Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

Classical bovine spongiform encephalopathy and chronic wasting disease:two sides of the prion coin

Transmissible spongiform encephalopathies(TSEs)are a group of progressive and ultimately fatal neurologic diseases of man and animals,all resulting from the propagated misfolding of the host's normal cellular prion protein.These diseases can be spontaneous,heritable,anthropogenic/iatrogenic,or in some cases horizontally transmissible,and include such notable TSEs as bovine spongiform encephalopathy(BSE)of cattle and chronic wasting disease(CWD)of cervids.Although they are both unequivocally protein misfolding disorders,they differ markedly in their pathogenesis,transmissibility,and zoonotic potential.While the BSE epidemic has largely abated over the past three decades following global feed bans on ruminant meat and bone meal,CWD,which is readily transmitted through various forms of excreta,has rapidly expanded from its original endemic zone to encompass much of North America,along with recently identified foci in Scandinavia.Most importantly,although the classical form of BSE has proven transmissible to humans consuming contaminated beef or beef products,so far there have been no conclusive reports on the zoonotic transmission of cWD to humans.The underlying basis for these differences-whether host or agent directed-are not well understood,though may be due to inherent differences in the three-dimensional structure of the misfolded BSE or CWD prion proteins or the expression levels and tissue distribution of respective cellular prion proteins.With the uncontrolled geographic spread of CWD,it is imperative that we improve our understanding of the factors governing prion disease pathogenesis,transmission,and zoonotic potential.

Nutrient Evaluation of Dining Center Food Waste and Comparison to Monogastric and Ruminant Feedstuffs

The objective of this study was to analyze the nutrient composition and variability of university dining hall food waste and compare it with common feedstuffs used in ruminant and monogastric diets. Food waste was categorized into two initial streams: mainstream (MS) from the serving line and vegetable preparation (VP) from the kitchen. Waste was collected from the Kramer Dining Center, Kansas State University, resulting in 30 daily samples. Waste was weighed and ground to homogenous particle size. Daily samples of MS and VP were analyzed for nutrient composition, where results were combined to calculate the nutrient profile of a hypothetical mixed food waste stream (MX) composited by total weight. Data were analyzed using R statistical software (v 4.2.2). Moisture and neutral detergent fiber (NDF) were greater in VP (P , while ether extract (EE) was less compared to MS and MX. Crude protein (CP) was greater (P < 0.05) in MS and MX streams compared to VP. The total digestible nutrients (TDN) and energy were greater in MS food waste than in MX, which was also greater than VP (P content, measured by standard deviation, was similar (P > 0.05) among streams for NDF, nitrogen-corrected neutral detergent fiber, acid detergent insoluble crude protein, CP, ash, lignin, and digestible and metabolizable energy. Dry matter and EE variation were greater (P < 0.05) in MS, whereas VP was less (P < 0.05) compared to MX. Standard deviation increased (P < 0.05) in MS and MX for neutral detergent insoluble crude protein, TDN, and gross energy when compared to VP. Despite having 70% - 80% moisture, dining hall food waste does have nutritive value and the potential to be included in ruminant and monogastric diets. Further research needs to be done to understand the value of including it in animal diets.

The MMTV-PyVT Transgenic Mouse as a Multistage Model for Mammary Carcinoma and the Efficacy of Antineoplastic Treatment

Animal models are commonly used to analyze the mechanism of carcinogenesis as well as the development and screening of potent drugs. Although numerous animal models of breast cancer have been used in research, few display multiple stages of tumorigenesis. The transgenic strain FVB/N-Tg(MMTV-PyVT) 634 Mul/J (also known as PyVT) was established to determine the effect of mammary gland-specific expression of the polyomavirus middle T antigen. Here the PyVT model with three distinct stages of tumor development (Pre, Early, and Late stages) was used as a model system for measuring tumor growth, tumor burden, and metastasis of mammary carcinomas. Additionally the expression profile of the molecular markers, survivin and Ki-67, was determined. Three antineoplastic compounds were tested over a 14-day period to determine their efficacy at attenuating tumor growth at each stage of development. Interestingly cisplatin and paclitaxel were determined to be ineffective anticancer drugs, while tamoxifen significantly reduced tumor growth in the Pre and Early stages of tumor formation.

Estimate of the energy value of soybean meal relative to corn based on growth performance of nursery pigs

