Riding apoptotic bodies for cell-cell transmission by African swine fever virus.

African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.

SARS-CoV-2 infection aggravates chronic comorbidities of cardiovascular diseases and diabetes in mice.

Background: Cardiovascular diseases (CVDs) and diabetes mellitus (DM) are top two chronic comorbidities that increase the severity and mortality of COVID-19. However, how SARS-CoV-2 alters the progression of chronic diseases remain unclear. Methods: We used adenovirus to deliver h-ACE2 to lung to enable SARS-CoV-2 infection in mice. SARS-CoV-2's impacts on pathogenesis of chronic diseases were studied through histopathological, virologic and molecular biology analysis. Results: Pre-existing CVDs resulted in viral invasion, ROS elevation and activation of apoptosis pathways contribute myocardial injury during SARS-CoV-2 infection. Viral infection increased fasting blood glucose and reduced insulin response in DM model. Bone mineral density decreased shortly after infection, which associated with impaired PI3K/AKT/mTOR signaling. Conclusion: We established mouse models mimicked the complex pathological symptoms of COVID-19 patients with chronic diseases. Pre-existing diseases could impair the inflammatory responses to SARS-CoV-2 infection, which further aggravated the pre-existing diseases. This work provided valuable information to better understand the interplay between the primary diseases and SARS-CoV-2 infection.

Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques.

Coronavirus disease 2019 (COVID-19), which is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. It is unclear whether convalescing patients have a risk of reinfection. We generated a rhesus macaque model of SARS-CoV-2 infection that was characterized by interstitial pneumonia and systemic viral dissemination mainly in the respiratory and gastrointestinal tracts. Rhesus macaques reinfected with the identical SARS-CoV-2 strain during the early recovery phase of the initial SARS-CoV-2 infection did not show detectable viral dissemination, clinical manifestations of viral disease, or histopathological changes. Comparing the humoral and cellular immunity between primary infection and rechallenge revealed notably enhanced neutralizing antibody and immune responses. Our results suggest that primary SARS-CoV-2 exposure protects against subsequent reinfection in rhesus macaques.

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.

Difference in pathogenicity of 2 strains of avian leukosis virus subgroup J in broiler chicken.

Avian leukosis virus subgroup J has been found to infect many types of chickens with various genetic backgrounds. The ALV-J strain NX0101, which was isolated from broiler breeders in 2001, mainly induces the formation of myeloid cell tumors. However, strain HN10PY01, which was recently isolated from laying hens, mainly induces the formation of myeloid cell tumors and hemangioma. In order to determine the difference in pathogenicity of the 2 strains in broiler chickens, 2 groups of chicken embryos were infected with NA0101 and HN10PY01 separately. A comparison was made of the mortality, oncogenicity, body weights, indexes for immune organs, levels of ALV group-specific antigen p27, and mRNA expression levels of the tumor-related gene, p53, in ALV-J-infected birds and immune organs of theses chickens in response to Newcastle Disease Virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. The results indicated that strain NX0101 was highly pathogenic in broiler chickens and led to a 30% mortality rate and 45% oncogenicity, compared with the HN10PY01-infected birds. Weight of chickens was also significantly lower after 15 wk (P < 0.05). In addition, the mRNA expression levels of tumor-related p53 in medulla, liver, and lung in broilers infected with strain NX0101 were significantly higher than those infected with strain HN10PY01 (P < 0.05). These results indicated that strain NX0101 had a higher replication ability in broiler chickens. The findings of this study will contribute to further elucidating the mechanisms underlying host susceptibility and tumor classification in ALV-J-infected chickens.

Viral proliferation and expression of tumor-related gene in different chicken embryo fibroblasts infected with different tumorigenic phenotypes of avian leukosis virus subgroup J.

Subgroup J avian leukosis virus (ALV-J) causes a neoplastic disease in infected chickens. The ALV-J strain NX0101, which was isolated from broiler breeders in 2001, mainly induced formation of myeloid cell tumors. However, strain HN10PY01, which was recently isolated from laying hens, mainly induces formation of myeloid cell tumors and hemangioma. To identify the molecular pathological mechanism underlying changes in host susceptibility and tumor classification induced by these two types of ALV-J strains, chicken embryo fibroblasts derived from chickens with different genetic backgrounds (broiler breeders and laying hens) and an immortalized chicken embryo fibroblasts (DF-1) were prepared and infected with strain NX0101 or HN10PY01, respectively. The 50% tissue culture infective dose (TCID50) and levels of ALV group-specific antigen p27 and heat shock protein 70 in the supernatant collected from the ALV-J infected cells were detected. Moreover, mRNA expression levels of tumor-related genes p53, c-myc, and Bcl-2 in ALV-J-infected cells were quantified. The results indicated that the infection of ALV-J could significantly increase mRNA expression levels of p53, c-myc, and Bcl-2 Strain HN10PY01 exhibited a greater influence on the three tumor-related genes in each of the three types of cells when compared with strain NX0101, and the TCID50 and p27 levels in the supernatant collected from HN10PY01-infected cells were higher than those collected from NX0101-infected cells. These results indicate that the infection of the two ALV-J strains influenced the gene expression levels in the infected cells, while the newly isolated strain HN10PY01 showed higher replication ability in cells and induced higher expression levels of tumor-related genes in infected cells. Furthermore, virus titers and expression levels of tumor-related genes and cellular stress responses of cells with different genetic backgrounds when infected with each of the two ALV-J strain were different, indicating that genetic backgrounds influenced the capabilities of the virus to infect and proliferate. The findings of this study provide useful data to further elucidate the mechanism underlying host susceptibility and tumor classification in ALV-J-infected chickens and cells.

Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015.

