Construction of Novel Thermostable Chimeric Vaccine Candidates for Genotype VII Newcastle Disease Virus.

Genotype VII Newcastle Disease Virus (NDV) has caused a pandemic in many countries and usually causes fatal consequences in infected chickens. Although current commercial attenuated NDV vaccines can provide an ideal protection against genotype VII NDV, they cannot completely prevent the infection and viral shedding, and the genotype of some vaccine strains cannot match with the prevalent strain. In this study, in order to construct a thermostable and genotype VII-matched live attenuated vaccine, we used a thermostable genotype VIII virulent HR09 strain as the backbone and replaced its F gene with that of the genotype VII DT-2014 strain. Meanwhile, the cleavage site of F gene of DT-2014 was mutated to that of class I F protein and avirulent class II F protein, respectively. The results showed that the two chimeric viruses, designated rcHR09-CI and rcHR09-CII, shared a similar growth kinetics and thermostability with their parental HR09 strain. Mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests showed that the two chimeric viruses were highly attenuated. Though both chimeric NDVs and La Sota vaccine strain could provide complete protection to immunized chickens against the challenge of virulent genotype VII ZJ1 strain, the two chimeric NDVs could induce a higher level of antibody response against ZJ1 strain and could significantly reduce the viral shedding compared with La Sota vaccine strain. In conclusion, our study constructed two chimeric thermostable genotype VII-matched NDV vaccine candidates, which provided complete protection against the challenge of virulent genotype VII NDV.

The role of PA-X C-terminal 20 residues of classical swine influenza virus in its replication and pathogenicity.

PA-X is a fusion protein encoded by a +1 frameshifted open reading frame (X-ORF) in PA gene. The X-ORF can be translated in full-length (61 amino acids, aa) or truncated (41 aa) form. However, the role of C-Terminal 20 aa of PA-X in virus function has not yet been fully elucidated. To this end, we constructed the contemporary influenza viruses with full and truncated PA-X by reverse genetics to compare their replication and pathogenicity. The full-length PA-X virus in MDCK and human A549 cells conferred 10- to 100-fold increase in viral replication, and more virulent and caused more severe inflammatory responses in mice relative to corresponding truncated PA-X virus, suggesting that the terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.

Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus.

Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a "cold-chain" is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315-375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.

Systematic evaluation of potential pathogenicity of Salmonella Indiana.

Salmonella Indiana has emerged in recent years as an important zoonotic pathogen, but its pathogenicity has not been fully elucidated. In this study, using in vivo and in vitro animal and cellular experimental model systems, we evaluated the pathogenicity of Salmonella Indiana (S. Indiana) compared with three other serotypes of Salmonella, S. Enteritidis, S. Typhimurium and S. Thompson. The animal experiments included observations of clinical symptoms, pathological changes and determination of median lethal dose in mice. The adhesion and invasiveness and intracellular proliferative capacity of Salmonella in vitro were measured with the murine macrophage-like cell line RAW264.7 cells and the human colon adenocarcinoma cell line Caco-2 cells. The results of animal experiments showed that S. Indiana, S. Enteritidis, S. Typhimurium and S. Thompson caused histopathological changes in most organs to varying degrees, primarily in the liver and intestine of mice. The gross lesions included white necrotic foci on the liver surface with different levels. The histopathological changes of monocyte/macrophage infiltration and coagulative necrosis were observed in the liver. Intestinal villi became short and were sloughed off, and lymphocyte infiltration was found in the submucosa. Compared with the other serotypes, the pathological changes caused by S. Indiana were slighter and had a relatively high median lethal dose in mice. The results of adhesion and invasion tests showed that the intracellular growth trend of most Salmonella strains was positively correlated with the number of pathogens adhering to and invading cells. Compared with the strains of the other three serotypes, most S. Indiana strains exhibited significantly lower adhesion and invasiveness to RAW264.7 and Caco-2 cells within 30 min. Most S. Indiana strains displayed twice to four times lower intracellular proliferation within 24 h in RAW264.7 cells. In conclusion, S. Indiana was pathogenic, but its pathogenicity was lower than that of S. Typhimurium and S. Enteritidis, and was similar to that of S. Thompson.

