Isolation and identification of Eurasian avian-like H1N1 swine influenza virus and evaluation of their pathogenicity and immune protective effects in pigs.

Swine influenza (SI) is a severe disease affecting pigs, with a huge economic impact on pig farmers. Currently, available SIV vaccines do not meet the requirements for Swine influenza prevention and control, indicating the need for vaccine development using predominant strains. Here, we isolated and identified the swine influenza virus in farms and slaughterhouses in nine provinces in China to determine the most prevalent strain. A total of 8383 samples were collected between 2013 and 2022, from which 87 swine influenza virus strains were isolated. Genome sequencing identified 62 strains of the H1N1 subtype, three strains of the H1N2 subtype, and 22 strains of the H3N2 subtype. The 521# strain virus possesses the viral ribonucleoprotein (vRNP) and matrix (M) genes from the pdm/09 lineage, the HA, NA from the original Eurasian avian-like (EA) H1N1 lineage, and the nonstructural (NS) gene from the triple-reassortant (TR) lineage. The 431# strain was also a TR, except its M-gene was derived from the original EA H1N1 lineage. The pathogenicity of two 431# strains and one typical 521# strain was evaluated in mice, and the 431# strain exhibited higher pathogenicity. Therefore, a new 521# strain was selected for vaccine production because it is the current circulating strain. The vaccine produced using the 521# strain and pre-evaluated adjuvants was effective against the homologous H05 strain, as evidenced by the normal body temperature of vaccinated pigs and low virus titer of nasal swabs. In contrast, infection with the H05 strain significantly increased the body temperature of unvaccinated pigs and increased the virus titer of nasal swabs. Notably, vaccination with the 521#-based vaccine conferred some level of protection against the heterologous B15 strain (H3N2 subtype), thus reducing the viral load in pigs.

The RNA-Splicing Ligase RTCB Promotes Influenza A Virus Replication by Suppressing Innate Immunity via Interaction with RNA Helicase DDX1.

The RNA-splicing ligase RNA 2',3'-cyclic phosphate and 5'-OH ligase (RTCB) is a catalytic subunit of the tRNA-splicing ligase complex, which plays an essential role in catalyzing tRNA splicing and modulating the unfolded protein response. However, the function of RTCB in influenza A virus (IAV) replication has not yet been described. In this study, RTCB was revealed to be an IAV-suppressed host factor that was significantly downregulated during influenza virus infection in several transformed cell lines, as well as in primary human type II alveolar epithelial cells, and its knockout impaired the propagation of the IAV. Mechanistically, RTCB depletion led to a robust elevation in the levels of type I and type III IFNs and proinflammatory cytokines in response to IAV infection, which was confirmed by RTCB overexpression studies. Lastly, RTCB was found to compete with DDX21 for RNA helicase DDX1 binding, attenuating the DDX21-DDX1 association and thus suppressing the expression of IFN and downstream IFN-stimulated genes. Our study indicates that RTCB plays a critical role in facilitating IAV replication and reveals that the RTCB-DDX1 binding interaction is an important innate immunomodulator for the host to counteract viral infection.

In vitro and in vivo effects of 3-indoleacetonitrile-A potential new broad-spectrum therapeutic agent for SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has resulted in significant global morbidity, mortality, and societal disruption. Currently, effective antiviral drugs for the treatment of SARS-CoV-2 infection are limited. Therefore, safe and effective antiviral drugs to combat COVID-19 are urgently required. In previous studies, we showed that 3-indoleacetonitrile, a plant growth hormone produced by cruciferous (Brassica) vegetables, is effective in treating influenza A virus infection. However, the molecular mechanisms underlying these effects remain unclear. Herein, we demonstrated that 3-indoleacetonitrile exhibits broad-spectrum antiviral activity and is effective against HSV-1 and VSV infections in vitro. This phenomenon prompted us to study its role in the anti-SARS-CoV-2 process. Interestingly, 3-indoleacetonitrile exhibited antiviral activity against SARS-CoV-2 in vitro. Importantly, tail vein injection of 3-indoleacetonitrile resulted in good antiviral activity in mouse models infected with WBP-1 (a mouse adaptation of the SARS-CoV-2 strain). Mechanistically, 3-indoleacetonitrile promoted the host interferon signalling pathway response and inhibited autophagic flux. Furthermore, we demonstrated that 3-indoleacetonitrile induced an increase in mitochondrial antiviral-signalling (MAVS) protein levels, which might be attributed to its inhibition of the interaction between MAVS and the selective autophagy receptor SQSTM1. Overall, our results demonstrate that 3-indoleacetonitrile is potently active against SARS-CoV-2 in vitro and in vivo, which may provide a foundation for further clinical testing for the treatment of COVID-19. In addition, considering its broad-spectrum antiviral effect, it should be explored whether it also has an effect on other viruses that threaten human health.

YWHAG inhibits influenza a virus replication by suppressing the release of viral M2 protein.

