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African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV–host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R , an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔ I73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔ I73R can be a potent live-attenuated vaccine candidate.

African swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by the African swine fever virus (ASFV). The current research on ASF vaccines focuses on the development of naturally attenuated, isolated, or genetically engineered live viruses that have been demonstrated to produce reliable immunity. As a result, a genetically engineered virus containing five genes deletion was synthesized based on ASFV Chinese strain GZ201801, named ASFV-GZΔI177LΔCD2vΔMGF. The five-gene-deleted ASFV was safe and fully attenuated in pigs and provides reliable protection against the parental ASFV strain challenge. This indicates that the five-gene-deleted ASFV is a potential candidate for a live attenuated vaccine that could control the spread of ASFV.

In 2018, a disease characterized by splenic hemorrhage and necrosis killed ducks in a duck farm in Guangxi province, China. A duck reovirus strain was isolated from the tissues of the dead ducks by inoculating duck embryos and BHK-21 cells. Electron microscopy of the cultured the isolate showed that the viral particles were nearly round in shape and approximately 70 nm in diameter, and they were designated DRV-GL18. Sequence analysis showed that the GL18 strain viral genome was 23,419 nt in length and had 10 dsRNA segments. Phylogenetic analysis of cDNA amplicons of segments encoding the protein σC which are outer capsid proteins showed that the isolate belongs to the branch of the epidemic strains of duck reovirus. The Recombination Detection Program (RDP) and SimPlot program analyses suggested potential genetic recombination events in the M2 segments. Pathogenicity experiments revealed that GL18 produced severe hemorrhaging in livers and necrosis in the spleen of infected SPF ducklings. A death rate of 50% in the experimental ducklings was calculated during the first 7 d, and the rest of the ducklings were observed to undergo spleen necrosis. These data suggested that GL18 is a duck reovirus isolate with severer pathogenicity, and it could be a candidate for development of vaccine. This is the first reported isolation of duck reovirus from mature ducks.

As the sand mass flux increases from zero at the leading edge of a saltating surface to the equilibrium mass flux at the critical fetch length, the wind flow is modified and then the relative contribution of aerodynamic and bombardment entrainment is changed. In the end the velocity, trajectory and mass flux profile will vary simultaneously. But how the transportation of different sand size groups varies with fetch distance is still unclear. Wind tunnel experiments were conducted to investigate the fetch effect on mass flux and its distribution with height of the total sand and each size group in transportation. The mass flux was measured at six fetch length locations (0.5, 1.2, 1.9, 2.6, 3.4 and 4.1 m) and at three free-stream wind velocities (8.8, 12.2 and 14.5 m/s). The results reveal that the total mass flux and the mass flux of each size group with height can be expressed by q=aexp(−bh), where q is the sand mass flux at height h, and a and b are regression coefficients. The coefficient b represents the relative decay rate. Both the relative decay rates of total mass flux and each size group are independent of fetch length after a quick decay over a short fetch. This is much shorter than that of mass flux. The equilibrium of the relative decay rate cannot be regarded as an equilibrium mass flux profile for aeolian sand transport. The mass fluxes of 176.0, 209.3 and 148.0 μm size groups increase more quickly than that of other size groups, which indicates strong size-selection of grains exists along the fetch length. The maximal size group in mass flux (176.0 μm) is smaller than the maximal size group of the bed grains (209.3 μm). The relative contribution of each size group to the total mass flux is not monotonically decreasing with grain size due to the lift-off of some small grains being reduced due to the protection by large grains. The results indicate that there are complex interactions among different size groups in the developmental process of aeolian sand transport and more attention should be focused on the fetch effect because it has different influences on the total mass flux, the mass flux profile and its relative decay rate.

