Generation and characterization of a nanobody against the avian influenza virus H7 subtype.

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 µg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Prevalence and genomic-based antimicrobial resistance analysis of Avibacterium paragallinarum isolates in Guangdong Province, China.

Infectious coryza (IC) is an acute infectious respiratory disease in chickens that is caused by Avibacterium paragallinarum (A. paragallinarum). A. paragallinarum poses a significant threat to poultry health due to its virulence and multidrug resistance. This study isolated and identified 21 A. paragallinarum isolates from Guangdong between 2022 and 2023. Biochemical tests showed that 100% of A. paragallinarum isolates fermented glucose but did not ferment alginate and galactose, and only YZ18 was nicotinamide adenine dinucleotide independent. To determine the genetic relatedness between these isolates and NCBI reference strains, whole-genome-based phylogenetic analysis was employed. In addition, analysis of the 2,000 bp-length hmtp210 gene showed that the hmtp210 gene was strongly associated with A. paragallinarum serotypes. Meanwhile, a PCR assay for serotyping A. paragallinarum was developed based on the hmtp210 gene, this assay has high sensitivity and specificity. The antimicrobial susceptibility of isolates was assessed using the disk diffusion method. The antibiotic resistance genes of isolates were analyzed using the genomic method. Phenotypic resistance to ampicillin (95.2%), streptomycin (95.2%), methotrexate-sulfamethoxazole (90.5%), and tetracycline (85.7%) was most frequent among the isolates. All of the isolates exhibited resistance to multiple drugs, and furthermore, the isolates possessed a collective total of 14 genes associated with antibiotic resistance. This study will contribute to advancing our knowledge of A. paragallinarum antibiotic resistance and provide a scientific basis for the prophylaxis and treatment of IC, and the subsequent rational design of potential clinical therapeutics.

Identification of Three Campylobacter Lysins and Enhancement of Their Anti-Escherichia coli Efficacy Using Colicin-Based Translocation and Receptor-Binding Domain Fusion.

The emergence of multidrug-resistant Escherichia coli, which poses a major threat to public health, has motivated the development of numerous alternative antimicrobials. Lysins are bacteriophage- and bacterium-derived peptidoglycan hydrolases that represent a new antibiotic treatment targeting bacterial cell walls. However, the bactericidal effect of native lysins on Gram-negative bacteria is restricted by the presence of an outer membrane. Here, we first evaluated the antibacterial activity of three Campylobacter-derived lysins (Clysins) against E. coli. To improve their transmembrane ability and antibacterial activities, six engineered Clysins were constructed by fusing with the translocation and receptor-binding (TRB) domains from two types of colicins (colicin A [TRBA] and colicin K [TRBK]), and their biological activities were determined. Notably, engineered lysin TRBK-Cly02 exhibited the highest bactericidal activity against the E. coli BL21 strain, with a reduction of 6.22 ± 0.34 log units of cells at a concentration of 60.1 µg/mL, and formed an observable inhibition zone even at a dose of 6.01 µg. Moreover, TRBK-Cly02 killed E. coli dose dependently and exhibited the strongest bactericidal activity at pH 6. It also exhibited potential bioactivity against multidrug-resistant E. coli clinical isolates. In summary, this study identified three lysins from Campylobacter strains against E. coli, and the enhancement of their antibacterial activities by TRB domains fusion may allow them to be developed as potential alternatives to antibiotics. IMPORTANCE Three lysins from Campylobacter, namely, Clysins, were investigated, and their antibacterial activities against E. coli were determined for the first time. To overcome the restriction of the outer membrane of Gram-negative bacteria, we combined the TRB domains of colicins with these Clysins. Moreover, we discovered that the Clysins fused with TRB domains from colicin K (TRBK) killed E. coli more effectively, and this provides a new foundation for the development of novel bioengineered lysins by employing TRBK constructs that target outer membrane receptor/transport systems. One of the designed lysins, TRBK-Cly02, exhibited potent bactericidal efficacy against E. coli strains and may be used for control of multidrug-resistant clinical isolates. The results suggest that TRBK-Cly02 can be considered a potential antibacterial agent against pathogenic E. coli.

Highly Prevalent Multidrug-Resistant Campylobacter spp. Isolated From a Yellow-Feathered Broiler Slaughterhouse in South China.

