Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

The role of wild goose(Anser) populations of Russia and theTibet Plateau in the spread of the avian influenza virus

Wild birds of the orders Anseriformes and Charadriiformes represent a natural reservoir of low pathogenic avian influenza(LPAI) viruses(family Orthomyxoviridae).Wild geese(order Anseriformes)relating to waterfowls undertake extensive migration flights reaching thousands of kilometers.Isolation of the avian influenza virus(AIV) from wild geese is quite low or absent.The aims of this study are to monitor the AIV in different wild goose species,nesting on Russian territory and the Tibet Plateau and to analyze the derived data for the purpose of determining the role of these wild bird species in spreading pathogens.In our study 3245 samples from nine wild goose species in nine regions of Russia and on the territory of the Tibet Plateau(the Xizang Autonomous Region) were tested and no AIV were detected.Our study shows the non-essential role of wild geese in the spread of the AIV over long distances and reaches theconclusion that geese are probably not natural reservoirs for the primary viruses.However,further inquiry of AIV in wild goose populations is required.Studies of wild geese and AIV ecology will allow us to obtainmore information about pathogen-host relationships and to make arrangements for the maintenance ofwild goose populations.

Study on Piezoelectric Immunosensor for the Detection of H9-subtype Avian Influenza Virus

[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo

This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

Genetic Variation Analysis on the Whole Genomic Sequence of a H9N2 Subtype Avian Influenza Virus Isolate

A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.

Strengthened Monitoring of H5 Avian Influenza Viruses in External Environment in Hubei, 2018

The contamination status of H5 avian influenza viruses and distribution of subtypes of H5N1 and H5N6 in poultry-related environment of Hubei areas were investigated.Urban and rural live poultry markets,poultry farms,intensive livestock farms and other monitoring types of 103 counties in 17 cities were selected in Hubei.Wiping samples from cage surface,wiping samples from chopping board,fecal specimens and other environmental samples were collected and tested by real-time RT-PCR using primers and probes of influenza A,avian influenza of H5,N1 and N6 from December 2017 to March 2018.The avian influenza virus positive rate was compared among different monitoring sites,samples,time and regions.Totally,7132 environmental samples were collected in 1634 monitoring points with a positive rate of 2.24%.The positive rate of H5 avian influenza virus was the highest in urban and rural live poultry markets(3.44%,x^2=61.329,P<0.05)in 6 monitoring sites and wiping samples from chopping board(5.46%,x^2=67.072,P<0.05)in 6 sample types.H5N6 avian influenza viruses were detected more in eastern than western Hubei,and H5N6 avian influenza viruses were detected only in Xiangyang city of western Hubei.There were important high-risk places of human infection with H5 avian influenza virus in urban and rural live poultry markets and the poultry slaughtering plants.H5N6 has been the predominant subtype of H5 avian influenza viruses in the eastern and western Hubei and H5N6 avian influenza viruses were still present in a few areas of Hubei.Outbreaks of human H5N1 and H5N6 avian influenza remain at risk in Hubei province.

The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

HA Gene Variation Analysis of H9N2 Sub-type Avian Influenza Virus from Three Strains in Different Times

HA Gene of H9N2. sub-type avian influenza virus from three strains in different times was amplified, purified and then sequenced. The results of its sequence analysis showed that the whole length of the amplified HA Gene was 1 683 bp, encoding 560 amino acids. The amino acid sequence of three virulent strains at cleavage site was R-S-S-R, which was low-pathogenicity strain. According to the amino acid sequence of the isolated strains, there were 7 potential glycosylation sites, and the receptor-binding site was the specific sequence of the avian-derived influenza virus. Amino acids on the left edge of receptor-binding site were all NGQQG, while amino acids on the right edge of receptor-binding site were GTSKA. From the comparative sequence analysis of HA Gene from some referenced strains, the results indicated that nucleotide and amino acid homology between isolated strains and referenced strains was higher. Evolutionary tree analysis showed that three strains were all Eurasian species, and there was a close relationship with the representative strains of A / duck / Hong Kong/Y280/97.

Distribution of Avian Influenza A Viruses in Poultry-Related Environment and Its Association with Human Infection in Henan, 2016 to 2017

Objective To survey avian influenza A viruses(AIVs) in the environment and explore the reasons for the surge in human H7 N9 cases.Methods A total of 1,045 samples were collected from routine surveillance on poultry-related environments and 307 samples from human H7 N9 cases-exposed environments in Henan from 2016 to2017. The nucleic acids of influenza A(Flu A), H5, H7, and H9 subtypes were detected by real-time polymerase chain reaction.Results A total of 27 H7 N9 cases were confirmed in Henan from 2016 to 2017, 24 had a history of live poultry exposure, and 15 had H7 N9 virus detected in the related live poultry markets(LPMs). About 96%(264/275) Flu A positive-environmental samples were from LPMs. H9 was the main AIV subtype(10.05%) from routine surveillance sites with only 1 H7-positive sample, whereas 21.17% samples were H7-positive in H7 N9 cases-exposed environments. Samples from H7 N9 cases-exposed LPMs(47.56%)had much higher AIVs positive rates than those from routine surveillance sites(12.34%). The H7+H9 combination of mixed infection was 78.18%(43/55) of H7-positive samples and 41.34%(43/104) of H9-positive samples.Conclusion The contamination status of AIVs in poultry-related environments is closely associated with the incidence of human infection caused by AIVs. Therefore, systematic surveillance of AIVs in LPMs in China is essential for the detection of novel reassortant viruses and their potential for interspecies transmission.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012

Objective In March 2012, an H7N7 subtype avian influenza virus （AIV） named A/wild goose/Dongting/PC0360/2022 （H7N7） （DT/PC0360） was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization. Methods RNA was extracted from environment samples （including fecal samples from wild bird or domestic ducks, and water samples） for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the HI-HI6 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed. Results Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in China's Mainland in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus. Conclusion Strengthening the surveillance of AlVs of wild waterfowl and poultry in this region is vita for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.

Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

Serological survey of avian influenza virus infection of unvaccinated backyard chickens in Mandlhakazi,Southern Mozambique

To investigate serologically the presence of avian influenza virus (AIV) in backyard chickens from Mandlhakazi district, Southern Mozambique.MethodsA total of 439 sera samples from unvaccinated and apparently healthy backyard chickens from 4 villages (Chidenguele, Macuacua, Chizavane, and Nwadjahane) were tested for the presence of AIV antibodies through commercial enzyme-linked immunoabsorbent assay (ELISA) kit used according to manufacturer instructions.ResultsAnti-AIV antibodies were detected in all villages surveyed. The overall seroprevalence obtained was 32.6% (95% CI 28.2%-37.0%). The highest prevalence of 51.3% (95% CI 42.3%-60.2%) was recorded in Macuacua village, while the lowest prevalence of 13.0% (95% CI 6.2%-19.9%) was found in Chizavane village. The results of logistic regression analyses suggested that chicken being located in Chizavane and Macuacua villages were more unlikely for getting the virus exposure (P < 0.05).ConclusionsOur findings suggested that AIV is widespread within backyard chickens in the studied villages. Further research is needed to identify the circulating virus genotypes and determine the potential role of backyard chickens in the zoonotic transmission of AIV in Mozambique.

Immune Efficacy of a Recombinant Fowlpox Virus Co-Expressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.