Duck adenoviruses (DAdVs) include serotype 1 (DAdV-1) in the genus Atadenovirus and serotypes 2-4 (DAdV-2, 3, and 4) in the genus Aviadenovirus. DAdV-3 was initially isolated from Chinese Muscovy ducks in 2014, whereby the infected ducks exhibited yellowing and hemorrhaging in the liver, along with slight pericardial effusion, swelling, and hemorrhaging in the kidneys. In recent years, duck adenovirus infections have appeared in Muscovy duck farms in Fujian, Zhejiang, Anhui, Guangdong, and other places in China. They have an incidence rate of 40 to 55% and a mortality rate of 35 to 43%, resulting in great losses to the duck breeding industry. In this study, 7 DAdV-3 strains, designated as TZ193, FJPT20161124, GX20170519, FJZZ, GDMM, AHAQ, and GDHS were isolated from Muscovy ducks in different provinces of China during 2016–2019, and their complete genomics were sequenced. Their genomes all exhibited significant deletions in ORF67, which also had G to A transitions at the 41st and 977th nt positions, resulting in a stop codon. The pathogenicity of TZ193, a novel isolate of DAdV-3, was investigated in Muscovy ducks. TZ193 caused characteristic lesions of swelling as well as hemorrhagic liver and kidney in the infected ducklings. Moreover, the mortality rate of TZ193 in 5-day-old domestic ducks was 100%. Our data provide concrete evidence for the identification of the DAdV-3 novel variant mutant in China, which effects increased mortality in ducks. This highlights the necessity for monitoring the specific molecular epidemiology of novel DAdV-3 mutants and the development of new vaccines in the future.

During epidemiological surveillance of Feline calicivirus (FCV) isolates in Shanghai, China, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat. The complete genome sequence of SH1909 was determined in this study. Sequence comparison and analysis showed that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909, which indicated that SH1909 was a novel and natural mutant of FCV. Interestingly, phylogenetic analysis based on LC-VP1 showed SH1909 could be clustered into an independent evolutionary branch with some Chinese isolates and was more distantly related to vaccine strains, indicating its potential to escape from vaccine-elicited immunity. According to the predicted B-cell epitopes of LC-VP1, amino acid mutation sites and positive selective sites, peptide C (aa sites: 445–460) and, peptide D (aa sites: 425–440) located in the hypervariable regions of LC-VP1, may result in the decreased immunological protection. Moreover, amino acid sites 439 and 449 may be responsible for the potential immune escape of SH1909. This study provides an important insight into genetic variations of FCV and vaccine development.

In this paper, the content and speciation of Cd, Cr, Pb, As, Cu, Zn and Ni in 87 groups of soil samples collected by grid method were determined by Inductively Coupled Plasma-Atomic Emission Spectrometry, to explore the speciation characteristics and ecological risk status of heavy metals in surface soil around Qinghai Lake. The results show that: Cd and Pb, compared with Cr, As, Cu, Zn and Ni, in the surface soil around Qinghai Lake are the most seriously polluted, Heavy metal speciation analysis showed that the proportion of weak acid soluble components of Cd was relatively high, with strong mobility and high environmental risk. The reducible components of Zn and Pb account for a relatively high proportion, while the oxidizable components of Cu, Cd and Pb account for a relatively high proportion. The high percentage of residual Cr, Ni and As indicates that they are relatively stable. The extractable content of Cd, Pb and Zn is relatively high, and their activities are strong, which have a great impact on the food chain. The results of Inductively Coupled Plasma-Atomic Emission Spectrometry and the ratio of secondary phase and primary phase showed that Cd and Pb had more serious ecological risk than other heavy metals. According to the results of soil speciation and ecological risk assessment of heavy metals around Chinese and foreign lakes, although the speciation characteristics of heavy metals are different, the pollution of Cd is relatively large. To sum up, heavy metal pollution in soil around Qinghai Lake area has occurred, and there is a serious ecological risk, so it is urgent to take corresponding monitoring and formulate treatment plan.

