With rapidly increasing animal pathogen surveillance requirements, new technologies are needed for a comprehensive understanding of the roles of pathogens in the occurrence and development of animal diseases. We applied metagenomic technology to avian virus surveillance to study the main viruses infecting six poultry farms in two provinces in eastern China. Cloacal/throat double swabs were collected from 60 birds at each farm according to a random sampling method. The results showed that the method could simultaneously detect major viruses infecting farms, including avian influenza virus, infectious bronchitis virus, Newcastle disease virus, rotavirus G, duck hepatitis B virus, and avian leukemia virus subgroup J in several farms. The test results were consistent with the results from traditional polymerase chain reaction (PCR) or reverse transcription-PCR analyses. Five H9N2 and one H3N8 avian influenza viruses were detected at the farms and were identified as low pathogenic avian influenza viruses according to HA cleavage sites analysis. One detected Newcastle disease virus was classified as Class II genotype I and avirulent type according to F0 cleavage sites analysis. Three avian infectious bronchitis viruses were identified as 4/91, CK/CH/LSC/99I and TC07-2 genotypes by phylogenetic analysis of S1 genes. The viral infection surveillance method using metagenomics technology enables the monitoring of multiple viral infections, which allows the detection of main infectious viruses.

The H5 highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, we constructed a replication-competent recombinant influenza A virus of clade 2.3.4.4 Н5N1 expressing the clade 2.3.2.1 H5 HA1 protein from a tricistronic NS segment. We used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. H5 HA1 and nuclear export information were fused in frame with a truncated NS1 open reading frame, separated by 2A self-processing sites. The resulting PR8-H5-NS1(73)H5 stably expressed clade 2.3.4.4 H5 HA and clade 2.3.2.1 H5 HA1 proteins and exhibited similar in vitro growth kinetics as the parental PR8-2344H5 virus. PR8-H5-NS1(73)H5 induced specific hemagglutination-inhibition (HI) antibody against clade 2.3.4.4 H5 that was comparable to that of the combination vaccine of PR8-2344H5 and PR8-2321H5. HI antibody titers were significantly lower against clade 2.3.2.1 H5 virus than with the combination vaccine. PR8-H5-NS1(73)H5 completely protected chickens from both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs challenge. Our results suggested that PR8-H5-NS1(73)H5 was highly immunogenic and efficacious against both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs in chickens.

The H5 and H7N9 subtypes of highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, a replication competent recombinant influenza A virus of the Í5N1 subtype expressing the H7 HA1 protein from a tri-cistronic NS segment was constructed. A heterologous dimerization domain was used to combine with the truncated NS1 protein of 73 amino acids to increase protein stability. H7 HA1, nuclear export protein coding region, and the truncated NS1 were fused in-frame into a single open reading frame via 2A self-cleaving peptides. The resulting PR8-H5-NS1(73)H7 stably expressed the H5 HA and H7 HA1 proteins, and exhibited similar growth kinetics as the parental PR8-H5 virus in vitro. PR8-H5-NS1(73)H7 induced specific hemagglutination inhibition (HI) antibody against H5, which was comparable to that of the combination vaccine of PR8-H5 and PR8-H7. The HI antibody titers against H7 virus were significantly lower than that by the combination vaccine. PR8-H5-NS1(73)H7 completely protected chickens from challenge with both H5 and H7 HPAIVs. These results suggest that PR8-H5-NS1(73)H7 is highly immunogenic and efficacious against both H5 and H7N9 HPAIVs in chickens. Highlights: - PR8-H5-NS1(73)H7 simultaneously expressed two HA proteins of different avian influenza virus subtypes. - PR8-H5-NS1(73)H7 was highly immunogenic in chickens. - PR8-H5-NS1(73)H7 provided complete protection against challenge with both H5 and H7N9 HPAIVs.

A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain.

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is spreading rapidly, which seriously impacts global public health and economy. Thus, developing effective drugs remains urgent. We identify two potent antibodies, nCoVmab1 and nCoVmab2, targeting the SARS-CoV-2 spike protein receptor-binding domain (RBD) with high affinities from a naïve human phage-displayed Fab library. nCoVmab1 and nCoVmab2 neutralize authentic SARS-CoV-2 with picomolar and nanomolar IC50 values, respectively. No detectable defects of nCoVmab1 and nCoVmab2 are found during the preliminary druggability evaluation. nCoVmab1 could reduce viral titer and lung injury when administered prophylactically and therapeutically in human angiotensin-converting enzyme II (hACE2)-transgenic mice. Therefore, phage display platform could be efficiently used for rapid development of neutralizing monoclonal antibodies (nmabs) with clinical potential against emerging infectious diseases. In addition, we determinate epitopes in RBD of these antibodies to elucidate the neutralizing mechanism. We also convert nCoVmab1 and nCoVmab2 to their germline formats for further analysis, which reveals the contribution of somatic hypermutation (SHM) during nCoVmab1 and nCoVmab2 maturation. Our findings not only provide two highly potent nmabs against SARS-CoV-2 as prophylactic and therapeutic candidates, but also give some clues for development of anti-SARS-CoV-2 agents (e.g., drugs and vaccines) targeting the RBD.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating cancers without effective treatments. Amplified in breast cancer 1 (AIB1) is a member of the steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors. While AIB1 is associated with the initiation and progression of multiple cancers, the mechanism by which AIB1 contributes to PDAC progression remains unknown. In this study, we aimed to explore the role of AIB1 in the progression of PDAC and elucidate the underlying mechanisms. Methods: The clinical significance and mRNA level of AIB1 in PDAC were studied by database analysis. To demonstrate whether AIB1 mediates the malignant features of PDAC cells, namely, proliferation, migration, invasion, we performed real-time PCR and Western blot analysis, established xenograft models and used in vivo metastasis assay. With insights into the mechanism of AIB1, we performed RNA sequencing (Seq), ChIP-Seq, luciferase reporter assays and pull-down assays. Furthermore, we analyzed the relationship between AIB1 expression and its target expression in PDAC cells and patients and explored whether PDAC cells with high AIB1 levels are sensitive to inhibitors of its target. Results: We found that AIB1 was significantly upregulated in PDAC and associated with its malignancy. Silencing AIB1 impaired hedgehog (Hh) activation by reducing the expression of smoothened (SMO), leading to cell cycle arrest and the inhibition of PDAC cell proliferation. In addition, AIB1, via upregulation of integrin αv (ITGAV) expression, promoted extracellular matrix (ECM) signaling, which played an important role in PDAC progression. Further studies showed that AIB1 preferably bound to AP-1 related elements and served as a coactivator for enhancing the transcriptional activity of MafB, which promoted the expression of SMO and ITGAV. PDAC cells with high AIB1 levels were sensitive to Hh signaling inhibitors, suggesting that blocking Hh activation is an effective treatment against PDAC with high AIB1 expression. Conclusions: These findings reveal that AIB1 is a crucial oncogenic regulator associated with PDAC progression via Hh and ECM signaling and suggest potential therapeutic targets for PDAC treatment.