Protective efficacy of a bivalent inactivated reassortant H1N1 influenza virus vaccine against European avian-like and classical swine influenza H1N1 viruses in mice.

The classical swine (CS) H1N1 swine influenza virus (SIVs) emerged in humans as a reassortant virus that caused the H1N1 influenza virus pandemic in 2009, and the European avian-like (EA) H1N1 SIVs has caused several human infections in European and Asian countries. Development of the influenza vaccines that could provide effective protective efficacy against SIVs remains a challenge. In this study, the bivalent reassortant inactivated vaccine comprised of SH1/PR8 and G11/PR8 arboring the hemagglutinin (HA) and neuraminidase (NA) genes from prevalent CS and EA H1N1 SIVs and six internal genes from the A/Puerto Rico/8/34(PR8) virus was developed. The protective efficacy of this bivalent vaccine was evaluated in mice challenged with the lethal doses of CS and EA H1N1 SIVs. The result showed that univalent inactivated vaccine elicited high-level antibody against homologous H1N1 viruses while cross-reactive antibody responses to heterologous H1N1 viruses were not fully effective. In a mouse model, the bivalent inactivated vaccine conferred complete protection against lethal challenge doses of EA SH1 virus or CS G11 virus, whereas the univalent inactivated vaccine only produced insufficient protection against heterologous SIVs. In conclusion, our data demonstrated that the reassortant bivalent inactivated vaccine comprised of SH1/PR8 and G11/PR8 could provide effective protection against the prevalent EA and CS H1N1 subtype SIVs in mice.

Generation and evaluation of a vaccine candidate of attenuated and heat-resistant genotype VIII Newcastle disease virus.

Newcastle disease, which is a highly contagious and fatal disease caused by the Newcastle disease virus (NDV), has harmed the poultry industry for decades. The administration of effective vaccines can control most outbreaks and epidemics of Newcastle disease in the world. However, vaccination failures of live attenuated vaccines becasue of storage and transportation problems have been reported. Hence, thermostable live vaccine strains, such as V4 and I-2 strains, are being used and welcomed in tropical regions such as Africa and Southeast Asia. In this study, a thermostable, attenuated vaccine candidate strain NDV/rHR09 was generated using the genotype VIII heat-resistant virulent NDV strain HR09 by the reverse genetics system. The results of the determination of the mean death time and intracerebral pathogenicity index indicated that NDV/rHR09 is lentogenic even after 15 serial passages in embryonated chicken eggs. The thermostability assessment showed that the NDV/rHR09 strain exhibited hemagglutination activity and infectivity when exposed to 56°C for 60 min. Compared with the commercially available La Sota and V4 vaccines, the NDV/rHR09 induced higher antibody titers in specific pathogen-free chickens. In addition, NDV/rHR09 conferred complete protection against virulent genotype VII NDV challenge and virus shedding from vaccinated chickens. These results suggest that NDV/rHR09 is a promising thermostable vaccine candidate strain.

Autophagy induced by avian reovirus enhances viral replication in chickens at the early stage of infection.

BACKGROUND: Avian reovirus (ARV) is an important pathogen that can cause serious disease in poultry. Though several in vitro studies revealed some molecular mechanisms that are responsible for ARV-induced autophagy, it is still largely unknown how ARV manipulates autophagy to promote its own propagation. RESULTS: In this study, we demonstrated that ARV infection triggered autophagy in chicken tissues, evident from the enhancement of LC3-I/-II conversion and the appearance of abundant autophagosomes. Moreover, viral replication and the expression of IL-1ß were coupled with the process of ARV-induced autophagy in the early stage of infection. Furthermore, regulation of autophagy affected the accumulation of LC3-II, the production of ARV and the expression of IL-1ß. CONCLUSIONS: Altogether, our data suggest that ARV induces autophagy, which benefits its replication and dissemination in chicken tissues at the early infection stage.