Duck short beak and dwarf syndrome (SBDS) is a viral infectious disease caused by novel duck parvovirus (NDPV). It has brought serious economic losses to the Chinese duck industry in recent years. Currently, there exists no effective vaccine against this disease. In this study, we developed an inactivated virus vaccine based on NDPV-DS15 for SBDS. Immune efficacy was evaluated in 112 ducks, which were randomly divided into vaccination, challenge control, vaccination-challenge, and blank control groups (n = 28 each). Clinical characteristics, antibodies, viral excretion, viremia, and pathological changes were monitored and analyzed. No morbidity or death was observed in the immunized ducks, which showed normal weight and good mental state. High levels of serum antibodies (OD450 nm: ~0.63) were detected in ducks immunized with inactivated vaccine at 7 days post-vaccination (dpv), and the amount of virus neutralizing antibodies increased from 1:23 to 1:28.5 from 7 dpv to 42 dpv. The anal swab, serum, and tissue viral load tests showed that vaccination could significantly inhibit the replication of NDPV in immunized ducks. Moreover, NDPV could not be isolated from the spleens of immunized or vaccination-challenged ducks. Our results show that the developed inactivated NDPV vaccine, administered in an oil emulsion adjuvant, possesses good immunogenicity and represents a potentially powerful tool for SBDS prevention and control.

The variability and the intrinsically high mutation rate of canine parvovirus type 2(CPV-2) increased the diversity of CPV-2 in canine populations. Since the first occurrence of CPV-2, three antigenic variants (2a, 2b and 2c) were detected and distributed worldwide. CPV-2c infection has been detected and increasingly reported in China. Here, a CPV-2c strain CPV-SH1516 was isolated and its complete genome sequence was first characterized. Compared with other CPV-2c isolates, CPV-2c isolates from China continued to evolve into divergent CPV-2c variants with specific unique amino acid substitutions under purifying selection. Emergence of CPV-2c isolates from China was driven by the unique gradual point mutations in key sites of VP2 rather than introduction from outside China. Combining sequence comparison with phylogenetic analysis based on the amino acid sequences of VP2, the vast majority of CPV-2c isolates from China formed a monophyletic cluster and CPV-SH1516 was a representative isolate of CPV-2c circulating in China. Overall, our study provides valuable insight into the evolutionary mechanism of CPV-2c.

During the period of the epidemiological surveillance of Feline calicivirus (FCV) isolates, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat in Shanghai, China. The complete genome sequence of SH1909 was also determined in this study. Sequence comparison and analysis show that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909 when compared with other FCV isolates. Phylogenetic analysis showed that SH1909 was assigned into a major evolutionary branch and distantly related to vaccine strains. These results indicated that SH1909 was a natural mutant of FCV circulating in China, which has the potential to escape from immunity of FCV vaccine.

In order to reveal the pollution characteristics and sources of heavy metals in surface soil of the region around the Qinghai Lake in Tibet Plateau, improve the prevention awareness and measures of local residents and urge the local government to implement necessary prevention and control measures, nine heavy metals (As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) in the surface soil samples of the region around the Qinghai Lake have been collected and analyzed. The methods such as statistic method, geo-accumulation index method, Nemerow index method, potential ecological risk index method, human health risk evaluation method and positive matrix factor analysis model (PMF) have been used to evaluate pollution characteristics and potential risks and analyze the sources of heavy metals. The results are shown below. First, the average contents of heavy metals (As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) in soil are 11.73 &plusmn, 3.78, 0.62 &plusmn, 1.40, 12.38 &plusmn, 3.68, 41.35 &plusmn, 13.01, 19.33 &plusmn, 8.92, 546.96 &plusmn, 159.28, 21.18 &plusmn, 7.04, 21.86 &plusmn, 6.61 and 63.51 &plusmn, 19.71 mg&middot, kg&minus, 1, respectively. Compared with the background values of the soil environment in Qinghai Province, it can be seen that there is an accumulation of these heavy metals to varying degrees, which is the most serious in Cd, Co and Pb. Second, the analysis of the geo-accumulation index and Nemerow index indicates that the heavy metals in the surface soil of the region around the Qinghai Lake have reached the level of heavy pollution, mainly polluted by Cd, and the accumulation of heavy metal pollution in the north, south, southwest and southeast of the study area is more serious. Third, the results of potential ecological risk evaluation show that the study area as a whole is classified as an area with high ecological risk, and Cd contributes the most to the overall risk. In fact, the heavy metals in the soil of the study area produce no noncarcinogenic and carcinogenic health risks to human health, and children and adults may be exposed to these risks by the mouth. Finally, the PMF results reveal that the sources of heavy metals in the study area include the sources of agricultural production, the nature, coal burning and transportation, with a contribution rate of 43.10%, 25.34%, 19.67% and 11.89%, respectively.