Influenza A virus (IAV) poses a serious threat to human life and property. The IAV matrix protein 2 (M2) is significant in viral budding. Increasing studies have proven the important roles of host factors in IAV replication. In this study, immunoprecipitation combined with mass spectrometry revealed that the host protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), which belongs to the 14-3-3 protein scaffold family, interacts with M2. Their interactions were further confirmed by co-immunoprecipitation (Co-IP), immunofluorescence, and confocal microscopy of virus-infected HeLa cells. Moreover, we constructed YWHAG-KO and YWHAG-overexpressing cells and found that YWHAG knockout significantly increased viral production, whereas its overexpression reduced the titer of virus progeny. Therefore, YWHAG is a negative regulatory factor during IAV infection. Further, YWHAG knockout or overexpression had no effect on the binding, entry, or viral RNA replication in the early stages of the virus life cycle. On the contrary, it impaired the release of virions at the plasma membrane as determined using transmission electron microscopy and suppressed the M2-mediated budding of the influenza virus. Importantly, the H158F mutation of YWHAG was found to affect interaction with M2 and its budding. Collectively, our work demonstrates that YWHAG is a novel cellular regulator that targets and mediates the interaction and release of M2.

PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.

The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

SARS-CoV-2 Infection Causes Hyperglycemia in Cats.

Isolated reports of new-onset diabetes in patients with coronavirus disease 2019 (COVID-19) have led researchers to hypothesize that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human exocrine and endocrine pancreatic cells ex vivo and in vivo. However, existing research lacks experimental evidence indicating that SARS-CoV-2 can infect pancreatic tissue. Here, we found that cats infected with a high dose of SARS-CoV-2 exhibited hyperglycemia. We also detected SARS-CoV-2 RNA in pancreatic tissues of these cats, and immunohistochemical staining revealed the presence of SARS-CoV-2 nucleocapsid protein (NP) in islet cells. SARS-CoV-2 NP and spike proteins were primarily detected in glucagon-positive cells, and most glucagon-positive cells expressed ACE2. Additionally, immune protection experiments conducted on cats showed that blood glucose levels of immunized cats did not increase postchallenge. Our data indicate cat pancreas as a SARS-CoV-2 target and suggest that the infection of glucagon-positive cells could contribute to the metabolic dysregulation observed in SARS-CoV-2-infected cats.

A serological survey of severe acute respiratory syndrome coronavirus 2 in dogs in Wuhan.

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis and economic losses. Although several cases of cats and dogs infected with SARS-CoV-2 have been reported during this outbreak, the prevalence of SARS-CoV-2 in dog and its transmission among other companion animals are still unknown. Here, we report an extensive serological study of SARS-CoV-2 infection in dogs in Wuhan and analyse the infection rates at different stages of the pandemic outbreak. A total of 946 dogs serum samples were collected from Wuhan, of which 36 samples were obtained prior to the pandemic outbreak. Indirect enzyme-linked immunosorbent assay (ELISA) showed that 16 sera collected during the outbreak were detected as positive through the receptor-binding domain (RBD) of SARS-CoV-2. Of these 16 sera, 10 exhibited measurable SARS-CoV-2-specific neutralizing antibodies whose titres ranged from 1/20 to 1/180. No serological cross-reactivity was detected between SARS-CoV-2 and canine coronavirus (CCV). Furthermore, with the effective control of the outbreak, a decrease in the SARS-CoV-2 seropositive dog number was observed. Our results suggest that SARS-CoV-2 has infected companion dogs during the outbreak, and that COVID-19 patient families have a higher risk of dog infection. Our findings deepen our understanding of the infection of SARS-CoV-2 in dogs and provide an important reference for prevention of COVID-19.

Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV.

African swine fever virus (ASFV) is an important viral pathogen infecting pigs worldwide throughout the pig industry. CD2v (an outer-membrane glycosylated protein of ASFV)-unexpressed lower-virulence mutants have appeared in China and other countries in recent years. Using OIE-recommended quantitative PCR and ELISA methods, people can accurately judge whether pigs are infected with wild-type ASFV. However, the strategy has failed to distinguish ΔCD2v lower-virulence mutants and wild-type ASFV infection. Here, we expressed and purified the CD2v and p30 proteins via CHO cells and successfully established a dual enzyme-linked immunosorbent assay (ELISA), which can be used to differentiate pigs infected with wild-type ASFV or with CD2v-unexpressed lower-virulence mutants. The dual ELISA showed excellent specificity without cross-reactions with antibodies of PRRSV, CSFV, JEV, PRV, or PPV. The dual ELISA could detect ASFV-infected positive serum samples up to dilutions of 5120 times, possessing high sensitivity. Therefore, the application of this dual ELISA approach can play an important role in ASFV epidemiology study and fill the gaps in differential diagnosis.

