Neonatal pigs are susceptible to experimental Zika virus infection

Emerging Microbes & Infections (2017) 6, e6; doi:10.1038/emi.2016.133; published online 15 February 201

Interference with major histocompatibility complex class I pathway by animal alphaherpesviruses

Bovine herpesvirus-1 (BHV-1), pseudorabies (PrV), and equine herpesvirus-1 (EHV-1) are three herpesviruses of veterinary importance. All three viruses down-regulate cell surface expression of major histocompatibility complex (MHC) class I molecules, in vitro. Previous studies have shown that interference with transporter associated with antigen processing (TAP) is one of the mechanisms of class I down-regulation by two of these viruses (BHV-1 & PrV). The objectives of this study were to extend these findings to further characterize mechanisms of class I down-regulation by these viruses, and to identify the vial proteins responsible. BHV-1 protein(s) responsible for TAP inhibition could be immunoprecipitated with the bovine TAP complex from infected cells. Lack of an antibody to, and low expression of, bovine TAP complex in epithelial cells hamper this approach. As the first step to overcome this problem, we have cloned, sequenced and characterized bovine TAP2. Virion host shutoff (vhs) activity of BHV-1 partially contributes to down-regulation of class I expression. In order to investigate whether this is true with PrV, we generated two PrV vhs gene deletion mutants. Both vhs mutants interfered with TAP activity and down-regulated expression of class I, indicating that inhibition of TAP function in PrV-infected cells is due to mechanism(s) specifically directed at class I pathway. Our studies with EHV-1 have revealed that this virus also interferes with TAP function. Furthermore, as with BHV-1 and PrV, an early protein(s) of EHV-1 inhibit(s) TAP function. In order to identify the PrV protein responsible for TAP inhibition, we have constructed a PrV-bacterial artificial chromosome transposon mutant library. Screening of this library should lead to identification of PrV proteins responsible for TAP inhibition. These studies shed light on the mechanisms of down-regulation of class I expression by these viruses. Furthermore, this dissertation research should expedite identification of the early protein(s) of BHV-1, PrV, and EHV-1 that mediate class I down-regulation

Varicella-Zoster Virus IE63, a Major Viral Latency Protein, Is Required To Inhibit the Alpha Interferon-Induced Antiviral Response▿

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is the most abundant transcript expressed during latency in human sensory ganglia. VZV with ORF63 deleted is impaired for replication in melanoma cells and fibroblasts and for latency in rodents. We found that replication of the ORF63 deletion mutant is fully complemented in U2OS cells, which have been shown to complement the growth of herpes simplex virus type 1 (HSV-1) ICP0 mutants. Since HSV-1 ICP0 mutants are hypersensitive to alpha interferon (IFN-α), we examined the effect of IFN-α on VZV replication. Replication of the ORF63 mutant in melanoma cells was severely inhibited in the presence of IFN-α, in contrast to other VZV mutants that were similarly impaired for replication or to parental virus. The VZV ORF63 mutant was not hypersensitive to IFN-γ. IFN-α inhibited viral-gene expression in cells infected with the ORF63 mutant at a posttranscriptional level. Since IFN-α stimulates gene products that can phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF-2α) and inhibit translation, we determined whether cells infected with the ORF63 mutant had increased phosphorylation of eIF-2α compared with cells infected with parental virus. While phosphorylated eIF-2α was undetectable in uninfected cells or cells infected with parental virus, it was present in cells infected with the ORF63 mutant. Conversely, expression of IE63 (encoded by ORF63) in the absence of other viral proteins inhibited phosphorylation of eIF-2α. Since IFN-α has been shown to limit VZV replication in human skin xenografts, the ability of VZV IE63 to block the effects of the cytokine may play a critical role in VZV pathogenesis

Outbreak of Rabbit Hemorrhagic Disease Virus 2 Infections, Ghana

In September 2019, high mortality in commercial rabbits was reported in the Greater Accra Region of Ghana. Rabbit hemorrhagic disease virus 2 phylogenetically related to isolates from 2015–2017 outbreaks in the Netherlands was confirmed as the causative agent. The virus has not yet been detected in native rabbits in Ghana