Background:Two experiments were conducted to determine the effects of increasing amounts of soybean meal(SBM)in swine diets and estimate the energy value of SBM.Methods:A total of 2233 pigs(PIC 337×1050,Hendersonville,TN)and 3796 pigs(PIC 359×C40),initially 11.0 kg and 17.6 kg body weight(BW),were used in Exp.1 and 2,respectively.In Exp.1,pigs were placed in 92 pens each containing 20 to 27 pigs.In Exp.2,pigs were placed in 84 pens each containing 37 to 43 pigs.Treatments were assigned in a randomized complete block design with BW as the blocking factor.Dietary treatments consisted of 21%,27%,33%,or 39%SBM in Exp.1 and 17.5%,22%,26.5%,31%,35.5%,or 40%SBM in Exp.2,obtained by changing the inclusion rate of feed-grade amino acids and corn grain.For Exp.1,representative samples of corn grain,SBM,and distillers dried grains with solubles were analyzed for total AA content prior to diet formulation.For Exp.2,diets were formulated using NRC(2012)nutrient loadings.Treatment diets were fed for 21 and 22 d(Exp.1 and 2)and there were 23 replicates in Exp.1 and 14 replicates in Exp.2.Pigs were weighed and feed disappearance measured weekly to calculate average daily gain(ADG),average daily feed intake(ADFI),gain-to-feed ratio(G:F),and caloric efficiency(CE).Data were analyzed with block as a random effect and treatment as a fixed effect,and contrasts were constructed to test the linear and quadratic effects of increasing SBM.Results:In Exp.1,there was a tendency(linear,P=0.092)for a decrease in ADFI as SBM increased.There was a tendency(P=0.090)for a quadratic response for ADG,with a decrease in ADG observed with 39%SBM inclusion.Pigs fed diets with increasing SBM had a tendency(quadratic,P=0.069)for an increase in G:F up to 33%SBM and an improvement(linear,P=0.001;quadratic,P=0.063)in CE with increasing SBM.Using CE to estimate the energy of SBM relative to corn,a value of 105.4%of corn energy or 2816 kcal/kg NE was determined using all data points.When removing the CE value of the 39%SBM treatment due to the quadratic tendency,SBM was estimated to have 121.1%of corn energy or 3236 kcal/kg NE.In Exp.2,there was a decrease(linear,P=0.001)in ADFI.Pigs fed increasing SBM had a tendency(linear,P=0.065)for reduced ADG but an improvement(linear,P=0.001)in G:F and CE as SBM increased.The energy value of SBM was estimated as 124.7%of corn energy or 3332 kcal/kg NE.Conclusions:The results suggest that feeding increasing levels of SBM improves G:F and CE.The energy value of SBM was estimated to be between 105%and 125%of corn,which is much greater than the NRC(2012)would indicate.

Development of an in vitro macrophage screening system on the immunomodulating effects of feed components

Background:While feed components capable of modulating the immune system are highly sought after and marketed,often little evidence is available to support functional immune response claims.Thus,a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects,using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments.This screening system utilized RAW 264.7 murine macrophages to assess live S.cerevisiae cells and S.cerevisiae-derived cell wall componentsβ-glucan,mannan,and zymosan(a crude cell wall preparation containing bothβ-glucan and mannan).D-mannose was also evaluated as the monomer of mannan.We also examined the effect of a saturated fatty acid(C12:0,lauric acid)and its esters(methyl laurate and glycerol monolaurate)on innate immune cell activation and cellular metabolism.RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factorκ-light-chain-enhancer of activated B cells(NFκB),a major inflammatory/immune transcription factor.RAW cells were incubated with 0.01,0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds.In a separate experiment,RAW cells were incubated with 0,0.5,2.5,12.5,62.5,and 312.5μmol/L of lauric acid,methyl laurate,or glycerol monolaurate alone,or RAW cells were challenged with LPS and then incubated with fatty acid treatments.Results:Treatment with zymosan orβ-glucan alone induced NFκB activation in a dose-dependent manner,whereas treatment with D-mannose,mannan,or live S.cerevisiae cells did not.Post-treatment with mannan after an LPS challenge decreased NFκB activation,suggesting that this treatment may ameliorate LPS-induced inflammation.Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS,yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge.Conclusions:Overall,this cell screening system using RAW macrophages was effective,high-throughput,and sensitive to feed components combined with LPS challenges,indicating modulation of innate immune signaling in vitro.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever(ASF)is a highly lethal disease of domestic and wild swine caused by African swine fever virus(ASFV).The disease currently circulates in Africa,Europe,Asia and on the island of Hispaniola.The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses.No effective vaccine is available for ASF,making rapid and accurate detection of ASFV essential for disease mitigation strategies.Currently available diagnostics for ASFV possess significant limitations related to assay performance,deployability,and/or turn-around time;therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity.A chromatographic lateral flow immunoassay(LFIA)was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein.The assay requires only water to perform and provides results in 25 min,making it well-suited for field use.The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection,though it was unable to detect a genotype X ASFV strain.Diagnostic sensitivity correlated with clinical disease severity,body temperature,and viral DNA levels,and was over 90%in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus.The LFIA also showed a robust diagnostic specificity of over 98%,which is essential to field testing for a high consequence to foregin animal disease.The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates,making it a promising candidate for use as a field-deployable antigen detection assay.Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

Obituary:Professor Dr.Bernhard F.Dietzschold(1940–2022)

We share with great sadness that our long-time friend and colleague,Prof.Dr.Bernhard Dietzschold,passed away surrounded by his family on May 2,2022,at the age of 81.With the passing of Bernhard Dietzschold,the worldwide virology and infectious disease community is losing an eminent representative who has shaped the scientific landscape of its discipline for decades.

