Let ’ s Get Vaccinated for Both Flu and COVID-19 : On the World Flu Day 2021

Introduction:  During the coronavirus disease 2019 (COVID-19)  pandemic,  the  circulation  of  seasonal influenza  virus  declined  globally  and  remained  below previous seasonal levels. We analyzed the results of the epidemiology,  antigenic,  and  genetic  characteristics, and  antiviral  susceptibilities  of  seasonal  influenza viruses  isolated  from  the  mainland  of  China  during October 5, 2020 through September 5, 2021, to better assess the risk of influenza during subsequent influenza season in 2021–2022. Methods:  Positive  rates  of  influenza  virus detection  during  this  period  were  based  on  real-time polymerase  chain  reaction  (PCR)  detection  by  the Chinese  National  Influenza  Surveillance  Network laboratories,  and  isolated  viruses  from  influenza positive  samples  were  submitted  to  the  Chinese National  Influenza  Center.  Antigenic  analyses  for influenza  viruses  were  conducted  using  the hemagglutination  inhibition  assay.  Next-generation sequencing was used for genetic analyses. Viruses were tested  for  resistance  to  antiviral  medications  using  a phenotypic assay and next-generation sequencing. Results:  In  southern  China,  the  influenza positivity rate was elevated especially after March 2021 and was higher than the same period the previous year with  the  COVID-19  pandemic.  In  northern  China, influenza positive rate peaked at Week 18 in 2021 and has declined since then. Nearly all isolated viruses were B/Victoria lineage viruses during the study period, and 37.3%  of  these  viruses  are  antigenically  similar  to  the reference  viruses  representing  the  vaccine  components for  the  2020–2021  and  2021–2022  Northern Hemisphere  influenza  season.  All  seasonal  influenza viruses  were  susceptible  to  neuraminidase  inhibitors and endonuclease inhibitors. Conclusions:  Influenza  activity  has  gradually increased in the mainland of China in 2021, although the  intensity  of  activity  is  still  lower  than  before  the COVID-19  pandemic.  The  diversity  of  circulating influenza  types/subtypes  decreased,  with  the  vast majority  being  B/Victoria  lineage  viruses.  The surveillance data from this study suggest that we should strengthen influenza surveillance during the upcoming traditional  influenza  season.  It  also  provided  evidence for  vaccine  recommendations  and  prevention  and control of influenza and clinical use of antiviral drugs.

