Improving poxvirus-mediated antitumor immune responses by deleting viral cGAMP-specific nuclease.

cGAMP-specific nucleases (poxins) are a recently described family of proteins dedicated to obstructing cyclic GMP-AMP synthase signaling (cGAS), an important sensor triggered by cytoplasmic viral replication that activates type I interferon (IFN) production. The B2R gene of vaccinia viruses (VACV) codes for one of these nucleases. Here, we evaluated the effects of inactivating the VACV B2 nuclease in the context of an oncolytic VACV. VACV are widely used as anti-cancer vectors due to their capacity to activate immune responses directed against tumor antigens. We aimed to elicit robust antitumor immunity by preventing viral inactivation of the cGAS/STING/IRF3 pathway after infection of cancer cells. Activation of such a pathway is associated with a dominant T helper 1 (Th1) cell differentiation of the response, which benefits antitumor outcomes. Deletion of the B2R gene resulted in enhanced IRF3 phosphorylation and type I IFN expression after infection of tumor cells, while effective VACV replication remained unimpaired, both in vitro and in vivo. In syngeneic mouse tumor models, the absence of the VACV cGAMP-specific nuclease translated into improved antitumor activity, which was associated with antitumor immunity directed against tumor epitopes.

Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis).

Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs-named BMMF (bovine meat and milk factors)-that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis.

Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR.

BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

N-terminal domain of Schmallenberg virus envelope protein Gc delivered by recombinant equine herpesvirus type 1 and modified vaccinia virus Ankara: Immunogenicity and protective efficacy in cattle.

Schmallenberg virus (SBV), which emerged in 2011 in Central Europe and subsequently spread very rapidly throughout the continent, affects predominantly ruminants. SBV is transmitted by insect vectors, and therefore vaccination is one of the major tools of disease control. Only recently, a domain connected to virus neutralization has been identified at the amino-terminal part of the viral envelope protein Gc. Here, this Gc domain delivered by recombinant EHV-1 or MVA vector viruses was tested in a vaccination-challenge trial in cattle, one of the major target species of SBV. The EHV-1-based vaccine conferred protection in two of four animals, whereas immunization using the MVA vector vaccine efficiently induced an SBV-specific antibody response and full protection against SBV challenge infection in all the vaccinated animals. Moreover, due to the absence of antibodies against SBVs N-protein, both vector vaccines enable the differentiation between vaccinated and field-infected animals making them to a promising tool to control SBV spread as well as to prevent disease in domestic ruminants.

Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains.

BACKGROUND: The occurrence of the novel porcine circovirus type 3 (PCV3) was reported from the Americas, Asia and Europe. Although this virus was detected in association with various clinical syndromes in pigs, its role as possible swine pathogen remains unclear. PCV3 was detected with high prevalence in Polish farms, but to date no genome sequences were available from European PCV3 strains. METHODS: We collected 1060 serum samples from piglets at the age of 20-24 weeks from 53 farms distributed all over Germany. PCV3 DNA was detected using a real-time PCR and subsequently complete PCV3 genome sequences were obtained after multiply primed rolling circle amplification and sequencing of overlapping PCR products. Phylogenetic analysis was performed by neighbor-joining method and maximum likelihood method. RESULTS: We obtained 15 complete PCV3 genome sequences as well as nine partial sequences including the putative ORFs 1, 2 and 3 from PCV3 viremic animals in German pig farms. Phylogenetic analysis of these German as well as 30 full genome sequences received from GenBank divided the PCV3 strains into two main groups and several subclusters. Furthermore, we were able to define group specific amino acid patterns in open reading frame 1 and 2. CONCLUSION: PCV3 is distributed with high prevalence in German pig industry. Phylogenetic analysis revealed two clearly separated groups of PCV3 strains, which might be considered as PCV3 genotypes. Specific nucleotide and amino acid marker positions may serve for easy and fast intraspecies classification and genotyping of PCV3 strains. No correlation between PCV3 variants with their geographical origin was evident. We found the same diversity of PCV3 strains in Germany as in other countries. We hypothesize that PCV3 is not a newly emerging virus in the German pig population. Future studies will have to show, if PCV3 genotype specific biological properties are evident.