Rabbit haemorrhagic virus (RHDV) is a highly infectious and fatal pathogen to rabbits. Classic RHDV is mainly pathogenic to adult rabbits; however, it is not lethal to young rabbits. Since 2010, a RHDV variant has become prevalent in Europe and the Australian continent. This variant, which was named RHDV2 (GI.2/RHDVb), is highly infectious and fatal to both adult and young rabbits; moreover, its host range is broader than that of classic RHDV (GI.1/G1-6). In May 2020, the first RHDV2 case in China was reported. Here, we report and describe the first RHDV2 case in China. We amplified its complete genome sequence (named SC20-01 strain). The phylogenetic tree showed that the SC20-01 strain is mostly related to two strains isolated in Europe, which indicated that it might have spread from Europe to China. Moreover, Recombination Detection Program software showed that the SC20-01 strain is a G6/RHDV2 recombinant strain. Animal experiments showed that the SC20-01 strain is a highly fatal pathogen to specific-pathogen-free (SPF) rabbits and induces typical clinical symptoms of RHD. Our findings highlight RHDV2 emergence in China and its potential to spread widely. There is a need for more attention on the RHDV2 threat in China and the development of new diagnostic methods and vaccines for preventing the spread of RHDV2.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is currently an infectious agent for ducks. Studies on NDRV replication and pathogenesis have been hampered by the lack of an available reverse-genetics system. In this study, a plasmid-based reverse-genetics system that is free of helper viruses has been developed. In this system, 10 full-length gene segments of wild-type NDRV TH11 strain are transfected into BSR-T7/5 cells that express bacteriophage T7 RNA polymerase. Production of infectious virus was shown by the inoculation of cell lysate derived from transfected cells into 10-day-old duck embryos. The in vivo growth kinetics and infectivity of the recombinant strains were identical to those of the wild-type strain. These viruses grew well and were genetically stable both in vitro and in vivo. Altogether, these results show the successful production of an infectious clone for NDRV. The infectious clone reported will be further used to elucidate the mechanisms of host tropism, viral replication and pathogenesis, as well as immunological changes induced by NDRV.

Currently, both goose astrovirus (GoAstV) and goose-origin Newcastle disease virus (NDV) are widely infectious agents for goslings. There is no vaccine for GoAstV. Capsid protein can elicit a neutralizing antibody in human astroviruses (HAstV). Molecular analysis of the genomic region encoding the capsid protein(ORF2) of goose astrovirus has revealed that it contains neutralizing epitopes. Goose-origin NDV is also an infectious agent. A wide range of NDV strains exist that can be commonly used as vaccine vectors. In the present study, the fusion protein cleavage site RRQKR↓F in a backbone of the virulent goose-origin NDV SH-12 was changed into an avirulent motif GRQGR↓L. The modified goose-origin NDV recombinant vaccine virus expressing the Capsid protein (Cap) of GoAstV was generated as a bivalent vaccine using a reverse-genetics approach. The recombinant virus, rNDV/GoAstV-Cap, was attenuated and similar growth dynamics, cytopathic effects, and virus titers in vitro were maintained when compared to the LaSota strain. Expression of the GoAstV-Cap protein in rNDV/GoAstV-Cap infected cells was detected by an immunofluorescence assay and Western blotting. Goslings inoculated with rNDV/GoAstV-Cap showed no apparent signs of disease and induced GoAstV-Cap-specific immune responses and NDV-specific serum antibody responses to a LaSota vaccination control. Complete protection against a pathogenic GoAstV challenge and avelogenic NDV challenge was conferred. The results of the study suggested that rNDV/GoAstV-Cap viruses have the potential to be the safe, stable, and effective bivalent vaccines.