This year marks the fourth World Flu Day on November 1. In 2018, on the occasion of centenary of the 1918 flu pandemic, the first World Flu Day was formally initiated and developed by leading influenza specialists in China and all over the world at a symposium in Shenzhen, China, with the theme to "Commemorate the Centenary of the 1918-19 Influenza Pandemic" (1). Scientists and representatives from different international associations and organizations, including the five World Health Organization (WHO) Collaborating Centers for Reference and Research on Influenza came together and advocated for raising public awareness of influenza, accelerating scientific innovation and international cooperation on influenza surveillance and basic research, and pushing for stronger global support on influenza prevention and control (2). The second and the third World Flu Day Symposia were held in Beijing with the themes as "Know Flu/Prevent Flu/Beat Flu" in 2019 and "Influenza Control and COVID-19 Pandemic Response" in 2020.
In the past century, humans had endured 5 influenza pandemics: "Spanish flu" (H1N1) in 1918, "Asian flu" (H2N2) in 1957, "Hong Kong flu" (H3N2) in 1968, and "Russian flu" (H1N1, "pseudopandemic") in 1977, and "Mexico Swine flu" (H1N1) in 2009 (3). With the emerging events of diverse human-infecting avian influenza viruses, i.e., H5N1, H5N6, H6N1, H7N9, H9N2, H10N8, H7N4, etc. in the recent decades, scientists estimated that the next pandemic may be caused by the influenza virus and the world seems to not be prepared for it. Meanwhile, a new respiratory tract infectious disease coronavirus disease 2019 (COVID-19) emerged in the end of 2019 and developed rapidly in the early months of 2020 as the first recognized pandemic caused by the coronavirus (4). Beyond all doubt, humans were not prepared for the pandemic and COVID-19 affected and even changed virtually everyone's lives (5). The relevant scientists and health workers all over the world have tried their best to understand and control the new disease (6). However, the etiology of COVID-19, severe acute respiratory syndrome (SARS-CoV-2; initially named 2019-NCIP and hCoV-19) is still wreaking havoc with the continuously emerging variants (7). Thus, in the coming seasons of influenza, we are facing the complexity and challenge of influenza and COVID-19 cocirculation. Currently, promoting vaccination coverage is still the prioritized strategy to efficiently control the current COVID-19 pandemic and to prevent seasonal influenza. Thus, for the World Flu Day 2021, we advocate the theme as "Flu and COVID-19, Let's Get Vaccinated" this year.
From early 2020, the seasonal influenza activity has had a dramatic drop with the implementation of nonpharmaceutical interventions (NPIs) and behavioral changes to mitigate COVID-19 (8)(9)(10). The 2019-2020 seasonal influenza activity was significantly reduced compared with the previous seasons in China, the United States, and other countries, and the decreases in influenza virus infection were also associated with the timing of NPIs (9). In actuality, this relief situation of influenza was sustained in the following seasons and one influenza lineage, i.e. B/Yamagata, has not been isolated from April 2020 to August 2021 and seems to have possibly become extinct (11). NPIs implemented against COVID-19 played an unexpected extra role in this process, including use of individual protection, e.g., wearing masks, watching distance, washing hands (3-W), and restriction of personal movement, e.g., canceling mass gatherings, closing public entertainment venues, closing schools, restricting domestic and international travel, and issuing stay-at-home orders (12).
In spite of the low prevalence of seasonal influenza viruses among humans, an undercurrent of avian influenza viruses still whirls among poultry populations. Since the end of 2019, highly pathogenic H5Ny avian influenza has frequently occurred in birds in Eurasia and Africa (13). Analyses of highly pathogenic avian influenza H5 viruses from poultry outbreaks and recent detections in migratory waterfowl across a wide Eurasian region revealed maintenance of A/H5 clade 2.3.4.4b, i.e., H5N8, H5N5, and H5N1, etc. (14), which raised a highly alarming threat to poultry production, veterinary disease control, and human public health. Especially in the spring of 2020, the 2.3.4.4b branch H5N8 avian influenza virus caused multiple outbreaks in Central and Eastern Europe, implying a new wave of highly pathogenic H5Ny avian influenza in the broad area (15). A detailed genetic and biological analysis of avian influenza outbreaks in wild birds in China over the decade before 2019 found that 5 out of 10 highly pathogenic H5Ny avian influenza could bind humantype receptors, all of which were from clade 2.3.4.4. Human infection of clade 2.3.4.4b influenza A(H5N8) virus with 7 cases was reported at a poultry farm in Astrakhan, Russia in December 2020 (16). Before the end of 2020, a total of 26 laboratory-confirmed cases of human infection with influenza A(H5N6) virus have been reported (17). However, in 2021 thus far, more than 20 sporadic human infections have been recorded in different provincial-level administrative divisions (18). All this evidence of human H5Ny infections indicate a sustained spillover of avian influenza virus and deserve close attention.
The pandemic of COVID-19 brings unprecedented pressure and challenges to global public health and changes the landscape of influenza prevalence. In front of the complexity and unpredictability of the influenza epidemic and its co-circulation with COVID-19, the concerns for seasonal influenza among humans and the avian influenza among birds should not be reduced. Herein, on the occasion of the fourth World Flu Day, we would like to lift up our voice to bring the attention of the public to influenza. Public understanding of science plays a pivotal role in the implementation of disease control and prevention strategies (19)(20). Historical lessons learnt from influenza and current experience obtained from COVID-19 should remain at the core of global efforts for pandemic preparedness. Commemorating World Flu Day together is not just a timely call for raising global awareness about this common and easily ignored disease, especially with the COVID-19 pandemic under the global spotlight, but it is also an important opportunity to advocate for the vaccination for the two pandemic-igniting viruses.
In this issue of the China CDC Weekly, we have reported the surveillance of influenza activity in the mainland of China in 2020-2021, with an intensity of activity lower than before COVID-19, but gradually increasing compared to 2020 (21); this issue also reported two human cases of H5N6 infection in 2021 in Guangxi Zhuang Autonomous Region, China, which provides evidence of a continuous threat to humans from H5Ny avian influenza viruses (22). We also included a review article on continuous circulation, frequent gene reassortment, and mammalian adaptation of H3 avian influenza viruses in the past two decades in the mainland of China (23). A review on the Global Influenza Surveillance and Response System (GISRS) was also included (24), which summarized the existing WHO influenza surveillance systems that have been leveraged for global responses to COVID-19 since the beginning of the pandemic and addressed future challenges and plans. Methods: Positive rates of influenza virus detection during this period were based on real-time polymerase chain reaction (PCR) detection by the Chinese National Influenza Surveillance Network laboratories, and isolated viruses from influenza positive samples were submitted to the Chinese National Influenza Center. Antigenic analyses for influenza viruses were conducted using the hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. Viruses were tested for resistance to antiviral medications using a phenotypic assay and next-generation sequencing.
Results: In southern China, the influenza positivity rate was elevated especially after March 2021 and was higher than the same period the previous year with the COVID-19 pandemic. In northern China, influenza positive rate peaked at Week 18 in 2021 and has declined since then. Nearly all isolated viruses were B/Victoria lineage viruses during the study period, and 37.3% of these viruses are antigenically similar to the reference viruses representing the vaccine components for the 2020-2021 and 2021-2022 Northern Hemisphere influenza season. All seasonal influenza viruses were susceptible to neuraminidase inhibitors and endonuclease inhibitors.
Conclusions: Influenza activity has gradually increased in the mainland of China in 2021, although the intensity of activity is still lower than before the COVID-19 pandemic. The diversity of circulating influenza types/subtypes decreased, with the vast majority being B/Victoria lineage viruses. The surveillance data from this study suggest that we should strengthen influenza surveillance during the upcoming traditional influenza season. It also provided evidence for vaccine recommendations and prevention and control of influenza and clinical use of antiviral drugs.

INTRODUCTION
Influenza is a respiratory illness that infects between 5%-15% of the global population annually during normal seasonal epidemics, and the World Health Organization (WHO) estimates that these infections result in 3-5 million cases of severe illness and about 290,000 to 650,000 respiratory deaths every year (1). Rapid evolution of influenza viruses creates difficulties in recognizing and predicting current and future epidemic threats (2).
Since the March 2020 co-incident with the exponential global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a dramatic decrease in influenza detection has been observed, despite the testing for influenza continuing at similar levels in many countries (3)(4). Influenza is traditionally prevalent in the Southern Hemisphere during April-July 2020, but the positive rate for influenza decreased dramatically from 13.7% in 2017-2019 to 0.06% in 2020 (5). The Northern Hemisphere has also experienced almost no influenza during the typical timing of the 2020-2021 season, compared to around 18%-23% in 2019-2020, the US and EU reporting only 0.15%-0.20% of samples as positive for influenza (6).
Influenza A(H1N1)pdm09, A(H3N2), and influenza B viruses circulated in low numbers and the predominant circulating viruses varied among reporting countries. Since September 2020 influenza activity was mostly reported from countries in the tropics and subtropics and some countries in the temperate zone of the Northern Hemisphere and Southern Hemisphere (7). It is possible that this continuous low-level virus circulation may allow influenza transmission following significant restarting of international travel. Now that we are approaching the influenza season in the mainland of China, it is important that we understand last year's influenza activity and surveillance results so that we can be better prepared for future influenza outbreaks. Therefore, we analyzed the antigenic and genetic characteristics and antiviral susceptibilities of influenza viruses isolated from the mainland of China in 2020-2021.