HP0487 contributes to the virulence of Streptococcus suis serotype 2 by mediating bacterial adhesion and anti-phagocytosis to neutrophils.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that poses a serious threat to human health and the swine industry. The survival and travel in the bloodstream are the important causes for SS2, contributing to bacteremia, septicemia even septic shock. However, the related mechanism remains largely unknown. Preliminary experiment demonstrated that SS2 could largely attach to the surface of neutrophils, implying that this phenomenon maybe contributed to the travel of SS2 in bloodstream and then influenced its pathogenicity. To confirm this hypothesis, using a previously established screening method that combines affinity chromatography (based on liquid chromatography-tandem mass spectrometry) with shotgun proteomics, three candidate proteins (HP0487, HP1765, and HP1111) were identified from SS2 that could interact with neutrophils. Next, by constructing the deletion mutations, we demonstrated that HP0487 of three proteins could significantly influence the adhesion of SS2 to neutrophils. Furthermore, HP0487 was shown to contribute to the anti-phagocytosis of SS2 to neutrophils and RAW264.7 cells. More importantly, the deletion of HP0487 significantly reduced lethality and bacterial loads in vivo of SS2. Thus, our findings demonstrate that HP0487 contributes to SS2 virulence by mediating the adhesion and anti-phagocytosis of SS2 to neutrophils, promoting a better understanding about the pathogenesis of SS2.

Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice.

BACKGROUND: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein . METHODS: SARS-CoV-2 was passaged in BALB/c mice to obtain mouse-adapted virus strain. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments determined the binding affinity of mouse-adapted SARS-CoV-2 WBP-1 RBD to mouse ACE2 and human ACE2. Finally, we tested whether TLR7/8 agonist Resiquimod (R848) could also inhibit the replication of WBP-1 in the mouse model. FINDINGS: The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, the study tentatively found that the TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge. Therefore, this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. INTERPRETATION: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies. This study also tentatively verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo. FUNDING: This research was funded by the National Key Research and Development Program of China (2020YFC0845600) and Emergency Science and Technology Project of Hubei Province (2020FCA046) and Robert A. Welch Foundation (C-1565).

SARS-CoV-2 rapidly adapts in aged BALB/c mice and induces typical pneumonia.

Age is a risk factor for coronavirus disease 2019 (COVID-19) associated morbidity and mortality in humans; hence, in this study, we compared the course of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in young and aged BALB/c mice. We found that SARS-CoV-2 isolates replicated in the respiratory tracts of 12-month-old (aged) mice and caused pathological features of pneumonia upon intranasal infection. In contrast, rapid viral clearance was observed 5 days following infection in 2-month-old (young) mice with no evidence of pathological changes in the lungs. Infection with SARS-CoV-2 elicited significantly upregulated production of cytokines, especially interleukin 6 and interferon gamma, in aged mice; whereas this response was much weaker in young mice. Subsequent challenge of infected aged BALB/c mice with SARS-CoV-2 resulted in neutralized antibody responses, a significantly reduced viral burden in the lungs, and inflammation mitigation. Deep sequencing showed a panel of mutations potentially associated with the enhanced infection in aged BALB/c mice, such as the Q498H mutations which are located at the receptor binding domain (RBD) of the spike (S) protein. We further found that the isolates can not only multiply in the respiratory tract of mice but also cause disease in aged mice. Overall, viral replication and rapid adaption in aged BALB/c mice were associated with pneumonia, confirming that the age-related susceptibility to SARS-CoV-2 in mice resembled that in humans.ImportanceAged BALB/c model are in use as a model of disease caused by SARS-CoV-2. Our research demonstrated SARS-CoV-2 can rapidly adapt in aged BALB/c mice through causing mutations at the RBD of the S protein. Moreover, SARS-CoV-2-infected aged BALB/c mice indicated that alveolar damage, interstitial pneumonia, and inflammatory immune responses were similar to the clinical manifestations of human infections. Therefore, our aged BALB/c challenge model will be useful for further understanding the pathogenesis of SARS-CoV-2 and for testing vaccines and antiviral agents.

A serological survey of SARS-CoV-2 in cat in Wuhan.

COVID-19 is a new respiratory illness caused by SARS-CoV-2, and has constituted a global public health emergency. Cat is susceptible to SARS-CoV-2. However, the prevalence of SARS-CoV-2 in cats remains largely unknown. Here, we investigated the infection of SARS-CoV-2 in cats during COVID-19 outbreak in Wuhan by serological detection methods. A cohort of serum samples were collected from cats in Wuhan, including 102 sampled after COVID-19 outbreak, and 39 prior to the outbreak. Fifteen sera collected after the outbreak were positive for the receptor binding domain (RBD) of SARS-CoV-2 by indirect enzyme linked immunosorbent assay (ELISA). Among them, 11 had SARS-CoV-2 neutralizing antibodies with a titer ranging from 1/20 to 1/1080. No serological cross-reactivity was detected between SARS-CoV-2 and type I or II feline infectious peritonitis virus (FIPV). In addition, we continuously monitored serum antibody dynamics of two positive cats every 10 days over 130 days. Their serum antibodies reached the peak at 10 days after first sampling, and declined to the limit of detection within 110 days. Our data demonstrated that SARS-CoV-2 has infected cats in Wuhan during the outbreak and described serum antibody dynamics in cats, providing an important reference for clinical treatment and prevention of COVID-19.