Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-α and IFN-γ. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants.

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is an acute fatal disease in elephants. Despite the fact that the underlying pathogenesis of EEHV-HD has been proposed, it remains undetermined as to what mechanisms drive these hemorrhagic and edematous lesions. In the present study, we have investigated and explained the pathogenesis of acute EEHV-HD using blood profiles of EEHV-HD and EEHV-infected cases, hematoxylin and eosin (H&E) stain, special stains, immunohistochemistry, quantitative polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). It was found that EEHV genomes were predominantly detected in various internal organs of EEHV-HD cases. Damage to endothelial cells, vasculitis and vascular thrombosis of the small blood vessels were also predominantly observed. Increases in platelet endothelial cell adhesion molecules-1 (PECAM-1)- and von Willebrand factor (vWF)-immunolabeling positive cells were significantly noticed in injured blood vessels. The expression of pro-inflammatory cytokine mRNA was significantly up-regulated in EEHV-HD cases when compared to EEHV-negative controls. We have hypothesized that this could be attributed to the systemic inflammation and disruption of small blood vessels, followed by the disseminated intravascular coagulopathy that enhanced hemorrhagic and edematous lesions in EEHV-HD cases. Our findings have brought attention to the potential application of effective preventive and therapeutic protocols to treat EEHV infection in Asian elephants.

The objective of this study was to evaluate the efficacy of two multivalent commercial porcine circovirus (PCV) vaccines against heterologous PCV2d challenges. A total of 24 crossbred male pigs aged 26 days selected from a specific pathogen-free herd were randomly divided into four groups (six pigs per group) and assigned as follows: negative control (unvaccinated/sham-challenge), vaccinated with chimeric PCV1-2a vaccine (PCV1-2a/PCV2d-challenge), vaccinated with chimeric PCV1-2a-2b vaccine (PCV1-2a-2b/PCV2d-challenge) and positive control (unvaccinated/PCV2d-challenge). At 21 days after vaccination, the pigs were intranasally and intramuscularly inoculated with either sham or field isolates of PCV2d (PCV2d/149/TH/2020). After being challenged, blood samples were obtained weekly and analyzed for levels of PCV2d viremia, neutralizing antibodies, and IgG against PCV2. At 30 days post-challenge (DPC), the pigs were euthanized and then subjected to pathological evaluations and molecular analysis. The results indicated that pigs in the PCV1-2a-2b/PCV2d-challenge and the PCV1-2a/PCV2d-challenge groups possessed significantly greater levels of PCV2d-neutralizing antibody titer when compared with the positive control group. Moreover, pigs in the PCV1-2a-2b/PCV2d-challenge group exhibited a lower degree of severity in terms of gross lesion scores and lower levels of PCV2 viremia when compared with the positive control group. This study demonstrated that vaccinating pigs with either the PCV1-2a or PCV1-2a-2b chimeric vaccines elicits a potent immune response against PCV2d infection and reduces viremia after PCV2d inoculation in pigs.

Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.