METHODS
The Chinese National Influenza Surveillance Network currently includes 410 laboratories and 554 sentinel hospitals. Influenza-like illness (ILI) cases were reported by sentinel hospitals to the Chinese National Influenza Surveillance Information System (CNISIS), and respiratory specimens were collected. Network laboratories determined whether the sample was influenza virus positive using real-time reverse transcription polymerase chain reaction (RT-PCR). Influenza positive specimens were propagated in Madin-Darby canine kidney (MDCK) cells and/or embryonated chicken eggs. The Chinese National Influenza Center (CNIC) received the viruses for further analysis.
Antigenic characterization was based primarily from results of hemagglutination inhibition (HI) assays, and genetic characterization was carried out based on nextgeneration sequencing. An HI titer less than or equal to 4 times that for the homologous virus was considered to be antigenically similar. Testing of seasonal influenza viruses for resistance to the neuraminidase and endonuclease inhibitors was performed at CNIC using next-generation sequencing analysis, a functional assay, or both. The viruses evaluated were isolated from specimens collected between Week 41 in 2020 (October 5, 2020) and Week 35 in 2021 (September 5, 2021).

RESULTS
Influenza activity in the mainland of China was below pre-pandemic COVID-19 levels during the period October 5, 2020 to September 5, 2021 (surveillance Week 41 in 2020 to Week 35 in 2021). The percentage of specimens testing positive for influenza each week ranged from 0.1% to 6.2% in southern China and ranged from 0% to 10.6% in northern China. The positivity rate of specimens collected from ILI cases slightly increased starting at Week 49 in 2020 and had been increasing in 2021 in southern China. The positivity rate in northern China began to increase in Week 10 of 2021, peaking at Week 18 of 2021, and declined since then (Figures 1  and 2).
Antigenic characterization of the 1,819 B/Victoria lineage viruses were conducted using HI tests. A total of 1,141 (62.7%) B/Victoria lineage viruses were not recognized well by the ferret antisera raised against B/Washington/02/2019, the egg-propagated reference virus representing the B/Victoria lineage component of the 2020-2021 and 2021-2022 Northern Hemisphere influenza vaccines (7)(8). Among the 797 viruses HA gene segments sequenced and phylogenetically analyzed, all belonged to genetic clade 1A, nearly all belonged to subclade 1A.3, which shared a three amino acid deletion in HA (positions 162-164) and the substitution K136E, and in which, 96.7% belong to group 1A.3a characterized by HA amino acid substitutions N150K, G184E, N197D, and R279K, and was further divided into subgroup 1A.3a1 (520, 65.2%), which had additional HA amino acid substitutions V220M and P241Q, and 1A.3a2 (251, 31.5%), which had HA amino acid substitutions A127T, P144L, and K203R.
No A(H3N2) and B/Yamagata lineage viruses were isolated and available for characterization during the study period.
Of the 2 A(H1N1)pdm09 viruses collected in the mainland of China, neither showed evidence of reduced inhibition by oseltamivir and zanamivir, nor to baloxavir. Of the 1,258 influenza B/Victoria lineage viruses screened for neuraminidase inhibitors susceptibility, all showed normal inhibition by oseltamivir and zanamivir. A total of 797 B/Victoria lineage viruses were screened for susceptibility to the endonuclease inhibitor, baloxavir, by genetic analysis, and none showed evidence of reduced susceptibility.

DISCUSSION
In the mainland of China, circulation of influenza

China CDC Weekly
virus was disrupted during the COVID-19 pandemic. During 2020, influenza virus circulation showed historically low levels (9). In 2021, influenza continues to circulate at low levels but higher than the equivalent period since March 2020 (9). The WHO meets twice a year to recommend influenza vaccines. In late February 2021, the WHO issued its recommendations for the 2021-2022 Northern Hemisphere influenza vaccines. Compared to the 2020-2021 Northern Hemisphere influenza vaccine components, the A(H1N1)pdm09 and A(H3N2) vaccine components were updated, except for the B/Yamagata and B/Victoria lineage (7)(8).
Influenza B/Victoria lineage viruses were predominant nationwide during the study period. Significant genetic diversity was seen in circulating viruses of the B/Victoria lineage. The viruses in the 1A.3a group were the most prevalent in the mainland of China. This 1A.3a HA group further diversified into 2 subgroups that 1A.3a1 had substitutions of either V220M and P241Q, which were predominant and seen almost exclusively in China, or 1A.3a2 with additional amino acid substitutions A127T, P144L, and K203R, for which the proportion has been increasing in recent months. Subgroup 1A.3a2 shows further genetic differentiation, with additional HA amino acid substitutions found in viruses from other global regions (10). The majority of group 1A.3a viruses showed reductions in inhibition by postinfection ferret antisera raised against egg-propagated 1A.3 viruses, such as B/Washington/02/2019 (2020-2021 and 2021-2022 Northern Hemisphere influenza vaccine components).
Only 2 A(H1N1)pdm09 viruses were available for antigenic analysis during this period. Ferret antisera raised against egg-propagated A/Victoria/2570/2019 (2021-2022 Northern Hemisphere influenza vaccine components), which belonged to phylogenetic group 6B.1A5A2, recognized the virus within 6B.1A5A2 virus well but showed poor recognition of 6B.1A5A virus. The global 6B.1A5A2 viruses increased rapidly in the early 2020s until it reached similar proportions to 6B.1A5A1, but 6B.1A5A1 viruses dominant from September 2020 and 6B.1A5A2 viruses were recently detected (10).
No A(H3N2) and B/Yamagata lineage viruses were available for characterization during the study period. Influenza A(H3N2) viruses were reported in most regions, including Africa, Asia, North America, Oceania and Europe, but the clades of A(H3N2) viruses circulating varied among reporting countries.
Based on our analysis, all tested viruses for susceptibility to therapeutics were susceptible to neuraminidase and endonuclease inhibitors. Antiinfluenza drugs were used as an effective means of postexposure prophylaxis and treatment of influenza virus infection. Continuous monitoring of circulating influenza viruses for antiviral resistance is still important.
Because of the common transmission route between SARS-CoV-2 and influenza, the same protective behaviors can greatly limit the spread of influenza (11)(12)(13). In the mainland of China, B/Victoria lineage viruses continued to be identified, but the diversity of types/subtypes co-circulating has decreased compared to recent seasons. Globally, few B/Yamagata lineage viruses have been found during the COVID-19 pandemic. The surveillance data presented here are a reminder that the low level of influenza circulation since early 2020 may not necessarily persist into the upcoming influenza season space. Given the long absence of sustained natural exposure to influenza viruses, the reduction in influenza virus transmission over the past year may affect the severity of the upcoming influenza season. Lower levels of population immunity, especially in young children and the elderly, may portend more widespread illness and potentially more severe epidemics when the influenza viruses reemerge (14)(15). The lack of influenza circulation in the previous season will reduce our confidence in the strains that may circulate in the next season and may result in mismatch between the vaccine and circulating strains. Maintaining surveillance and outbreak response is essential to track geographic spread of the virus and its variation characteristics so that the vaccine remains optimized for circulating influenza viruses, and influenza surveillance should continue to be strengthened.