As ferrite nanoparticles (MFe2O4) have been widely used in biomedical field, their safety evaluation has been paid great attention both in vitro and in vivo. In this paper, the ultra-small MFe2O4 (M = Fe, Mn, Co) nanoparticles with the average size less than 5 nm were prepared by thermal decomposition method and characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HRTEM), and vibrating sample magnetometry (VSM). The toxic effect on human lung epithelial A549 cells treated with MFe2O4 nanoparticles at the different concentrations was evaluated in vitro. Mitochondrial function (MTT assay), cellular morphology, reactive oxygen species (ROS), superoxide dismutase (SOD), membrane lipid peroxidation (LPO) and glutathione (GSH) were assessed as toxicity end points. The results showed that the cytotoxicity of ultra-small MFe2O4 nanoparticles was in a dose- and time-dependent manner. Moreover, ultra-small Fe3O4 nanoparticles were found to be nearly non-toxic in A549 cells, while MnFe2O4 nanoparticles exhibited cytotoxic effects, and CoFe2O4 nanoparticles exerted higher cytotoxic effects among the three studied particles at the same concentration.

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48–72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro. Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

Cancer theranostics, the ability to simultaneously diagnose and treat cancer, has become one of the major driving forces in nanobiotechnology. In the present work, a multifunctional system, methylene blue-incorporated folate-functionalized Fe3O4/mesoporous silica core–shell magnetic nanoparticles (MNPs), for simultaneous near-infrared (NIR) fluorescence imaging and targeting photodynamic therapy (PDT) has been developed. The core Fe3O4 MNPs offers the function of magnetically guided drug delivery, the mesoporous silica shell acts as an efficient drug loaded carrier, the photosensitizer methylene blue (MB) exhibits excellent NIR fluorescence imaging and PDT efficiency, and the folic acid (FA) can effectively enhance the delivery of MB to the targeting cancer cells, which overexpress the folate receptor. The results indicated that the multifunctional system could effectively be used in NIR fluorescence imaging. Moreover, it exhibited a synergistic effect of magnetic targeted PDT of cancer under NIR laser irradiation. Thus, the multifunctional system is promising for simultaneous cancer diagnosis and therapy.

Novel duck reovirus (NDRV) disease emerged in China in 2011 and continues to cause high morbidity and about 5.0 to 50% mortality in ducklings. Currently there are no approved vaccines for the virus. This study aimed to assess the efficacy of a new vaccine created from the baculovirus and sigma C gene against NDRV. In this study, a recombinant baculovirus containing the sigma C gene was constructed, and the purified protein was used as a vaccine candidate in ducklings. The efficacy of sigma C vaccine was estimated according to humoral immune responses, cellular immune response and protection against NDRV challenge. The results showed that sigma C was highly expressed in Sf9 cells. Robust humoral and cellular immune responses were induced in all ducklings immunized with the recombinant sigma C protein. Moreover, 100% protection against lethal challenge with NDRV TH11 strain was observed. Summary, the recombinant sigma C protein could be utilized as a good candidate against NDRV infection.

Newcastle disease virus (NDV) causes a devastating poultry disease worldwide. Frequent outbreaks of NDV in chickens vaccinated with conventional live vaccines suggest a need to develop new vaccines that are genetically matched against circulating NDV strains, such as the genotype V virulent strains currently circulating in Mexico and Central America. In this study, a reverse genetics system was developed for the virulent NDV strain Mexico/01/10 strain and used to generate highly attenuated vaccine candidates by individually modifying the cleavage site sequence of fusion (F) protein. The cleavage site sequence of parental virus was individually changed to those of the avirulent NDV strain LaSota and other serotypes of avian paramyxoviruses (APMV serotype-2, -3, -4, -6, -7, -8, and -9). In general, these mutations affected cell-to-cell fusion activity in vitro and the efficiency of the F protein cleavage and made recombinant Mexico/01/10 (rMex) virus highly attenuated in chickens. When chickens were immunized with the rMex mutant viruses and challenged with the virulent parent virus, there was reduced challenge virus shedding compared to birds immunized with the heterologous vaccine strain LaSota. Among the vaccine candidates, rMex containing the cleavage site sequence of APMV-2 induced the highest neutralizing antibody titer and completely protected chickens from challenge virus shedding. These results show the role of the F protein cleavage site sequence of each APMV type in generating genotype V-matched vaccines and the efficacy of matched vaccine strains to provide better protection against NDV strains currently circulating in Mexico.

