Newcastle disease virus-induced autophagy mediates antiapoptotic signaling responses in vitro and in vivo.

In this study, we investigated the role of autophagy and apoptosis in Newcastle disease virus (NDV)-infected chicken cells and tissues. NDV-infected and starvation-induced chick embryo fibroblasts (CEF) cells showed higher autophagosome formation than mock-infected CEF cells on transmission electron microscopy. The NDV-infected CEF cells showed enhanced conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of p62/SQSTM1. The diminished conversion of LC3-I to LC3-II and cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in ultraviolet-inactivated NDV-infected cells suggested that autophagosome formation was necessary for NDV replication. Inhibition of autophagy by chloroquine (CQ) enhanced apoptosis resulting in increased cleavage of caspase 3 and PARP and AnnexinV/propidium iodide staining. Autophagy induction by rapamycin resulted in upregulation of all autophagy-related genes except Beclin 1, anti-apoptosis factors, and proinflammatory cytokines in the NDV-infected spleen and lung tissues. Subsequently, decreased apoptosis was observed in NDV-infected spleens and lungs than mock-infected organs. The pan-caspase inhibitor ZVAD-FMK promoted conversion of LC3-I to LC3-II, the degradation of p62/SQSTM1, NDV replication and cell viability by inhibiting apoptosis. Our study demonstrates that apoptosis inhibition enhances autophagy and promoted cell survival and NDV replication.

Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses.

Viral infection activates a host's cellular phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which is involved in cell differentiation, growth, survival, and apoptosis. To elucidate molecular mechanisms in the pathogenesis of Newcastle disease virus (NDV), we demonstrated that NDV transiently activates the PI3K/Akt pathway in chicken cells at an early phase of infection. Its activation was observed as early as 15 min post-infection and gradually weakened after 24 h. Incubating cells with a PI3K inhibitor, LY294002 or wortmannin, prior to NDV infection decreased NDV progeny yields and suppressed Akt phosphorylation at early times post-infection. Akt activation is triggered by NDV-GM or NDV-F48E9 and is abolished by methyl ß-cyclodextrin and chlorpromazine. Treatment following NDV-La Sota infection had no obvious effect. However, inhibiting PI3K activation promoted apoptotic responses during an early stage of NDV infection. The pan caspase inhibitor ZVAD-FMK mitigated the reduction in Akt phosphorylation by inhibiting PI3K activation, which indicates the signaling pathway promotes cell survival and, in turn, facilitates viral replication. By suppressing premature apoptosis upon NDV infection, the PI3K/Akt pathway enhances the anti-apoptotic response.

Highly pathogenic H5N6 influenza A viruses recovered from wild birds in Guangdong, southern China, 2014-2015.

Since 2013, highly pathogenic (HP) H5N6 influenza A viruses (IAVs) have emerged in poultry in Asia, especially Southeast Asia. These viruses have also caused sporadic infections in humans within the same geographic areas. Active IAV surveillance in wild birds sampled in Guangdong province, China from August 2014 through February 2015 resulted in the recovery of three H5N6 IAVs. These H5N6 IAV isolates possess the basic amino acid motif at the HA1-HA2 cleavage site that is associated with highly pathogenic IAVs infecting chickens. Noteworthy findings include: (1) the HP H5N6 IAV isolates were recovered from three species of apparently healthy wild birds (most other isolates of HP H5N6 IAV in Asia are recovered from dead wild birds or fecal samples in the environment) and (2) these isolates were apparently the first recoveries of HP H5N6 IAV for two of the three species thus expanding the demonstrated natural host range for these lineages of virus. This investigation provides additional insight into the natural history of HP H5N6 IAVs and identifies the occurrence of non-lethal, HP H5N6 IAV infections in wild birds thereby demonstrating the value of active IAV surveillance in wild birds.

Newcastle disease virus infection in chicken embryonic fibroblasts but not duck embryonic fibroblasts is associated with elevated host innate immune response.

BACKGROUND: Chickens and ducks are major hosts of Newcastle disease virus (NDV) with distinct responses to infection. However, whereas ducks are generally asymptomatic or exhibit only mild symptoms following NDV infection and are thus regarded as potential long-term reservoirs of the virus, chickens exhibit severe clinical lesions, transient infections and even death due to NDV infection. These differences may in part result from the host innate immune response to NDV infection. METHODS: To better understand the host innate immune response to NDV infection in avian species, by using the quantitative real-time polymerase chain reaction method we examined the messenger RNA expression levels of immune-related genes in chicken embryonic fibroblasts (CEFs) and duck embryonic fibroblasts (DEFs) when infected with NDV of different pathogenicities. RESULTS: Gene expression profiles showed that the expression of IL-1beta, TNF-α-like factor (LITAF) and interferon (IFN)-beta was upregulated in both CEFs and DEFs infected with SS-10 and NH-10 viruses or treated with polyinosinic:polycytidylic acid [poly(I:C)], as well as that expression levels were greater in CEFs than in DEFs. The expression of TLR3, TLR7, IL-6, IFN-alpha, IFN-gamma, MHC-I and MHC-II, except for IL-8, were also greater in CEFs than in DEFs in response to infection to both viruses or treatment with poly(I:C). However, unlike moderate virulent NH-10, highly virulent SS-10 induced greater pattern recognition receptors and cytokines, except for IFNs, in CEFs and DEFs. CONCLUSION: Results show distinct expression patterns of cytokines, Toll-like receptors and IFNs associated with inflammatory immune responses to NDV between species and by virulence.

Phylogenetic and Pathotypic Characterization of Newcastle Disease Viruses Circulating in South China and Transmission in Different Birds.

Although Newcastle disease virus (NDV) with high pathogenicity has frequently been isolated in poultry in China since 1948, the mode of its transmission among avian species remains largely unknown. Given that various wild bird species have been implicated as sources of transmission, in this study we genotypically and pathotypically characterized 23 NDV isolates collected from chickens, ducks, and pigeons in live bird markets (LBMs) in South China as part of an H7N9 surveillance program during December 2013-February 2014. To simulate the natural transmission of different kinds of animals in LBMs, we selected three representative NDVs-namely, GM, YF18, and GZ289-isolated from different birds to evaluate the pathogenicity and transmission of the indicated viruses in chickens, ducks, and pigeons. Furthermore, to investigate the replication and shedding of NDV in poultry, we inoculated the chickens, ducks, and pigeons with 10(6) EID50 of each virus via intraocular and intranasal routes. Eight hour after infection, the naïve contact groups were housed with those inoculated with each of the viruses as a means to monitor contact transmission. Our results indicated that genetically diverse viruses circulate in LBMs in South China's Guangdong Province and that NDV from different birds have different tissue tropisms and host ranges when transmitted in different birds. We therefore propose the continuous epidemiological surveillance of LBMs to support the prevention of the spread of these viruses in different birds, especially chickens, and highlight the need for studies of the virus-host relationship.