INVESTIGATION AND RESULTS
We reported 2 cases infected with the H5N6 virus belonging to genetic clade 2.3.4.4b and clade 2.3.4.4h in Guangxi, China in 2021. These 2 cases occurred in two different cities. A case was admitted to the intensive care unit (ICU) due to severe clinical symptoms and was subsequently tested positive for H5N6. The other case was detected from the influenza-like illness (ILI) surveillance system. When a case is found, the local CDC immediately conducts epidemiological and environmental investigations.
On February 16, 2021, a 50-year-old male (Patient A) developed a fever with chest tightness, tightness of breath, headache, cough, sputum, and pneumonia, and was transferred to the Respiratory Department of Hechi People's Hospital for hospitalization on February 21. Patient A had suffered from rheumatoid arthritis for more than 10 years, and he was found to be H5N6 influenza virus positive on February 26. He had a history of exposure to infected poultry 9 days before the onset of illness and died on March 2.
On July 6, 2021, a 61-year-old female (patient B) developed a fever with a maximum temperature of 38.5 °C and was admitted to the Second People's Hospital of Guangxi for treatment on July 9. After 6 days, she was confirmed to be positive for H5N6 by Guangxi CDC. She had a history of chronic gastritis and denied having contact with live poultry.
Viral RNA from the throat swabs of the two patients was extracted using the QIAamp ® Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Specific real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays with specific primer and probe sets for detecting avian influenza A (H5N6). The viral genomes were reversetranscribed and amplified using Easy-Fast FluA whole genome amplification one-step kit (Xinlihechuang Technology Co., Ltd.). The sequencing libraries were prepared using the Illumina Nextera ® XT Library Prep Kit. The final viral-enriched libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, USA). The whole genome sequences of the 2 avian influenza A (H5N6) strains were assembled and obtained using CLC Genomics Workbench 9.5.2, and the 2 strains were designated A/GX-hechi/01/ 2021(H5N6) (GX01) from Patient A and A/GXguilin/11151/2021(H5N6) (GX11151) from Patient B. The genomes were aligned with the reference genomes downloaded from GISAID (www.gisaid.org) using MAFFT v7.037b (https://mafft.cbrc.jp/ alignment/software/). The genetic and evolutionary analyses were conducted in MEGA 7.0.14 (https://www. megasoftware.net/). Through sequencing and splicing, 8 gene fragments of the virus were successfully extracted. Some mutations related to viral replication, receptor-binding, mammalian virulence-related markers and drugresistance related markers were detected. The PB2 and MP genes of GX01 virus showed high homology with H9N2 viruses, and the following genes showed high homology with H5N6 viruses. The HA and NP genes of GX11151 virus showed high homology with H5N8 and H9N2 viruses, respectively, and the following genes showed high homology with H5N6 viruses (Table 1).
Phylogenetic analysis of HA gene was constructed according to the World Health Organization (WHO) reference sequence. Their nucleotide sequence showed a similarity of 90.7% between each other. The HA gene of GX01 falls into clade 2.3.4.4h, while GX11151falls into clade2.3.4.4b (Figure 1).
The HA cleavage site of two viruses possessed a multiple basic amino acids motif, indicating potentially high pathogenicity in chickens. The receptor-binding site at the 222-224 motif was QGG of GX01 virus and QRG of GX11151 virus, respectively, suggesting that these viruses preferred binding to avian-like receptors (α 2,3 SA) (5). However, D94N (6), S133A (7), and T156A (8) mutations in GX01 HA gene, S133A, D155N (8), T156A, and T188I (7)  A263T mutation in HA gene was detected in both strains, suggesting that the virulence was enhanced. However, there was no mutation associated with resistance to NA inhibitors in the NA gene of the two strains. K389R, V598T/I mutations of PB2 gene, and N409S mutation of PA gene, which could increase virus replicative ability in mammals, were observed in both strains. Some mutations increasing virulence in

DISCUSSION
The previous study showed H5N6 has replaced H5N1 as one of the dominant AV subtypes in southern China (9). The avian influenza A (H5N6) virus continues to threaten human life and health.  I  T  T  T  T  T  V   PB1   I368V  I  I  I  I  I  V  Transmissible among

Increased virulence in mice T139A
A  (12)(13). Fortunately, these mutations were not found in our virus. Therefore, using NA inhibitors to treat the 2 cases infected with avian influenza A (H5N6) was still a good choice. The median age of the two cases was over 50 years old. Like other studies previously reported, the elderly people may be more vulnerable to avian influenza (3). One case was reported through ILI surveillance system (14), indicating that the ILI system was beneficial for the detection of avian influenza cases to a certain extent. However, this may be just the tip of the iceberg, and perhaps many mild cases have not been detected.
This study has certain limitations. Sampling of the environment exposed by the cases, and poultry around the living environment were missing, resulting in lacking of laboratory tests. One reason was that the period from onset to reporting was too long for sampling, in spite of dead poultry found in epidemiology survey, and another reason was that patient B had no clear history of exposure to poultry.

