Genome cloning and genetic evolution analysis of eight duck-sourced novel goose parvovirus strains in China in 2023.

Introduction: There are three major categories of waterfowl parvoviruses, namely goose parvovirus (GPV), Muscovy duck parvovirus, and novel goose parvovirus (NGPV). NGPV can infect both Cherry Valley ducks and mule ducks, resulting in short beaks and dwarfism syndrome, and the incidence of short beaks and dwarfism syndrome rises annually, posing a significant threat to the waterfowl breeding and the animal husbandry. Therefore, clarifying the biological characteristics and genetic evolution of NGPV is very important for the prevention and control of NGPV. Methods: Ducks with short beaks and dwarfism syndrome from Shandong and Henan Province were investigated by dissection and the tissue samples were collected for study. The NGPV genome was amplified by PCR, and the genome was analyzed for genetic evolution. Results: Eight strains of NGPV were isolated, which were designated as HZ0512, HZ0527, HZ0714, HZ0723, HZ0726, HZ0811, HZ0815, and HN0403. The nucleotide homology among these strains ranged from 99.9% to 100%. The eight strains, along with other NGPVs, belong to GPV. The eight strains showed a 92.5%-98.9% nucleotide homology with the classical GPV, while a 96.0%-99.9% homology with NGPV.Therefore, it can be deduced that there have been no major mutations of NGPV in Shandong and Henan provinces in recent years. Discussion: This study lays a theoretical foundation for further studying the genetic evolution and pathogenicity of NGPV, thereby facilitating the prevention and control of NGPV.

Effects of different laying periods on airborne bacterial diversity and antibiotic resistance genes in layer hen houses.

Poultry farms are a complex environment for close contact between humans and animals. Accumulating evidence has indicated that pathogens and drug resistance genes in chicken houses may pose a serious threat to public health and economic concerns. However, insufficient knowledge of the indoor aerosol microbiome and resistome profiles of layer hen houses hampers the understanding of their health effects. Environmental surveillance of antibiotic resistance may contribute to a better understanding and management of the human exposure risk of bioaerosols under the environmental conditions of chicken houses. In addition, the chicken house has a long operation cycle, and the bacterial diversity and antibiotic resistance genes of aerosols in different periods may be different. In this study, air samples were collected from 18 chicken houses on three farms, including the early laying period (EL), peak laying period (PL), and late laying period (LL). 16S rRNA gene sequencing and metagenomics were used to study the composition of the bacteria and resistome in aerosols of layer hen houses and the results showed that they varied with laying period. The highest alpha diversity of bacteria was observed in PL bioaerosols. The dominant bacterial phyla included Firmicutes, Bacteroidetes and Proteobacteria. Three potential pathogenic bacterial genera (Bacteroides, Corynebacterium and Fusobacterium) were found. The most abundant ARG type was aminoglycosides in all laying periods. In total, 22 possible ARG host genera were detected. ARG subtypes and abundance were both higher in LL. Network analysis also showed higher co-occurrence patterns between the bacteria and resistome in bioaerosols. The laying period plays an important role in the bacterial community and resistome in layer house aerosols.

Real-Time Reverse Transcription Recombinase-Aided Amplification Assay for Rapid Amplification of the N Gene of SARS-CoV-2.

COVID-19 was officially declared a global pandemic disease on 11 March 2020, with severe implications for healthcare systems, economic activity, and human life worldwide. Fast and sensitive amplification of the severe acute respiratory syndrome virus 2 (SARS-CoV-2) nucleic acids is critical for controlling the spread of this disease. Here, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay, targeting conserved positions in the nucleocapsid protein gene (N gene) of SARS-CoV-2, was successfully established for SARS-CoV-2. The assay was specific to SARS-CoV-2, and there was no cross-reaction with other important viruses. The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability. Furthermore, 100% concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 72 clinical specimens. Further linear regression analysis indicated a significant correlation between the real-time RT-RAA and RT-qPCR assays with an R2 value of 0.8149 (p < 0.0001). In addition, the amplicons of the real-time RT-RAA assay could be directly visualized by a portable blue light instrument, making it suitable for the rapid amplification of SARS-CoV-2 in resource-limited settings. Therefore, the real-time RT-RAA method allows the specific, sensitive, simple, rapid, and reliable detection of SARS-CoV-2.