An unknown infectious disease in Cherry Valley ducks (Anas platyrhynchos domesticus) characterized by short beak and strong growth retardation occurred in China during 2015. The causative agent of this disease, tentatively named duck short beak and dwarfism syndrome (DSBDS), as well as the evolutionary relationships between this causative agent and all currently known avian-origin parvoviruses were clarified by virus isolation, transmission electron microscope (TEM) observation, analysis of nuclear acid type, (RT-)PCR identification, whole genome sequencing, and NS1 protein sequences-based phylogenetic analyses. The results indicated that the causative agent of DSBDS is closely related with the goose parvovirus-like virus, which is divergent from all currently known avian-origin parvoviruses and should be a novel duck parvovirus (NDPV).

We investigated the ability of the rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) to interact specifically with the minor structural protein VP10, using an in vivo cell-based CheckMate™ Mammalian Two-Hybrid System. RHDV VP60 protein interacted specifically with VP10. Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes. However, when VP60 was divided into two fragments, the interaction between VP60 and VP10 was impaired dramatically. These results will be helpful for further investigating the mechanism of RHDV particle assembly.

Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5′UTR and 2 in 3′UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5′/3′UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone. Electronic supplementary material The online version of this article (doi:10.1007/s11262-012-0767-0) contains supplementary material, which is available to authorized users.

Background Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR3 is involved in the recognition of double-stranded RNA. This study was designed to explore the relationship between duTLR3 and duck reovirus (DRV) infection. Methods In this study, we cloned and performed a molecular characterization of the complete sequence of Peking duck TLR3 (duTLR3). The expression level of duTLR3 was also determined, along with the relative levels of Mx and IFN-α mRNA after DRV infection. Results The duTLR3 gene is 2776-bp long and encodes an 895-amino-acid-long protein. Sequence analysis of the product revealed the complete transcript of Peking duck TLR3, including the 88-bp 5′UTR, the 2688-bp coding sequence (ORF), and the 76-bp 3′UTR and poly(A) tail. DuTLR3 was found to share a high amino acid sequence similarity with TLR3 from Jing ding duck (99.6 %), Muscovy duck (97.1 %) and chicken (86.3 %). Additionally, the tissue distribution of duTLR3 suggested that it was abundantly expressed in various tissues, especially in the trachea, esophagus and pancreatic gland. Duck reovirus (DRV) infection resulted in high mRNA expression levels of duTLR3 in the spleen, liver, lung and brain. Conclusion These results suggest that duTLR3 may play an important role in anti-viral defense mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0434-x) contains supplementary material, which is available to authorized users.

A Neisseria strain, Neisseria sp. AH-N10, was isolated from liver of a Chinese Peking duck and characterized using a number of phenotypic and genotypic approaches. Based on scanning electron microscopy examination, the isolated strain has the typical structure of Neisseria species. Sequence comparison of 16S rRNA gene and phylogenetic analysis suggest that Neisseria sp. AH-N10 is closely related to Neisseria canis, which was previously isolated from a human dog bite wound. Animal infection experiments demonstrated that the isolated Neisseria sp. AH-N10 is pathogenic in ducks and mice. The pathogenicity to humans and evolutional origin of this Neisseria strain should be further investigated.

Lysosome is a highly membrane-bound organelle which possesses a sequence of biological functions including protein degradation, cell signal transduction, plasma membrane repairment, homoeostasis, and autophagy. The lysosome contains more than 50 soluble acid hydrolases, and the acidification of lysosome is the most important biological characteristic. The integrity of lysosome is of vital importance. During the past few years, it was reported that the destabilization of lysosomal membrane can result in the release of lysosomal contents into cytosol and trigger cell death in a caspase-dependent or caspase-independent pathway. Lysosome functions at the late stage of autophagy and degrades cellular components delivered by autophagosome, which is a complicated process. The present article will summarize the current knowledge on the role of lysosome in cell death regulation and the underlying mechanisms.