Practical starter pig amino acid requirements in relation to immunity,gut health and growth performance

Immune system activation begins a host of physiological responses. Infectious agents are recognized by monocytes and macrophages which in turn stimulate cytokine production. It is the hormone-like factors called cytokines that orchestrate the immune response. The classic responses observed with immune system activation and cytokine production include： anorexia, fever, lethargy, recruitment of other immune cells, and phagocytosis. While production of immune system components is known to require some amino acids, increases in amino acid requirements are more than offset by the associated decrease in protein accretion and increased muscle protein degradation that also accompanies immune system activation. However, the biggest impact of cytokine production is a decrease in feed intake. Therefore, as feed intake decreases, the energy needed to drive protein synthesis is also decreased. This suggests that diets should still be formulated on a similar calorie：lysine ratio as those formulated for non-immune challenged pigs. The evidence is sparse or equivocal for increasing nutrient requirements during an immune challenge. Nutritionists and swine producers should resist the pressure to alter the diet, limit feed, or add expensive feed additives during an immune challenge. While immune stimulation does not necessitate changes in diet formulation, when pigs are challenged with non-pathogenic diarrhea there are potential advantages on gut health with the increased use of crystalline amino acids rather than intact protein sources （i.e., soybean meal）. This is because reducing crude protein decreases the quantity of fermentable protein entering the large intestine, which lowers post weaning diarrhea. It also lowers the requirement for expensive specialty protein sources or other protein sources such as soybean meal that present immunological challenges to the gut. The objective of this review is two-fold. The first is to discuss immunity by nutrition interactions, or lack thereof, and secondly, to review amino acid requirement estimates for nursery pigs.

Montanide^TMGel01 ST Adjuvant Enhances PRRS Modified Live Vaccine Efficacy by Regulating Porcine Humoral and Cellular Immune Responses

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease caused by the PRRS virus. The MontanideTM class of flexible polymeric adjuvants has recently been shown to enhance protective immunity against PRRSV infection in piglets when used in combination with PRRS modified live vaccines (MLV). In this study, we explored the efficacy and immunological mechanisms of protection of MontanideTM Gel01 ST (Gel01) adjuvanted modified live PRRSV vaccine in pigs challenged with two genetically distinct strains of PRRSV. Gel01-MLV reduced lymph node pathology scores in pigs challenged with VR-2332 (parental strain of MLV vaccine) but not that in pigs challenged with MN184A (heterologous strain), when compared to that in pigs vaccinated with un-adjuvanted MLV. Pigs vaccinated with Gel01-MLV had higher levels of PRRS-specific antibodies, as measured by IDEXX ELISA and virus neutralizing antibodies, after vaccination and VR-2332 challenge. In addition, pigs vaccinated with Gel01-MLV had decreased levels of IFN-γ, IL-10, and T-regulatory lymphocytes in the blood as compared to that in pigs vaccinated with MLV alone. Interestingly, we found that addition of Gel01 did not change the profile of other T lymphocyte populations after PRRSV challenge. These results demonstrate that the MLV adjuvanted with Gel01 provides enhanced protection against homologous PRRSV infection, possibly by regulating the production of PRRSV-specific antibodies and cytokines involved in the development of T-regulatory cells. Thus, Gel01 ST is a promising adjuvant that can be formulated with PRRSV MLV vaccines to reduce disease severity and tissue damage caused by PRRSV infection in pigs.

Phylogeny and Homologous Recombination Occurring in Classical Swine Fever Viruses

Classical swine fever virus(CSFV) is the causative agent of classical swine fever, a highly contagious disease of pigs. But there is little information on the recombination in natural populations of CSFVs. Therefore, a phylogenetic analysis of 62 fulllength genome CSFV strains, isolated from all over the world, was performed to detect potential recombination events, with the recombinant sequences being analyzed with the SimPlot and RDP programs. The results identified a mosaic virus, Chinese CSFV HCLV(2)(AF091507.1), which is the one naturally emerged recombinant CSFV with two recombination breakpoints at 2 484 and 2 900 bp of the genome alignment. Its two putative parental-like strains were CSFV Shimen(AF092448.2) and CSFV strain C/HVRI(AY805221.1). This work demonstrated that homologous recombination did occur in natural CSFV populations. It had significant implications for understanding the molecular epidemiology of CSFV, and revealed that recombination was an important factor for high genetic diversities of CSFV.

3C^(pro)of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation

Foot-and-mouth disease virus(FMDV)has developed various strategies to antagonize the host innate immunity.FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms.The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γsignaling transduction.Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown.In this study,it was shown that FMDV replication was resistant to IFN-γtreatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes(ISGs).We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation.3C^(pro)expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences(GAS)promoter activity,without affecting the protein level,tyrosine phosphorylation,and homodimerization of STAT1.Finally,we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities.Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

Corrigendum to“3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation”[Virol Sin 38(2023)387–397]

Due to our negligence,the original version of this article,published online on Mar 14,2023,contained some mistakes in several Figs.In Fig.2D,the positions of the bands for protein 3A and 3B were incorrectly shifted.This has been modified in corrected Fig.2 as shown below.