The purpose of this study was to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Campylobacter spp. along the yellow-feathered broiler slaughtering line in Southern China from December 2018 to June 2019. A total of 157 Campylobacter spp. isolates were identified from 1,102 samples (including 53.6% (75/140) of live chicken anal swab samples, 27.5% (44/160) of defeathering samples, 18.1% (29/160) of evisceration samples, 2.1% (3/140) of washing samples, 1.4% (2/140) of chilling samples, and 1.1% (4/362) of environmental samples). The prevalence of Campylobacter spp. was 14.2%, including 43.9% Campylobacter jejuni, 53.5% Campylobacter coli, and 2.5% other Campylobacter species. The highest antimicrobial resistance rate was found to be against sulfamethoxazole (138/157, 87.9%), and 90.4% (142/157) of the isolates were multidrug resistant (MDR). Examination of resistance-related genes revealed the double base mutated Thr-86-Ile, which informed ACA-TTA, with an Arg-79-Lys substitution in gyrA. Eleven virulence-associated genes (cadF, cdtA, cdtB, ciaB, flaA, imaA, dnaJ, plaA, virB11, racR, and cdtC) were also detected by a polymerase chain reaction (PCR) analysis, and cadF (81.5%) was the most prevalent. Based on an analysis of pulsed-field gel electrophoresis (PFGE) results, we found that Campylobacter spp. could be cross-contaminated throughout the entire slaughtering line. These results show that it is imperative to study the Campylobacter spp. from the yellow-feathered broiler along the slaughtering line in China to develop preventative and treatment measures for the poultry industry, as well as food safety and public health.

Genetic characterization of an H5N6 avian influenza virus with multiple origins from a chicken in southern China, October 2019.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China. RESULTS: Nucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified. CONCLUSIONS: Our results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health.

Effect of the Recombinant Adenovirus-Mediated HIF-1 Alpha on the Expression of VEGF in the Hypoxic Brain Microvascular Endothelial Cells of Rats.

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated HIF-1 alpha (HIF-1α) on the expression of vascular endothelial growth factor (VEGFA) and HIF-1α in hypoxic brain microvascular endothelial cells (BMEC) in rats. METHODS: Primary cultured rat BMEC in vitro were treated without or with either recombinant adenovirus-mediated hypoxia-inducible factor-1 alpha (AdHIF-1α) or recombinant adenovirus empty vector (Ad) in the presence of CoCl2 (simulating hypoxia conditions), or were grown under normoxia conditions. The expression of VEGFA and HIF-1α was analyzed at 12h, 24h, 48h and 72h incubation time, respectively. We also accessed a GEO dataset of stroke to analyze in vivo the alteration of HIF-1α and VEGFA expression, and the correlations between HIF-1α, VEGFA and CD31 mRNA levels in vascular vessels after stroke. RESULTS: VEGFA and HIF-1α expression were significantly higher in at each time point in the AdHIF-1α than other groups (p<0.05), whereas the Ad group and hypoxia group, showed no statistically significant difference (p>0.05). Moreover, VEGFA and HIF-1α levels were significantly higher in BMEC under hypoxia conditions than normoxia conditions (p <0.05). Both HIF-1α and VEGFA expression significantly increased after stroke in vivo with 1.30 and 1.57 fold-change in log2, respectively. There were significantly positive associations between HIF-1α, VEGFA and CD31 mRNA levels in vivo after stroke. CONCLUSION: Hypoxia-induced HIF-1α and VEGFA expression in vascular vessels, and recombinant AdHIF-1α could up-regulate VEGFA, and enhance HIF-1ααlevels in BMEC in vitro, which may play an important role in the recovery of stroke.

Genetic, Molecular, and Pathogenic Characterization of the H9N2 Avian Influenza Viruses Currently Circulating in South China.

The prevalence and variation of the H9N2 avian influenza virus (AIV) pose a threat to public health. A total of eight viruses isolated from farmed poultry in South China during 2017-2018 were selected as representative strains for further systematic study. Phylogenetic analyses indicated that these prevalent viruses belong to the Y280-like lineage and that the internal genes are highly similar to those of recently circulating human H7N9 viruses. The receptor-binding assay showed that most of the H9N2 isolates preferentially bound to the human-like receptor, increasing the risk of them crossing the species barrier and causing human infection. Our in vitro, multi-step growth curve results indicate these viruses can effectively replicate in mammalian cells. Infection in mice showed that three viruses effectively replicated in the lung of mice. Infection in swine revealed that the viruses readily replicated in the upper respiratory tract of pig and effectively induced viral shedding. Our findings suggested that the H9N2 AIVs circulating in poultry recently acquired an enhanced ability to transmit from avian to mammalians, including humans. Based on our findings, we propose that it is essential to strengthen the efforts to surveil and test the pathogenicity of H9N2 AIVs.