It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. IMPORTANCE An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

Mitochondria as the critical powerhouse of eukaryotic cells play important roles in regulating cell survival or cell death. Under numerous stimuli, impaired mitochondria will generate massive reactive oxygen species (ROS) which participate in the regulation of vital signals and could even determine the fate of cancer cells. While the roles of mitochondria in radiation-induced autophagic cell death still need to be elucidated. Human cervical cancer cell line, Hela, was used, and the SOD2 silencing model (SOD2-Ri) was established by gene engineering. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assays, MitoTracker Green staining was used to detect mitochondrial mass, Western blot was used to detect protein expression, and the level of ROS, autophagy, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. Ionizing radiation (IR) could induce the increase of MAPLC3-II/MAPLC3-I ratio, Beclin1 expression, and ROS generation but decrease the MMP in a time-dependent manner. After SOD2 silencing, the IR-induced changes of ROS and the MMP were significantly enhanced. Moreover, both the radio sensitivity and autophagy increased in SOD2-Ri cells. Whereas, compared with SOD2-Ri, the opposite results were obtained by NAC, an antioxidant. After the treatment with the inhibitor of mitochondrial electron-transport chain complex II, thenoyltrifluoroacetone (TTFA), the rate of autophagy, ROS, and the total cell death induced by IR increased. In addition, the decrease of MMP was more obvious. However, these results were reversed by cyclosporine A (CsA). IR could induce ROS generation and mitochondrial damage which lead to autophagic cell death in Hela cells.

Nowadays, heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has become the target of antitumor drugs as a result of its close relationship with the occurrence and development, biological behavior, and prognosis of a tumor. Autophagy has attracted big attention recently for its paradoxical roles in cell survival and cell death, especially in the pathogenesis and treatment of cancer. Moreover, it has been verified that Hsp90 plays a role in autophagy via regulating the stability and activity of signaling proteins, and some Hsp90 inhibitors can induce autophagy. However, the underlying mechanisms for these important processes have not been clarified so far. In this study, we focus on the roles of Hsp90 in the regulation of autophagy, such as toll-like receptor (TLR)-mediated autophagy, Ulk1-mediated mitophagy, and chaperone-mediated autophagy (CMA). The roles of Hsp90 inhibitors in cancer therapy will also be elucidated.

Amino acid alignment of Peking duck, Anas platyrhynchos, Cairina moschata, Gallus, Homo sapiens, and Mus musculus TLR3. Alignment was performed using the CLUSTAW program and edited with BOXSHADE. Black boxes indicate amino acid identity; gray boxes indicates similarity (50 % threshold). LRR, leucine rich repeat. TIR, toll-interleukin 1 receptor signaling domain. The TLR3 sequences are shown for Muscovy duck (Cai), human (Hom), mouse (Mus), chicken (Gal), Jinding duck (Ana), and peking duck (Pek). (DOC 51Â kb)

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.

