence was determined and analyzed by the neighbor-joining method for construction of the phylogenetic tree. Phylogenetic analysis based on the 5'-UTR revealed that the two positive samples were grouped into the same subtype BVDV-1b in the BVDV-1 genotype, but the infected cattle belonged to two different farms. In conclusion, this is the first study to identify the presence of BVDV in a slaughterhouse survey in Kyushu. These findings suggest that a slaughterhouse survey is a useful tool for developing a surveillance system for monitoring infectious diseases in cattle.

Endless variety for bovine virus diarrhea viruses: new members of a novel subgroup into Pestivirus A from Turkey.

As ubiquitous pathogens, bovine virus diarrhea viruses (BVDVs) in cattle have been reported several times in Turkey. Over time, the frequency and importance of this infection has increased for the livestock industries. A total of 1291 animals were sampled from a dairy herd in Turkey suspected of BVDV clinical signs, for instance, reproductive failures (abortion, congenital malformations in calves, repeat breeding, etc.) and interdigital phlegmon in adult animals. Reverse transcription polymerase chain reactions (RT-PCRs) were made by using targeted 5' untranslated region (UTR), Npro, E2, and NS2-3 pestiviral gene region primers for antigen ELISA-positive samples (n = 20). The obtained amplicons were sequenced. Sequence results showed the presence of a new subgroup in Pestivirus A species. This paper describes the nucleotide sequences of a new BVDV 1 (BVDV 1-v) subgroup member.

Who's who in the Bovine viral diarrhea virus type 1 species: Genotypes L and R.

The bovine viral diarrhea virus type 1 species is responsible for cosmopolitan diseases affecting cattle and other ruminants, with relevant impact on animal production. The species presents high genomic heterogeneity, with implications on control and prophylactic programs. Genomic traits of different genetic groups are often related to geographic origin. Atypical sequences have been reported from Pestivirus isolates originated from cattle in Turkey. Based on phylogenetic analysis of 5' untranslated region and Npro and secondary structure analysis of the 5'-UTR RNA, Turkish isolates have been segregated in two distinct genotypes. Out of the twenty-three identified BVDV-1 genotypes, the Turkish clusters, named L and R or 1.16 and 1.14, according to palindromic nucleotide substitution genotyping method, represent genomic clusters so far, not described elsewhere, suggesting geographic segregation. In order to avoid confusion in the current taxonomy of the species, nomenclature of described homonymous genotypes, referred to different genomic clusters, should be corrected.

Bovine viral diarrhea virus type 1 current taxonomy according to palindromic nucleotide substitutions method.

Pestivirus bovine viral diarrhea virus type 1 species is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, four hundred-eighty-two genomic sequences have been evaluated applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure alignment and computing genetic distance among strains in the 5' untranslated region of Pestivirus RNA, the current taxonomy of the species was reviewed. Twenty-two genotypes have been identified, applying a nomenclature based on divergence in the genus.

HoBi-like is the most prevalent ruminant pestivirus in Northeastern Brazil.

The ruminant pestiviral species BVDV-1, BVDV-2 and BDV, along with the putative species HoBi-like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT-PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi-like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi-like viruses. The increasing reports of HoBi-like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi-like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi-like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.

Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate.

Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS: showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.

Analysis of bovine viral diarrhoea virus: Biobank and sequence database to support eradication in Scotland.

Samples from bovine viral diarrhoea virus (BVDV)-positive cattle were gathered by Scottish diagnostic laboratories and used to produce a Biobank of samples with associated location and identification data in support of the Scottish BVDV eradication scheme. The samples were subject to direct amplification and sequencing of the 5'-untranslated region (5'-UTR) to define the viral types and subtypes present. From 2693 samples collected prior to 2016, approximately 2300 sequences were obtained, representing 8 BVDV type 1 subtypes. No BVDV type 2 samples were detected. The samples came from all regions of the UK but 66 per cent were from Scotland. Analysis of the sequences showed great diversity in the 5'-UTR, with 1206 different sequences. Many samples carried virus with identical 5'-UTR sequences; often from single locations, but there were also examples of the same sequence being obtained from samples at several different locations. This work provides a resource that can be used to analyse the movement of BVDV strains both within Scotland and between Scotland and other nations, particularly in the latter stages of the Scottish eradication programme, and so inform the advice available to both livestock keepers and policymakers.