Epidemiological and Genetic Characteristics of the H3 Subtype Avian Influenza Viruses in China
Jiaying Yang 1,2 ; Lei Yang 2 ; Wenfei Zhu 2 ; Dayan Wang 2 ; Yuelong Shu 1,2,# Avian influenza viruses (AIVs) are naturally preserved in waterfowls and sometimes spill over to infect humans and mammalian animals. The H3 subtype is one of the most prevalent influenza virus subtypes in waterfowls. Since 2000, H3 subtype AIVs have been continuously isolated from poultry and wild birds in the mainland of China, which implied a dynamic spread in large-scale geography and multiple species. Combinations of H3 with N1-N8 subtypes were reported, among which H3N2 and H3N8 subtypes predominated. Frequent mutations and reassortments increased the genetic diversity associated with altered virus virulence, transmission, and mammalian adaptation of H3 AIVs, posing a potential threat to animal and human health. This study systematically analyzed the epidemiology, genetic characteristics, and mammalian adaptation-related mutations of H3 subtype AIVs in the mainland of China, facilitating the H3 subtype AIVs research and risk assessment.
Avian influenza virus (AIV) belongs to the Orthomyxoviridae family Influenza virus A genus, packaged with 8 negative-sense and single-strand RNA segments, which encode a total of 14 proteins (1). Based on the antigenic diversity of surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), AIV can be classified into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) (2)(3). Except for the H17N10 and H18N11 subtypes found in bats in recent years, all other subtypes of influenza A virus can be found in wild waterfowls. Therefore, wild waterfowls are known as the natural reservoir and gene pool of influenza A virus, and AIV is considered to be the source of other animal influenza viruses (4). According to the pathogenicity of AIV to chickens, it can be divided into highly pathogenic AIV (HPAIV) and lowly pathogenic AIV (LPAIV) (5). Most avian influenza viruses exhibit low pathogenicity. To date, only partial proportions of H5 and H7 subtype AIVs have developed high pathogenicity, causing high mortality in wild birds, poultry, and even humans.
Among influenza A virus subtypes, the H3 subtype has a wide range of hosts. In addition to circulating in wild birds and poultry, it can infect multiple species of mammals such as humans, pigs, dogs, cats, horses, and even seals (6). The H3 subtype AIV is LPAIV and one of the influenza virus subtypes with the highest isolation rate among ducks (proportion up to 91.76%) (7). Surveillance data showed that the H3 subtype AIV was widely prevalent in domestic ducks in the live poultry markets (LPMs) in China and reassortants were continuously being detected (8)(9). Genetically, the HA genes of H3 subtype AIVs could be divided into Eurasian and North American lineages. Both H3 lineages were able to cross the species barrier to infect swine, while Eurasian lineage was identified to infect a wider range of hosts (swine, equine, canine, and human) than North American lineage (swine and seals) (10). In China, only the Eurasian lineage has been detected in domestic poultry (11). The interspecies transmissibility of H3 AIVs historically caused the H3N2 influenza pandemic in 1968 (12), which has posed a threat to human health (13).
Here, we systematically reviewed the current situation of the H3 subtype AIVs in China using surveillance data from 82 published studies and summarized the epidemiological and genetic characteristics and the mammalian adaptation of H3 AIVs in the first 20 years of 21st century in the mainland of China.

EPIDEMIOLOGY OF H3 AIVS IN CHINA
Since the 1970s, China has reported isolation of AIVs (14). After Hong Kong Special Administrative Region (SAR) reported 18 human infections and 6 deaths caused by HPAIV (H5N1) in 1997 (15), avian influenza surveillance has received global attention for pandemic preparedness. During 2000-2019, AIV surveillance had been conducted in LPMs, poultry farms, and wild birds habitats in the mainland of China. Extensive and continuous active surveillance work included collecting throat and cloacal samples from birds and bird-related environmental specimens such as feces, water, and cage surfaces (11,(16)(17)(18)(19)(20)(21). Since 2009, surveillance has been intensified in all of 31 provincial-level administrative divisions (PLADs), with a sampling frequency from 2-4 weeks a time to once a year. H3 AIVs were subtyped by RT-PCR or sequencing directly from the samples or AIV isolates.
To summarize the activities of H3 AIVs in the mainland of China, we have counted each H3Nx subtype at the provincial scale each year. For example, if one or more studies reported that H3N2 AIV was found in Heilongjiang Province in 2010, it would be an activity of H3N2 AIV. Since 2000, 214 activities of H3 AIVs have been detected in 26 PLADs in the mainland of China except for the PLADs of Xizang (Tibet), Gansu, Shaanxi, Shanxi, and Hainan ( Figure 1A). Surveillance indicated that H3 viruses have become enzootic in domestic ducks in southern China * , where LPMs bring together numerous host species in a high-density setting, creating an ideal environment for viral reassortment and interspecies transmission (23). In eastern China † and northeastern China § , H3 AIVs could affect both domestic poultry and wild birds, except for Fujian, Shandong and Zhejiang provinces where H3 viruses were only detected in domestic poultry ( Figure 1A). Previous studies have reported that H3N8 circulated in the Poyang Lake area around Jiangxi Province (18) and the wetlands of Jiangsu Province (28), which are along the wild fowl migratory flyway. In northeastern China, H3 AIVs with multiple NA subtypes (including N2-N8) were detected in wild birds in Heilongjiang Province ( Figure 1A), where the complicated bird migration network lies (29). Researchers have continuously detected novel H3N8 subtype AIVs in wild ducks in their habitats such as the Heilongjiang Sanjiang Nature Reserve (30). H3 subtype AIVs were mainly detected in wild birds in northwestern China ¶ , while in southwestern China ** they were mainly found in domestic poultry ( Figure 1A).
In the last decade, increased detection of H3 AIVs were reported throughout the country. Notably, H3N2 and H3N8 have been consistently detected ( Figure 1B). An epidemiological survey showed that poultry infection with H3N2 AIV occurred throughout the year in southern China, with higher detection rates in winter and spring and a lower detection rate in summer (31). In the wetlands of northeastern China, virus isolation rate of H3 subtype AIVs varied among seasons, and the highest rate was in autumn, which might due to the migration of wild birds (32).
In summary, H3N2 subtype AIV was the main prevalent subtype in poultry farms and LPMs from China, while H3N8 subtype AIV was widespread among wild birds in this region. Therefore, the following focuses on the H3N2 subtype and H3N8 subtype AIVs.