Influenza D Virus Infection in Feral Swine Populations, United States.

Influenza D virus (IDV) has been identified in domestic cattle, swine, camelid, and small ruminant populations across North America, Europe, Asia, South America, and Africa. Our study investigated seroprevalence and transmissibility of IDV in feral swine. During 2012-2013, we evaluated feral swine populations in 4 US states; of 256 swine tested, 57 (19.1%) were IDV seropositive. Among 96 archived influenza A virus-seropositive feral swine samples collected from 16 US states during 2010-2013, 41 (42.7%) were IDV seropositive. Infection studies demonstrated that IDV-inoculated feral swine shed virus 3-5 days postinoculation and seroconverted at 21 days postinoculation; 50% of in-contact naive feral swine shed virus, seroconverted, or both. Immunohistochemical staining showed viral antigen within epithelial cells of the respiratory tract, including trachea, soft palate, and lungs. Our findings suggest that feral swine might serve an important role in the ecology of IDV.

The genetic and phylogenetic analysis of a highly pathogenic influenza A H5N6 virus from a heron, southern China, 2013.

The H5N6 highly pathogenic avian influenza viruses (HPAIVs) have circulated within poultry in China since 2013. Infections of H5N6 in wild birds were reported since 2014. In order to investigate the infection history of H5N6 in wild birds, we conducted a retrospective analysis of H5 positive wild bird samples collected in 2013, the year H5N6 was discovered in poultry. We isolated a new HPAI H5N6 virus from a dead heron collected in 2013. The virus had high identity in all eight gene sequences to those collected from poultry in 2013 (for example, A/chicken/Shenzhen/1845/2013, 99.1%-99.7%). Our findings revealed that H5N6 HPAIVs infected wild birds in southern China since the emergence of H5N6 in poultry in 2013. The co-circulation of H5N6 between wild birds and poultry is very close, and should raise our attention more.

Up-regulated expression of phospholipase C, ß1 is associated with tumor cell proliferation and poor prognosis in hepatocellular carcinoma.

BACKGROUND: Phospholipase C, ß1 (PLCB1) plays critical roles in intracellular transduction and regulating signal activation which are important to tumorigenesis. However, the mechanism of PLCB1 in hepatocellular carcinoma (HCC) is still unknown. This study aims to investigate whether its expression is associated with the clinicopathological parameters and prognosis of the patients with HCC. METHODS: Immunohistochemistry on two tissue microarrays containing 141 cases of HCC tissues and adjacent non-tumorous tissues were performed to analyze the correlation between PLCB1 expression and clinicopathological features. Kaplan-Meier analysis and Cox multivariate analysis were performed to determine the PLCB1 expression in HCC prognosis. Furthermore, effects of PLCB1 on proliferation of HCC cells were explored using a colony formation assay and apoptosis assay. RESULTS: We identified that PLCB1 expression was significantly higher in tumor tissues than that in adjacent non-tumorous tissues and associated with advanced tumor stage. Kaplan-Meier survival analysis showed that patients with PLCB1-positive tumors had poorer survival than the patients with PLCB1-negative tumors. In multivariate analyses, PLCB1 expression was an independent prognostic factor. Moreover, overexpression of PLCB1 in HCC cells promoted cell proliferation and inhibited apoptosis, while knocking down PLCB1 reduced cell viability in vitro. Further investigation found that activation of ERK signaling might involve in PLCB1-mediated cell growth. CONCLUSION: Our study suggests that PLCB1 promotes the progression of HCC and can be served as an independent prognostic factor and a promising therapeutic target in HCC.

D701N mutation in the PB2 protein contributes to the pathogenicity of H5N1 avian influenza viruses but not transmissibility in guinea pigs.