A synthetic human cytomegalovirus pp65-IE1 fusion antigen efficiently induces and expands virus specific T cells.

Infection with human cytomegalovirus (HCMV) can cause severe complications in newborns and immunocompromised patients, and a prophylactic or therapeutic vaccine against HCMV is not available. Here, we generated a HCMV vaccine candidate fulfilling the regulatory requirements for GMP-compliant production and clinical testing. A novel synthetic fusion gene consisting of the coding sequences of HCMV pp65 and IE1 having a deleted nuclear localization sequence and STAT2 binding domain was introduced into the genome of the attenuated vaccinia virus strain MVA. This recombinant MVA, MVA-syn65_IE1, allowed for the production of a stable ∼120kDa syn65_IE1 fusion protein upon tissue culture infection. MVA-syn65_IE1 infected CD40-activated B cells activated and expanded pp65- and IE1-specific T cells derived from HCMV-seropositive donors to at least equal levels as control recombinant MVA expressing single genes for pp65 or IE1. Additionally, we show that MVA-syn65_IE1 induced HCMV pp65- and IE1-epitope specific T cells in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Thus, MVA-syn65_IE1 represents a promising vaccine candidate against HCMV and constitutes a basis for the generation of a multivalent vaccine targeting relevant pathogens in immunocompromised patients.

Sus scrofa papillomavirus 2 - genetic characterization of a novel suid papillomavirus from wild boar in Germany.

We identified a novel papillomavirus, Sus scrofa papillomavirus 2 (SsPV2), which is the first papillomavirus associated with papillomas in pigs. In skin alterations of a German wild boar, showing typical gross and histological appearance of papillomas, papillomavirus-like particles were demonstrated by electron microscopy. Degenerate papillomavirus-specific primers were used to amplify and sequence parts of the viral DNA. Subsequently, the complete genomic DNA was cloned and sequenced. The SsPV2 genome had a length of 8218 bp, encoded the early proteins E6, E7, E1 and E2, the late proteins L1 and L2 and contained an upstream regulatory region. Genomic characterization demonstrated papillomavirus-typical characteristics as well as unique features. For example, the E2 protein was significantly larger than in every other known papillomavirus species. Phylogenetic analysis was not able to relate SsPV2 unambiguously with other papillomavirus species or existing genera. Therefore, it might be representative of a new papillomavirus genus.

An evaluation of pediatric residency education in conveying immunization knowledge.

BACKGROUND: The Accreditation Council of Graduate Medical Education now requires all pediatric residency training programs assess medical knowledge competency. PURPOSE: The goal of this project was to determine whether pediatric residency training using patient-based/experiential teaching made residents competent in the area of immunization knowledge or whether additional teaching strategies might need to be developed. METHODS: Cross-sectional and longitudinal study designs were used to determine improvement in immunization knowledge on a multiple-choice quiz over the 3 years of residency training. RESULTS: Both the cross-sectional and longitudinal data showed a statistically significant improvement in performance between residency training Years 1 and 2 but not between Years 2 and 3 on the quiz. This statistically significant relationship by year of training was seen despite the modest reliability of the short quiz and the sample size. CONCLUSIONS: This study shows that pediatric residency education using patient-based/experiential teaching is effective in teaching first year residents about immunization knowledge but is not as effective for 2nd- and 3rd-year residents. Other instructional methods such as computer-based cases could be employed during the 2nd and 3rd years.

Healthy Steps: resident's perceptions.

The objective of this study is to determine pediatric residents' perception of Healthy Steps for Young Children, Healthy Steps Iowa, and Healthy Steps specialists. Twenty-nine of 37 (78%) pediatric residents participated in this survey. The Healthy Steps specialists facilitated pediatric residents' learning of anticipatory guidance (69%), and assured the patients received information efficiently (69%). Sixty-two percent reported that the Healthy Steps specialists did not interfere with their relationship as the patient's primary care provider. Seventy-six percent would consider using Healthy Steps specialists in their future practice. Program improvement areas include improving clinic efficiency with the incorporation of Healthy Steps specialists; increasing the pediatric residents' awareness of family violence, mental illness, and substance abuse; and improving their method of referral to community contacts.