GENETIC EVOLUTION OF H3 AIVS IN CHINA H3N2 Subtype AIV
Phylogenetic analysis showed that all of the H3N2 subtype AIVs in China belonged to the Eurasian lineage (11). The HA gene may be derived from viruses circulating in different regions of China and neighbouring countries in East and Southeast Asia (7,17,33), while the NA gene may be derived from other AIVs such as H5 (34)(35), H7 (9,36), H9 (23,36), and H11 (34) subtype AIVs in birds in East and Southeast Asia. The prevalence of H3N2 AIVs in birds and frequent reassortments with other AIVs also led to the emergence of various genotypes. Most of the reassortants came from LPMs along the bird migration routes, which suggests that wild bird migration might have a great impact on poultry AIV infection (8,18,23,37). Studies have found that the PA gene of H3N2 subtype AIV of Chinese ducks was highly homologous to the HPAIV H7N9 in Korean wild ducks (33). The H9N2 viruses that were prevalent in poultry in eastern China [Jiangxi (23), Jiangsu (38), Zhejiang (39)] also provided internal genes for H3N2  AIVs. The internal genes of some H3N2 AIVs were closely related to HPAI H5 AIVs, which may suggest reassortment with H5N1, H5N2, H5N3, H5N6, and H5N8 subtype AIVs (8,32,34,40).

H3N8 Subtype AIV
Molecular epidemiological analysis revealed that the H3N8 subtype AIVs had complex sources. Studies have shown that the HA gene of H3N8 subtype AIV might come from the H3 subtype AIV that is prevalent in poultry in East Asia, Southeast Asia, and Europe (41)(42), and the NA gene might come from East Asia, North America, and Europe (43)(44). The NA gene of H3N8 subtype AIV could be divided into Eurasian lineage and North American lineage (45). The Eurasian lineage was widely detected in the whole country, while the North American lineage was mainly detected in eastern China (8,11,45), indicating that gene reassortments have occurred between AIVs from the Eurasian lineage and the North American lineage (8,(45)(46)(47). These events most likely occurred in LPMs and could be transmitted from ducks to chickens (48). The internal genes of H3N8 AIVs were derived from a variety of AIVs from wild birds or domestic ducks (24,(49)(50), including HPAI H5 viruses (28,43,(51)(52).
The H3N8 subtype AIVs might be potential gene sources of AIVs causing interspecies transmission. During 2004-2005, researchers detected an H3N8 virus in domestic ducks in Beijing LPMs, of which HA gene was highly similar to the H3N8 virus that caused the 1989-1990 outbreaks in equine populations in northern China (43). In 2018, researchers found that the NA gene of the H3N8 virus isolated from a Guangdong LPM was highly homologous to H10N8 AIV and speculated that the N8 gene originated from the same lineage that caused human infections in 2014 (48).

Other H3 Subtypes AIV
Except for the AIVs of H3N2 and H3N8 subtypes, other AIVs of H3 subtypes were scattered in various regions of China ( Figure 1) and had a high degree of genetic diversity and origin. A study indicated that the H5N1 subtype AIV in domestic ducks in Anhui Province and Fujian Province might be involved in the gene reassortment of H3N1 subtype AIV (53). The internal genes of H3N3 might be derived from viruses of waterfowl origin (mainly ducks) in North America (Alaska) and East Asia (Japan and the Republic of Korea) (21)(22)(23)39). The HA gene of H3N5 detected in wild birds in Khanka Lake was closely related to poultry-origin AIVs isolated from southern China (21). Studies have shown that H3N6 frequently reassorted with H5N6 subtype AIVs (18,(54)(55), while PB2 and NP genes were also derived from H9N2 AIV that circulated in the same period (23). Experimental results showed that the novel H3N6 reassortant could effectively replicate in mammalian cells (54). The gene of H3N7 subtype AIV came from a variety of LPAIVs and had greater antigenic differences compared with the H3 subtype AIV strains isolated from previous studies (56).