H5N1 highly pathogenic avian influenza virus (HPAIV) of clade 2.3.2 has been circulating in waterfowl in Southern China since 2003. Our previous studies showed that certain H5N1 HPAIV isolates within clade 2.3.2 from Southern China had high pathogenicity in different birds. Guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts. However, few studies have reported pathogenicity and transmissibility of H5N1 HPAIVs of this clade in guinea pigs. In this study, we selected an H5N1 HPAIV isolate, A/duck/Guangdong/357/2008, to investigate the pathogenicity and transmissibility of the virus in guinea pigs. The virus had high pathogenicity in mice; additionally, it only replicated in some tissues of the guinea pigs without production of clinical signs, but was transmissible among guinea pigs. Interestingly, virus isolates from co-caged guinea pigs had the D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs but did not demonstrate enhanced the transmissibility among guinea pigs. These findings indicate the transmission of the H5N1 virus between mammals could induce virus mutations, and the mutant viruses might have higher pathogenicity in mammals without higher transmissibility. Therefore, the continued evaluation of the pathogenicity and transmissibility of avian influenza virus (AIVs) in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains.

Antimicrobial susceptibility and molecular typing of salmonella agona isolated from humans and other sources.

Salmonella enterica serotype Agona (Salmonella Agona) has been among the top 10 serotypes that cause human diarrheal diseases in China. A total of 95 Salmonella Agona (67 from humans, and 28 from animals, food of animal origins, and environmental sources) recovered in Shanghai, China from 2005 to 2011 were subjected to antimicrobial susceptibility testing and molecular subtyping using pulsed-field gel electrophoresis (PFGE). Approximately 68.4% of the Salmonella Agona isolates were pansusceptible to 15 antimicrobial agents tested, and 4 isolates (4.21%) were resistant to at least 3 antimicrobials. PFGE analysis resulted in 41 unique patterns, of which 4 major PFGE patterns (X3, X4, X5, and X6) were grouped together at 96.1% similarity. Isolates of the four patterns included those from food (pork, beef, and chicken) and humans. Our findings showed that the same clones of Salmonella Agona were recovered from human patients and food, and that food of animal origin was potentially a major vehicle of Salmonella Agona in human salmonellosis in Shanghai.

An analysis of China's physician salary payment system.

Physician payment system (PPS) is a principal incentive system to motivate doctors to provide excellent care for patients. During the past decade, physician remuneration in China has not been in proportional to physician's average work load and massive responsibilities. This paper reviewed the constitution of the PPS in China, and further discussed the problems and issues to be addressed with respect to pay for performance. Our study indicated that the lower basic salary and bonus distribution tied to "profits" was the major contributor to the physician's profit-driven incentive and the potential cause for the speedy growth of health expenditures. We recommend that government funding to hospitals should be increased to fully cover physicians' basic salary, a flexible human resource and talent management mechanism needs to be established that severs personal interest between physicians and hospitals, and modern performance assessment and multiplexed payment systems should be piloted to encourage physicians to get the more legitimate compensation.

Complete genome sequence of an H5N2 avian influenza virus isolated from a parrot in southern China.

We report the complete genome sequence of an H5N2 avian influenza virus (AIV) that was first isolated from a parrot in Guangdong in southern China in 2004. Genomic sequence and phylogenetic analyses showed that it was highly homologous with the North American H5N2 viruses and all eight genes of this virus belonged to the North American gene lineage. These data will help in the investigation of the epidemiology and host range of AIVs in southern China.

Complete genomic sequence of an H5N1 influenza virus from a parrot in southern China.

An H5N1 avian influenza virus (AIV) designated A/Parrot/Guangdong/C99/2005 (H5N1) was first isolated from a sick parrot in Guangdong in southern China in 2005. The complete genome of this strain was analyzed. Genome sequence analysis showed that all 8 gene segments of the virus nucleotide had 99.0% homology to A/chicken/Henan/12/2004 (H5N1). Phylogenetic analysis demonstrated that all 8 gene segments of the virus were derived from the Eurasian lineage. The availability of genome sequences is useful to investigate the host range and genetic evolution of the H5N1 avian influenza virus in Southern China.

Complete genome sequence of an H10N8 avian influenza virus isolated from a live bird market in Southern China.

An H10N8 avian influenza virus (AIV), designated A/Duck/Guangdong/E1/2012 (H10N8), was isolated from a duck in January 2012. This is first report that this subtype of AIV was isolated from a live bird market (LBM) in Guangdong Province in southern China. Furthermore, the complete genome of this strain was analyzed. The availability of genome sequences is helpful to further investigations of epidemiology and molecular characteristics of AIV in southern China.