Prevention of fetal infection in heifers challenged with bovine viral diarrhoea virus type 1a by vaccination with a type 1c or type 1a vaccine.

AIMS: To evaluate a vaccine containing type 1c bovine viral diarrhoea (BVD) virus for prevention of fetal infection in pregnant heifers when challenged with New Zealand BVD virus type 1a 6 months after vaccination, compared to unvaccinated heifers and heifers vaccinated with a vaccine containing type 1a BVD virus. METHODS: Fifty five crossbred Friesian heifers, free from BVD virus and antibody, were randomly allocated to three groups. Twenty five heifers were vaccinated twice with a vaccine containing type 1c BVD virus (T1c group), and 10 heifers with a vaccine containing type 1a BVD virus (T1a group), and 20 heifers were unvaccinated (NC group). After oestrus synchronisation the heifers were bred by artificial insemination followed by natural bull mating. Six months after booster vaccination 15 heifers from the T1c group, eight from the T1a group, and 15 from the NC group, were exposed to four calves that were persistently infected with type 1a BVD virus, for 4 weeks. At the beginning of the challenge phase 36/38 heifers were 72-74 days pregnant and 2/38 heifers were approximately 53 days pregnant. Approximately 52 days after the start of the challenge the heifers were subjected to euthanasia and fetal tissues were collected for the detection of BVD virus by ELISA in fetal heart blood and PCR in fetal tissues. RESULTS: Based on PCR results, BVD virus was detected in 15/15 fetuses in the NC group, compared to 4/14 fetuses in the T1c group and 3/8 fetuses in the T1a group. The proportion of BVD virus-positive fetuses was lower in both vaccinated groups compared to the NC group (p<0.002), but there was no difference in proportions between the vaccinated groups (p=1.00). Fetal protection, expressed as the prevented fraction, was 71.4 (95% CI=41.9-91.6)% and 62.5 (95% CI=24.5-91.5)% for the T1c and T1a groups, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The vaccines containing killed type 1c and type 1a BVD viruses significantly reduced fetal infection following challenge with a New Zealand type 1a BVD virus. Prevention of fetal infection by vaccination may not be 100%, and the risk of persistently infected calves being born to some vaccinated cattle should be acknowledged and managed as part of a BVD control programme.

Circulation of multiple subtypes of bovine viral diarrhoea virus type 1 with no evidence for HoBi-like pestivirus in cattle herds of southern Italy.

Pestiviruses of cattle include bovine viral diarrhoea 1 (BVDV-1) and 2 (BVDV-2) plus an emerging group, named HoBi-like pestivirus. In the present paper, the results of an epidemiological survey for pestiviruses circulating in cattle in southern Italy are presented. Molecular assays carried out on a total of 924 bovine samples detected 74 BVDV strains, including 73 BVDV-1 and 1 BVDV-2 viruses. Phylogenetic analysis carried out on partial 5'UTR and Npro sequences revealed the presence of 6 different subtypes of BVDV-1 and a single BVDV-2c strain. BVDV-1 displayed a high level of genetic heterogeneity, which can have both prophylactic and diagnostic implications. In addition, the detection of BVDV-2c highlights the need for a continuous surveillance for the emergence of new pestivirus strains in cattle farms in southern Italy.

Experimental acute infection of alpacas with Bovine viral diarrhea virus 1 subgenotype b alters peripheral blood and GALT leukocyte subsets.

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αß T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.