MAMMALIAN ADAPTIONS OF H3 SUBTYPE AIV
In 1968, a novel H3N2 subtype virus contained genes from human-derived H2N2 influenza virus and avian-derived H3 subtype influenza virus was firstly reported in Hong Kong SAR and caused the pandemic globally (12). It highlighted the pandemic potential of H3 subtype AIVs. Mutations that increased replicative ability and transmissibility of the virus in mammals may facilitate the interspecies transmission (57)(58)(59). Previous studies have experimentally demonstrated the relation between some molecular mutations and receptor binding, transmission, replication, pathogenicity, and drug resistance of the H3 subtype AIV in mammals. Here, we have summarized the mammalian adaptation molecular markers in Table 1.
Receptor binding is the initial process of virus life circle. AIVs usually show higher binding preference for α2,3-Gal sialic acid receptors (avian-type), and increased binding capacity of α2,6-Gal sialic acid receptors (human-type) indicates increased adaptability in mammals. Studies have shown that H3N2 and H3N8 subtype AIVs could simultaneously bind to both avian-type and human-type receptors (17,22,60). Overall, 2 residues, 226 and 228, of HA of the H3 subtype were well known to be important for the host range restriction (61). Recently, researchers found that residue 155T in the HA protein could enhance the ability of H3N2 subtype AIV to bind to human-type receptors, and the virus could replicate effectively in the lungs and turbinates of mice (9). The G225D mutation of HA could increase the thermal stability of H3N2 AIV, which could probably lead to increased virus replication on Madin-Darby canine kidney (MDCK) cells (7). The H3N2 subtype AIV with Q226R and G228S mutations in HA could enhance the replicative ability and mammalian adaptability in ferrets (17). The Q226L mutation of HA could promote the airborne transmission of the H3N8 virus in ferrets (45).
The RNA polymerase of the influenza virus is composed of three subunits including PB2, PB1, and PA proteins, which are related to the virus's hostspecificity and replication. Dong et al. found that E627K and D701N mutations in the PB2 protein of H3N8 AIV (51) may enhance the adaptability of the virus in mammalian cells and enable AIV to replicate efficiently in mammals (62)(63)(64)(65). Another study found that the PB1 S524G mutation of wild bird-origin H3N8 virus could enhance the virulence and fitness for transmission in mammals (45).
Mutations that increase pathogenicity were also found. The N30D and T215A mutations found in the M1 protein could increase the pathogenicity of the H3N8 subtype AIV in mice (51). The residue at 42 of the NS1 protein in the H3N2 subtype AIV changed from P to S, which might increase virus replication and virulence in mice (7).
For drug resistance, the D198N and G119P mutations of NA protein have been identified in the natural isolates of H3N6 and H3N8, respectively. These mutations were likely to cause the H3 subtype AIV with reduced sensitivity of NA inhibitors (28,53). In addition, many studies have found that the V27I and S31N mutations of the matrix protein (M) of H3N2 subtype AIV caused the virus to be resistant to amantadine (8,11,18,32).

DISCUSSION
The H3 subtype AIVs have continuously circulated in wild birds and poultry in the mainland of China. Wild bird migration and live poultry trade play important roles for virus transmission. Frequent gene reassortments were observed between viruses from poultry and wild birds (52,(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66), which increased the genetic diversity of H3 AIVs and contributed genes to other subtype AIVs such as HPAIV H5 (67)(68). Despite the relatively improved surveillance studies having been performed in recent years in China, our understanding of the H3 AIVs transmission and evolution is still limited, especially in intercontinental migratory birds. The H3 viruses could acquire mammalian adaptation mutations during replication. Considering the pandemic history and the potential

Leveraging Global Influenza Surveillance and Response System for the COVID-19 Pandemic Response and Beyond INTRODUCTION
Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, existing influenza surveillance systems, platforms, and capacities have been leveraged for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus detection, reporting, risk assessment, and sharing of virus materials and data. This article reviews the contributions made and addresses some of the future challenges and plans for the Global Influenza Surveillance and Response System (GISRS) and associated influenza surveillance systems.

DESCRIPTION
Since 1952, the world has benefited from an international collaboration of laboratories, public health institutions, and other partners to protect people from influenza. Beginning with 26 laboratories, the World Health Organization (WHO) Global Influenza Surveillance Network (GISN), renamed as the "Global Influenza Surveillance and Response System (GISRS)" in 2011, has expanded in number and scope to now 158 institutions in 127 countries. The network has adapted to the needs and changing capacities in countries as well as events and scientific advancements, with trust and country ownership key to its success (1-2).

SCOPE
From the early years of the network, there has been an emphasis on the sharing of influenza virus specimens and related information from Member States within the network and on the development and dissemination of technical guidance and standardized protocols. Member States continue to regularly share virus materials, alongside virologic and epidemiologic influenza-related information year-round. Through web-based data platforms such as FluNet (3) and FluID (4), there has been a steady increase in the number of samples tested for influenza since 2011. Since 2015, surveillance of respiratory syncytial virus (RSV) (5) based on GISRS has been piloted for integration into the influenza surveillance system. WHO, through the Global Influenza Programme (6), regularly updates technical guidance on laboratory techniques, influenza surveillance standards, influenza vaccine virus strains, and other pharmaceutical and non-pharmaceutical measures to mitigate influenzarelated morbidity and mortality.

HISTORICAL PERSPECTIVE
Ever since its inception, GISRS has played key roles in the response to 3 influenza pandemics (in 1957, 1968, and 2009), outbreaks of animal influenza viruses, and the occasional spill-over of these viruses into humans, also including other non-influenza events such as the SARS outbreak in 2002 and the current SARS-CoV-2 pandemic. These events have highlighted the important international coordinating role of WHO and GISRS in pandemic preparedness and response (7).
GISRS continues to monitor the influenza viruses circulating in animal populations that may be a risk to humans. Working closely with international animal health partners, such as the Food and Agriculture Organization (FAO) (http://www.fao.org) and the World Organisation for Animal Health (OIE) (8), GISRS works to detect and respond to the sporadic spill-over of animal influenza viruses into humans (zoonotic transmission events).
In 2011, WHO's Member States and Executive Board called upon WHO's Global Influenza Programme and GISRS to expand its work on pandemic preparedness and response with the adoption of the Pandemic Influenza Preparedness Framework for the Sharing of Influenza Viruses and Access to Vaccines and Other Benefits (PIP Framework). The objective of the framework was to improve influenza pandemic preparedness and response by improving and strengthening GISRS (9). With partners globally, the PIP Framework assists Member States with building national and international capacities to better detect and respond to inevitable future pandemics of influenza and beyond.