Pathogenicity and Transmissibility of Goose-Origin H5N6 Avian Influenza Virus Clade 2.3.4.4h in Mammals.

Throughout the last decade, H5N6 avian influenza viruses (AIVs) circulating in poultry and infecting humans have caused increasing global concerns that they might become a pandemic threat to global health. Since AIVs could occasionally cause asymptomatic infections in geese, virus monitoring in such a host should be critical to the control of cross-species infection. In addition, previous studies showed that clade 2.3.4.4h H5N6 AIVs could infect mammals without adaptation. However, the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals remain unknown. In this study, two H5N6 AIVs were isolated from a domestic chicken (A/chicken/Hebei CK05/2019 (H5N6)) and a goose (A/goose/Hebei/GD07/2019(H5N6)). This study is the first to evaluate the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals by comparison with chicken-origin 2.3.4.4h H5N6 AIVs. The CK05 virus had an affinity for α-2,3-receptors, while the GD07 virus had an affinity for both α-2,3-and α-2,6-receptors. The GD07 virus had a higher replication capacity in vitro and more severe pathogenicity in mice than the CK05 virus. The CK05 virus could not be transmitted effectively among guinea pigs, whereas the GD07 virus could be transmitted through direct contact among guinea pigs. The results of this study indicated the potential health threat of clade 2.3.4.4h H5N6 AIVs to mammals and emphasized the importance of continuous monitoring of H5N6 AIVs, especially in waterfowl.

Pathogenicity and Transmissibility of Clade 2.3.4.4h H5N6 Avian Influenza Viruses in Mammals.

Avian influenza viruses (AIVs) have the potential for cross-species transmission and pandemics. In recent years, clade 2.3.4.4 H5N6 AIVs are prevalent in domestic poultry, posing a threat to the domestic poultry industry and public health. In this study, two strains of H5N6 AIVs were isolated from chickens in Hebei, China, in 2019: A/chicken/Hebei/HB1907/2019(H5N6) and A/chicken/Hebei/HB1905/2019(H5N6). Phylogenetic analysis showed that both viral HA genes clustered in the 2.3.4.4h clade. Receptor binding analysis showed that the HB1905 strain preferentially binds to α-2,3-linked sialic acid (SA) receptors, while the HB1907 strain preferentially binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors. During early infection, the HB1907 strain is highly replicable in MDCK cells, more so than the HB1905 strain. Pathogenicity assays in mice showed that both viruses could replicate in the lungs without prior adaptation, with HB1907 being more highly pathogenic in mice than the HB1905 strain. Significantly, both the HB1905 and HB1907 strains can be transmitted through direct contact among guinea pigs, but the transmission efficiency of the HB1907 strain through contact between guinea pigs is much greater than that of the HB1905 strain. These results strengthen the need for ongoing surveillance and early warning of H5N6 AIVs in poultry.

Epidemiological Investigation of Porcine Pseudorabies Virus in Hebei Province, China, 2017-2018.

Pseudorabies (PR) is a serious disease affecting the pig industry in China, and it is very important to understand the epidemiology of pseudorabies virus (PRV). In the present study, 693 clinical samples were collected from Bartha-K61 vaccinated pigs with symptoms of suspected PRV infection between January 2017 and December 2018. All cases were referred for full clinical autopsy with detailed examination of histopathological examination, virus isolation and genetic evolution analysis of the PRV glycoprotein E (gE) gene. In addition, PRV gE antibodies in 3,449 serum samples were detected by the enzyme-linked immunosorbent assay (ELISA). The clinical data revealed that abortion and stillbirth are the most frequent appearances in pregnant sows of those cases. Histopathological examination exhibited a variety of pathological lesions, such as lobar pneumonia, hepatitis, lymphadenitis, nephritis, and typical nonsuppurative encephalitis. A total of 248 cases tested positive for the PRV gE gene. 11 PRV variants were isolated and confirmed by gE gene sequencing and phylogenetic analysis. These strains had 97.1%-100.0% nucleotide homology with the PRV reference strains. Notably, the isolated strains were highly homologous and clustered in the same branch as HSD-1/2019, which caused human acute encephalitis. Serological tests showed that the positive rate of PRV gE antibody in the 3449 serum samples collected from the Hebei Province was 46.27%. In conclusion, PRV variant strains Are high prevalence in the Hebei Province, which not only causes huge economic losses to the breeding industry but also potentially poses a threat to public health.