Protective efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the European Avian-like H1N1 swine influenza virus in mice and pigs.

Swine influenza A viruses (SIVs) causing outbreaks of acute, highly contagious respiratory disease in pigs also pose a potential threat to public health. European avian-like H1N1 (EA H1N1) SIVs are the predominant circulating viruses in pigs in China and also occasionally cause human infection. In this study, a high-growth reassortant virus (SH1/PR8), with HA and NA genes from a representative EA H1N1 isolate A/Swine/Shanghai/1/2014 (SH1) in China and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus, was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of inactivated vaccine. The protective efficacy of inactivated SH1/PR8 was evaluated in mice and pigs challenged with wild-type SH1 virus. After primer and boost vaccination, the SH1/PR8 vaccine induced high-level hemagglutination inhibiting (HI) antibodies, IgG antibodies, and neutralization antibodies in mice and pigs. Mice and pigs in the vaccinated group showed less clinical phenomena and pathological changes than those in the unvaccinated group. In conclusion, the inactivated high-growth reassortant vaccine SH1/PR8 could induce high antibody levels and complete protection is expected against SH1 wild type SIV, and protection against heterologous EA H1N1 SIV needs further evaluation.

The PB2-K627E mutation attenuates H3N2 swine influenza virus in cultured cells and in mice.

PB2-627K is an important amino acid that determines the virulence of some influenza A viruses. However, it has not been experimentally investigated in the H3N2 swine influenza virus. To explore the potential role of PB2-K627E substitution in H3N2 swine influenza virus, the growth properties and pathogenicity between H3N2 swine influenza virus and its PB2-K627E mutant were compared. For the first time, our results showed that PB2-K627E mutation attenuates H3N2 swine influenza virus in mammalian cells and in mice, suggesting that PB2-627K is required for viral replication and pathogenicity of H3N2 swine influenza virus.

Avian infectious bronchitis virus disrupts the melanoma differentiation associated gene 5 (MDA5) signaling pathway by cleavage of the adaptor protein MAVS.

BACKGROUND: Melanoma differentiation associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) selectively sense cytoplasmic viral RNA to induce an antiviral immune response. Infectious bronchitis virus (IBV) is one of the most important infectious agents in chickens, and in chicken cells, it can be recognized by MDA5 to activate interferon production. RIG-I is considered to be absent in chickens. However, the absence of RIG-I in chickens raises the question of whether this protein influences the antiviral immune response against IBV infection. RESULTS: Here, we showed that chicken cells transfected with domestic goose RIG-I (dgRIG-I) exhibited increased IFN-ß activity after IBV infection. We also found that IBV can cleave MAVS, an adaptor protein downstream of RIG-I and MDA5 that acts as a platform for antiviral innate immunity at an early stage of infection. CONCLUSIONS: Although chicken MDA5 (chMDA5) is functionally active during IBV infection, the absence of RIG-I may increase the susceptibility of chickens to IBV infection, and IBV may disrupt the activation of the host antiviral response through the cleavage of MAVS.

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus.

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China.

Pigs are susceptible to both human and avian influenza viruses and therefore have been proposed to be mixing vessels for the generation of pandemic influenza viruses through reassortment. In this study, for the first time, we report the isolation and genetic analyses of three novel triple-reassortant H1N1 swine influenza viruses from pigs in Tianjin, Northern China. Phylogenetic analysis showed that these novel viruses contained genes from the 2009 pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. This indicated that the reassortment among the 2009 pandemic H1N1, Eurasian swine and triple-reassortant swine influenza viruses had taken place in pigs in Tianjin and resulted in the generation of new viruses. Furthermore, three human-like H1N1, two classical swine H1N1 and two Eurasian swine H1N1 viruses were also isolated during the swine influenza virus surveillance from 2009 to 2013, which indicated that multiple genetic lineages of swine H1N1 viruses were co-circulating in the swine population in Tianjin, China. The emergence of novel triple-reassortant H1N1 swine influenza viruses may be a potential threat to human health and emphasizes the importance of further continuous surveillance.