There have been many outbreaks of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, in Chinese chicken farms since June 2015. HPS was mainly found in miscellaneous meat-type chickens, Ma chickens, layer chicks and Three-yellow chickens, while it was occasionally found in white broilers. To determine the specific causative pathogen and pathogenicity of HPS in chickens, we collected 25 suspected cases and performed clinical pathology and aetiology analyses. The results showed that the 25 cases exhibited multifocal hepatitis with intra-nuclear inclusion bodies and 70 nm-latticed viral particles in the cell nuclei. All samples were positive for fowl adenovirus (FAdV), and sequencing results showed that the hexon gene shared the highest nucleotide similarities with the hexon gene of group 1 serotype 4 (FAdV-4). FAdV-4 was highly pathogenic to embryos and specific pathogen-free chickens, causing 100 and 70 % mortality rates, respectively. Thus, FAdV-4 is associated with HPS outbreaks in China.

Distribution of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in different stages of gestation sows: HP-PRRSV distribution in gestation sows.

Highly pathogenic PRRSV (HP-PRRSV) emerged in China in 2006 and caused severe reproductive losses, particularly in late-term sows. To determine whether these reproductive failures were related to the susceptibility of late-term sows to HP-PRRV, 60- and 90-days of gestation sows were infected with HP-PRRSV isolate TA-12 (GenBank accession HQ417620). A monoclonal antibody specific to the C-terminal of the nucleocapsid protein was used to evaluate viral distribution by IHC. This showed that HP-PRRSV had a similar distribution in both sets of sows. However, HP-PRRSV infection led to dramatically decreased serum levels of luteinizing hormone (LH) and 17-ß-estradiol (E2) in late-term sows, while only E2 was decreased in the 60-day sows. These results indicate that HP-PRRSV-induced reproductive failure is more likely due to reproductive hormone level imbalances rather than tissue tropism differences.

Toxicity to the hematopoietic and lymphoid organs of piglets treated with a therapeutic dose of florfenicol.

Florfenicol (FLO) is a broad-spectrum antibacterial agent for treatment of bacteriosis of piglets in veterinary practice. To study the toxicity to the hematopoietic and lymphoid organs of piglets treated with a therapeutic dose of FLO, 20 healthy weaned piglets were selected and randomly divided into two groups. Piglets in the FLO group were fed with fodder supplemented with 30mg/kg BW of FLO twice a day for 10 days. Blood samples were drawn at four time points: 1 day before FLO administration and 1, 7, and 14 days post-withdrawal. Three or four piglets were euthanized at each time point post-withdrawal and tissue samples (bone marrow, thymus and spleen) were collected for fixation and cryostorage. The levels of classical swine fever virus (CSFV) antibody against the vaccine, the concentrations of Hsp70 and IL-6 in serum and Hsp70 in tissues, and the mRNA expression levels of B-cell lymphoma 2 (bcl-2) and tumor suppressor p53 were detected, the hematology of the piglets were analyzed, and the histopathology and the status of apoptosis of the hematopoietic and lymphoid organs was examined. The results showed changes in several indicators in the FLO group 1 day post-withdrawal: the concentration of red blood cells (RBCs) was decreased, and that of platelets (PLTs) was significantly lower (p<0.05); the volumes of RBC and PLT were increased; the sum of blood lymphocytes was statistically decreased (p<0.05); the concentration of IL-6 was significantly increased (p<0.05); the concentrations of Hsp70 in serum and tissues were increased; obvious atrophy of the hematopoietic cell lines and partial replacement by fat cells were observed in bone marrow; thymus and spleen tissues showed lower concentrations and sparser arrangement of lymphocytes in the thymic medulla and white pulp of the spleen respectively; and the mRNA expression levels of bcl-2 in the three tissues were up-regulated, while that of p53 was down-regulated. With time after cessation of FLO administration, the indicators of the FLO group gradually returned to close to that of the control group and the histological lesions of the tissues gradually recovered, and the differences in the densities of lymphocytes and cell arrangements in the tissues between two groups gradually decreased. In conclusion, a therapeutic dose of FLO induces temporary toxicity in the hematopoietic and lymphoid organs of piglets to some extent, and influences hemopoiesis and immune function. These effects gradually decrease after cessation of FLO administration.

Skin involvement in lymphomas caused by Marek's disease virus infection in Silkie chickens.

The Silkie is a typical Chinese breed of chicken. In 2012, batches of Silkies were found to have diffuse tumor-like nodules on their skin after feather removal, when they were slaughtered at about 60 days old. Gross examination showed no visible neoplastic lesions on the visceral organs and peripheral nerves, except slight splenomegaly in individual chickens. The disease was prevalent, with high condemnation rates for skin lesions, which caused great economic losses to the company. Tissues, including skin, visceral organs, and peripheral nerves, were collected for histologic examination. Heparinized blood samples were collected for virus isolation and identification. Marek's disease virus (MDV), Reticuloendotheliosis virus (REV), and Avian leukosis virus (ALV) were analyzed, using polymerase chain reaction (PCR) tests. Histologic examination showed that all of the tumor-like nodules on the skin were lymphomas. Lymphoproliferative lesions occurred mostly on the skin and only a few on the viscera, including the liver and proventriculus. Infected chick embryo fibroblasts showed clear cytopathic effects; indirect fluorescent antibody test for envelope glycoprotein B was positive. In addition, PCR indicated the presence of MDV serotype 1 infection without REV and ALV. A phylogenetic tree of the Meq gene showed that the isolate (SD121201) and Chinese reference strains, which are very virulent MDVs, are in the same clade. It was concluded that the Silkies tested were infected with MDV serotype 1. The Marek's disease epidemic has been controlled using CVI988/Rispens vaccines.