Emergence and genomic analysis of a novel ostrich-origin GPV-related parvovirus in China.

In recent years, ostrich disease characterized by paralysis and diarrhea has been circulating in some regions of China, causing huge economic losses to the ostrich breeding industry. In our study, clinical samples from diseased ostriches were collected, and only parvovirus was detected. The virus distribution analysis by histopathology and quantitative real-time PCR assays indicated that the virus had a wide range of tissue tropisms. The full-length genome of the ostrich parvovirus (OsPV) was sequenced and comprehensively analyzed. Interestingly, the phylogenetic and alignment results indicated that the OsPV and the goose parvovirus (GPV) form a separate branch. In contrast to GPV strains, OsPV showed 2 new 14 nucleotide deletions in the inverted terminal repeat (ITR) region. Furthermore, recombination analysis indicated that OsPV was a recombination strain between the vaccine strain SYG61v and the virulent strain B strain, with the major parent of OsPV as the SYG61v strain and the minor parent as the B strain. The 14 nucleotide deletions in the ITR region as well as recombination may be some of the reasons for the cross-species transmission of parvovirus from goose to ostrich. The above data will contribute to a better understanding of the molecular biology of the novel OsPV and help to develop the vaccine candidate strain.

Anti-Influenza Effect and Mechanisms of Lentinan in an ICR Mouse Model.

Influenza virus is a serious threat to global human health and public health security. There is an urgent need to develop new anti-influenza drugs. Lentinan (LNT) has attracted increasing attention in recent years. As potential protective agent, LNT has been shown to have anti-tumor, anti-inflammatory, and antiviral properties. However, there has been no further research into the anti-influenza action of lentinan in vivo, and the mechanism is still not fully understood. In this study, the anti-influenza effect and mechanism of Lentinan were studied in the Institute of Cancer Research (ICR) mouse model. The results showed that Lentinan had a high degree of protection in mice against infection with influenza A virus, delayed the emergence of clinical manifestations, improved the survival rate of mice, significantly prolonged the middle survival days, attenuated the weight loss, and reduced the lung coefficient of mice. It alleviated the pathological damage of mice infected with the influenza virus and improved blood indices. Lentinan treatment considerably inhibited inflammatory cytokine (TNF-α, IL-1ß, IL-4, IL-5, IL-6) levels in the serum and lung and improved IFN-γ cytokine levels, which reduced cytokine storms caused by influenza virus infection. The underlying mechanisms of action involved Lentinan inhibiting the inflammatory response by regulating the TLR4/MyD88 signaling pathway. This study provides a foundation for the clinical application of Lentinan, and provides new insight into the development of novel immunomodulators.

The Distribution Characteristics of Aerosol Bacteria in Different Types of Pig Houses.

With the development of modern pig raising technology, the increasing density of animals in pig houses leads to the accumulation of microbial aerosols in pig houses. It is an important prerequisite to grasp the characteristics of bacteria in aerosols in different pig houses to solve the problems of air pollution and disease prevention and control in different pig houses. This work investigated the effects of growth stages on bacterial aerosol concentrations and bacterial communities in pig houses. Three traditional types of closed pig houses were studied: farrowing (FAR) houses, weaning (WEA) houses, and fattening (FAT) houses. The Andersen six-stage sampler and high-volume air sampler were used to assess the concentrations and size distribution of airborne bacteria, and 16S rRNA gene sequencing was used to identify the bacterial communities. We found that the airborne bacterial concentration, community richness, and diversity index increased with pig age. We found that Acinetobacter, Erysipelothrix, Streptococcus, Moraxella, and Aerococcus in the microbial aerosols of pig houses have the potential risk of causing disease. These differences lead us to believe that disinfection strategies for pig houses should involve a situational focus on environmental aerosol composition on a case-by-case basis.