Superficial Inguinal Lymph Nodes for Screening Dead Pigs for African Swine Fever

African swine fever (ASF) has spread across the globe and has reached closer to North America since being reported in the Dominican Republic and Haiti. As a result, surveillance measures have been heightened and the utility of alternative samples for herd-level monitoring and dead pig sampling have been investigated. Passive surveillance based on the investigation of dead pigs, both domestic and wild, plays a pivotal role in the early detection of an ASF incursion. The World Organization for Animal Health (OIE)-recommended samples for dead pigs are spleen, lymph nodes, bone marrow, lung, tonsil and kidney. However, obtaining these samples requires opening up the carcasses, which is time-consuming, requires skilled labour and often leads to contamination of the premises. As a result, we investigated the suitability of superficial inguinal lymph nodes (SILNs) for surveillance of dead animals. SILNs can be collected in minutes with no to minimum environmental contamination. Here, we demonstrate that the ASF virus (ASFV) genome copy numbers in SILNs highly correlate with those in the spleen and, by sampling SILN, we can detect all pigs that succumb to highly virulent and moderately virulent ASFV strains (100% sensitivity). ASFV was isolated from all positive SILN samples. Thus, sampling SILNs could be useful for routine surveillance of dead pigs on commercial and backyard farms, holding pens and dead on arrival at slaughter houses, as well as during massive die-offs of pigs due to unknown causes

Validation of an Effective Protocol for Culicoides Latreille (Diptera: Ceratopogonidae) Detection Using eDNA Metabarcoding

eDNA metabarcoding is an effective molecular-based identification method for the biosurveillance of flighted insects. An eDNA surveillance approach maintains specimens for secondary morphological identification useful for regulatory applications. This study identified Culicoides species using eDNA metabarcoding and compared these results to morphological identifications of trapped specimens. Insects were collected using ultraviolet (UV) lighted fan traps containing a saturated salt (NaCl) solution from two locations in Guelph, Ontario, Canada. There were forty-two Culicoides specimens collected in total. Molecular identification detected four species, C. biguttatus, C. stellifer, C. obsoletus, and C. mulrennani. Using morphological identification, two out of these four taxonomic ranks were confirmed at the species level (C. biguttatus and C. stellifer) and one was confirmed at the subgenus level (Avaritia [C. obsoletus]). No molecular detection of Culicoides species occurred in traps with an abundance of less than three individuals per taxon. The inconsistency in identifying Culicoides specimens to the species level punctuates the need for curated DNA reference libraries for Culicoides. In conclusion, the saturated salt (NaCl) solution preserved the Culicoides’ morphological characteristics and the eDNA

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible

Development and Validation of Competitive ELISA for Detection of H5 Hemagglutinin Antibodies

Influenza A viruses (IAVs) belonging to the goose/Guangdong (Gs/GD)-lineage H5Nx remain a major concern for the global poultry industry, wildlife, and humans. The hemagglutinin (HA) protein is the dominant antigenic epitope carrier within IAV, which in turn triggers substantial immunogenic responses in the infected host. The current study describes the development and validation of a highly sensitive competitive H5 ELISA (cELISA) based on a novel monoclonal antibody developed in mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/1966). The cELISA is capable of detecting the H5 antibody response to a wider range of H5-subtype viruses belonging to both North American and Eurasia lineages, including the Gs/GD H5Nx from clade 2.3.4.4b that is currently causing the highly pathogenic avian influenza outbreaks in Eurasia, Africa, and Latin and North America. The developed H5 cELISA provides a specific, sensitive, and species-independent serological assay for the rapid detection of H5 antibodies. The assay is more robust and more sensitive than the hemagglutination inhibition assay, which is the “Gold standard”. The assay can be used in serological diagnosis, serosurveillance, and vaccine monitoring of serum samples collected from different species of animals

A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups

The African swine fever virus (ASFV) is currently causing a world-wide pandemic of a highly lethal disease in domestic swine and wild boar. Currently, recombinant ASF live-attenuated vaccines based on a genotype II virus strain are commercially available in Vietnam. With 25 reported ASFV genotypes in the literature, it is important to understand the molecular basis and usefulness of ASFV genotyping, as well as the true significance of genotypes in the epidemiology, transmission, evolution, control, and prevention of ASFV. Historically, genotyping of ASFV was used for the epidemiological tracking of the disease and was based on the analysis of small fragments that represent less than 1% of the viral genome. The predominant method for genotyping ASFV relies on the sequencing of a fragment within the gene encoding the structural p72 protein. Genotype assignment has been accomplished through automated phylogenetic trees or by comparing the target sequence to the most closely related genotyped p72 gene. To evaluate its appropriateness for the classification of genotypes by p72, we reanalyzed all available genomic data for ASFV. We conclude that the majority of p72-based genotypes, when initially created, were neither identified under any specific methodological criteria nor correctly compared with the already existing ASFV genotypes. Based on our analysis of the p72 protein sequences, we propose that the current twenty-five genotypes, created exclusively based on the p72 sequence, should be reduced to only six genotypes. To help differentiate between the new and old genotype classification systems, we propose that Arabic numerals (1, 2, 8, 9, 15, and 23) be used instead of the previously used Roman numerals. Furthermore, we discuss the usefulness of genotyping ASFV isolates based only on the p72 gene sequence