To the Editor: In 2011, an unidentified disease in Pekin ducks (Anas platyrhynchos) was reported in People’s Republic of China. The infection caused death in 40% of ducks of various age and 35%–40% mortality in different flocks. Clinical signs included unstable gait, weakness in legs, and diarrhea. At necropsy, large necrotic foci were observed in the spleens. All classical endemic and emerging viruses, such as duck enteritis virus, duck hepatitis virus, duck flavivirus, duck parvovirus, and avian influenza virus, could be excluded as the causative agent by PCR and serologic methods. To identify the cause of the disease, we tested tissue from affected ducks and subsequently isolated a novel duck-pathogenic orthoreovirus from the livers of affected ducks. Avian orthoreoviruses (ARVs) belong to the family Reoviridae, genus Orthoreovirus (1). The virions are nonenveloped, with icosahedral symmetry and a double capsid containing 10 double-stranded RNA segments that can be separated by polyacrylamide gel electrophoresis into 3 size classes: large (L1–L3), medium (M1–M3), and small (S1–S4) (2,3). ARVs cause a range of diseases in chicken, including viral arthritis/tenosynovitis, and are associated with respiratory disease, enteric disease, inclusion body hepatitis, hydropericardium, runting stunting syndrome, malabsorption syndrome, and sudden death. ARVs also have been isolated from the Muscovy duck (Cairina moschata). Muscovy duck reovirus infection caused illness in 30% and death in 20% of ducks on poultry farms in Israel (4). In China, reovirus infection has been reported in Muscovy ducklings, with a resulting death rate of 10%–30% since 1997 (5). The isolated reovirus was highly pathogenic to 1-day-old Muscovy ducklings by experimental infection. However, the Muscovy duck reovirus isolate was nonpathogenic for Pekin ducks when inoculated subcutaneously (4). Since 2007, three isolates of orthoreovirus were confirmed in Pekin ducks from several duck farms in China. However, experiment infection with the isolates did not cause death (6). In 2011, farmers and veterinarians in China reported to the Animal Health Services and National Research Institutes an unidentified disease in ducks that spread rapidly around the county. We conducted further investigation to identify the causative agent of this disease. The diseased ducks showed depression and leg weakness. Large necrotic foci were observed in the spleens of the dead ducks. Histopathologic examination showed necrotic foci and granulomas in the spleen. Focal hepatic necrosis and proliferation of bile ducts were seen in the liver. Virus isolation from liver homogenate was conducted in duck embryo fibroblast cultures. At 48 hours after infection, a strong cytopathic effect was observed, including syncytium formation. All duck embryos experimentally infected with the isolate died within 48–72 hours after infection. The dead embryos showed swollen livers with petechial hemorrhages. Spherical, spiked virus particles, consistent with those of members of the family Reoviridae, were observed by electron microscopy. As reported (7), the diameter of the particles was ≈85 nm (Figure A1). The RNA extracted from DRV-infected duck embryo fibroblast cultures showed 10 dsRNA segments in 3 size classes (L1–3, M1–3, and S1–4) on polyacrylamide gel electrophoresis. The isolate was designated as novel duck reovirus, DRV-TH11. The pathogenicity of DRV-TH11 was tested by infecting 10-day-old Pekin ducks subcutaneously at a dose of 4 × 104.5 50% tissue culture infective dose. Experimental infection caused death on day 3 after infection. The clinical signs and histopathologic examination show the same features as the naturally infected ducks. For phylogenetic analyses, the S2 gene was amplified by reverse transcription PCR with avian reovirus–specific primers. The complete sequence of the S2 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JQ664689","term_id":"387862751","term_text":"JQ664689"}}JQ664689) was aligned with 30 published orthoreovirus sequences, including data on all 3 newly obtained sequences from Pekin duck reovirus in China in 2008 and 2011. Phylogenetic relationship was assessed by using the neighbor-joining method based on a Tamura 3-parameter model and bootstrap analysis (1,000 replicates) as implemented in MEGA5 (8). The phylogenetic tree shows that the complete sequence of S2 gene is distinct but clusters closely with sequences from all 3 Pekin duck isolates within the ARVs serogroup, which suggests that the novel virus is an ARV-like virus within the genus Orthoreovirus (Figure). Figure Phylogenetic relationship between DRV-TH11 isolate and orthoreovirus of the avian orthoreovirus (ARV) and mammalian orthoreovirus (MRV). ARV includes chicken reovirus, Muscovy duck reovirus, and Pekin duck reovirus. GenBank accession numbers of the sequences ... In summary, we isolated a novel duck-pathogenic orthoreovirus from the liver of affected Pekin ducks. The regression test in its natural host animal showed that the newly isolated virus caused their deaths. This finding highlights the need to prevent and control this highly transmissible infectious agent. Further study is needed to determine what role the newly isolated DRV played in the 2011 outbreaks on many of the duck farms in China.

Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1., This study was funded by grants from the Chinese Natural Sciences Foundation (30870114), National Key Laboratory of Veterinary Biotechnology (SKLVBF201004), the Special Fund for Agro-scientific Research in the Public Interest (No.201003012), the National advanced Technology Research and Development Program of China (863 Program) (No. 2011AA10A200).