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R.

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (2VEPREQFFQDLLSAV16) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

The Indirect ELISA and Monoclonal Antibody against African Swine Fever Virus p17 Revealed Efficient Detection and Application Prospects.

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (3TETSPLLSH11) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Risk of Environmental Exposure to H7N9 Influenza Virus via Airborne and Surface Routes in a Live Poultry Market in Hebei, China.

Environmental transmission of viruses to humans has become an early warning for potential epidemic outbreaks, such as SARS-CoV-2 and influenza virus outbreaks. Recently, an H7N9 virus, A/environment/Hebei/621/2019 (H7N9), was isolated by environmental swabs from a live poultry market in Hebei, China. We found that this isolate could be transmitted by direct contact and aerosol in mammals. More importantly, after 5 passages in mice, the virus acquired two adaptive mutations, PB1-H115Q and B2-E627K, exhibiting increased virulence and aerosol transmissibility. These results suggest that this H7N9 virus might potentially be transmitted between humans through environmental or airborne routes.

Pathogenicity and transmissibility assessment of two strains of human influenza virus isolated in China in 2018.

OBJECTIVE: Influenza season occurs every year in China, but its presentation was unusual in the period from December 2017 to early 2018. During this period, influenza activity was increasing across the country and was much greater than during the same period in previous years, with great harm to people's health. METHODS: In this study, we isolated two human influenza virus strains-A/Hebei/F076/2018(H1N1) and B/Hebei/16275B/2018-from patients with severe influenza in Hebei, China, during the flu season in January 2018, and explored their genetic characteristics, pathogenicity, and transmissibility. RESULTS: A/Hebei/F076/2018(H1N1) belongs to the human-like H1N1 influenza virus lineage, whereas B/Hebei/16275B/2018 belongs to the Victoria lineage and is closely related to the World Health Organization reference strain B/Brisbane/60/2008. Pathogenicity tests revealed that A/Hebei/F076/2018(H1N1) replicated much more strongly in mice, with mice exhibiting 40% mortality, whereas B/Hebei/16275B/2018 was not lethal. Both viruses could be transmitted through direct contact and by the aerosol route between guinea pigs, but the H1N1 strain exhibited higher airborne transmissibility. CONCLUSIONS: These results may contribute to the monitoring of influenza mutation and the prevention of an influenza outbreak.

Identification of cellular microRNA miR-188-3p with broad-spectrum anti-influenza A virus activity.

BACKGROUND: Influenza A virus (IAV) continues to pose serious threats to public health. The current prophylaxis and therapeutic interventions for IAV requires frequent changes due to the continuous antigenic drift and antigenic shift of IAV. Emerging evidence indicates that the host microRNAs (miRNAs) play critical roles in intricate host-pathogen interaction networks. Cellular miRNAs may directly target virus to inhibit its infection and be developed as potential anti-virus drugs. METHODS: In this study, we established a broad-spectrum anti-IAV miRNA screening method using miRanda software. The screened miRNAs were further verified by luciferase assay, viral protein expression assay and virus replication assay. RESULTS: Five cellular miRNAs (miR-188-3p, miR-345-5p, miR-3183, miR-15-3p and miR-769-3p), targeting 99.96, 95.31, 92.9, 94.58 and 97.24% of human IAV strains recorded in NCBI, respectively, were chosen for further experimental verification. Finally, we found that miR-188-3p downregulated PB2 expression at both mRNA and protein levels by directly targeted the predicted sites on PB2 and effectively inhibited the replication of IAV (H1N1, H5N6 and H7N9) in A549 cells. CONCLUSIONS: This is the first report screening cellular miRNAs that broad-spectrum inhibiting IAV infection. These findings suggested that cellular miR-188-3p could be used for RNAi-mediated anti-IAV therapeutic strategies.

Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.