ACHIEVEMENTS
The achievements of GISRS and WHO's Global Influenza Programme since its inception, notably the timely response to the 2009 influenza pandemic, have been demonstrated and documented elsewhere (7,(10)(11). However, it is worth outlining how GISRS and associated influenza surveillance systems have also significantly contributed to the response to COVID-19 pandemic, caused by a non-influenza respiratory virus.

National Influenza Centres were Crucial to Initial Laboratory Response During COVID-19
National Influenza Centres (NICs) are the backbone of GISRS, collecting clinical specimens from sentinel and non-sentinel sources in their country, testing them for influenza and other respiratory viruses, performing preliminary analyses on influenza viruses detected, and sharing representative specimens or viruses with WHO Collaborating Centres (CCs) for advanced antigenic and genetic analyses. NICs are designated by national Ministries of Health and recognized by WHO. Given the capacities and expertise in these laboratories of the GISRS network, they became COVID-19 testing centres in many countries early in the SARS-CoV-2 pandemic. As of July 2020, approximately 85% of over 220 national COVID-19 reference or testing laboratories globally were either NICs or closely associated with GISRS (12).

National Influenza Centres have Demonstrated Expertise of Virus Detection
The first External Quality Assessment Project (EQAP) for SARS-CoV-2, which was critical to ensure national capacity of laboratory detection of the novel virus, was undertaken through the GISRS annual influenza EQAP mechanism, coordinated by WHO's GIP (13). A total of 233 laboratories (including 130 NICs) in 164 countries, areas, and territories participated, with the survey dispatched to labs from April to June 2020, despite disruptions caused by the pandemic. Overall, 94% of laboratories received a 100% pass rate. Of the 233 laboratories that participated in the SARS-CoV-2 EQAP, 68% (159/233) also participated in the influenza EQAP in 2019, indicating the substantial capacity for influenza testing that was utilized for testing during the COVID-19 response. Among laboratories that participated in the 2019 influenza EQAP, 94% of the labs scored 100% on the 2020 SARS-CoV-2 EQA.

Surveillance Data Generating and Sharing to Global Platforms
Data is key to understanding the dynamics of and informing the response to seasonal epidemics and pandemics. A key component of GISRS is the global data platform FluMart, in place since 1997, where countries report data on samples processed for influenza, such as the number of positive samples and subtype, on a weekly basis to a dataset called FluNet. Outputs of the data are visible to the public on WHO's website (14). Since the emergence of SARS-CoV-2, WHO has requested that Member States regularly report counts of COVID-19 cases and deaths at different levels of aggregation for global situational awareness. FluMart formed the basis of the data collection platform for COVID-19 data (COVMart) since early in the pandemic. Globally, as of September 10, 2021, there have been 223,022,538 confirmed cases of COVID-19, including 4,602,882 deaths, reported to WHO through COVMart and displayed on the WHO COVID-19 dashboard (15).

Genomic Sequencing and Genetic Sequence Data Sharing
The sharing of virus genetic sequence data is essential, especially in the early stages of the response to an outbreak of a novel virus. Sequence data allows for the development of laboratory protocols to enable countries to detect and monitor novel viruses. GISAID (available at: https://www.gisaid.org/), which was launched in 2008 to enable rapid and open access to epidemic and pandemic influenza virus genetic data and associated clinical and geographical information through its EpiFlu platform, quickly became the host of the genetic sequence data of SARS-CoV-2. In fact, it published the first SARS-CoV-2 sequence data hours after it became available. GISAID has been an important partner of GISRS with confidence from countries for sharing their data through its data sharing mechanism. The development of the EpiCoV database and associated tools has been instrumental in the COVID-19 pandemic response. As of September 12, 2021, since the emergence of SARS-CoV-2, nearly 3.5 million complete genome sequences of SARS-CoV-2 have been uploaded to GISAID. About 80% of GISRS laboratories uploaded complete genome sequence data of SARS-CoV-2 to GISAID EpiCoV. In 2021, GISAID has also expanded to collect and share information on RSV, aside from influenza and SARS-CoV-2, though its EpiRSV data platform.

LEVERAGING EXISTING SURVEILLANCE SYSTEMS FOR MONITORING COVID-19
Sentinel surveillance is an efficient way to collect high-quality data in a timely manner systematically (using a consistent case definition and sampling strategy) and routinely from representatives of the population under surveillance so that the information gathered can be applied to the population or among subpopulations at higher risk of developing severe disease. Sentinel surveillance for COVID-19 using GISRS complements COVID-19 surveillance activities under the overall COVID-19 pandemic response. It is a cost-effective way to meet pandemic response objectives, including monitoring the geographic spread, intensity of transmission, and severity trends of community transmission of COVID-19 over time; understanding the risk factors for disease; systematically monitoring the genetic evolution of the COVID-19 virus; and assessing the impact on health systems. In addition, understanding the relative contribution of SARS-CoV-2 to the data captured in syndromic disease surveillance systems is critical to guide national responses and interpret the burden of disease caused by SARS-CoV-2 and influenza viruses. The data gathered can be used as a baseline for future evaluations of COVID-19 interventions such as vaccination, as has been already undertaken for influenza (12).
Since March of 2020, GISRS has incorporated SARS-CoV-2 into laboratory algorithms for testing specimens obtained from sentinel surveillance to monitor trends in co-circulation of the influenza and SARS-CoV-2 viruses and has provided guidance on the reporting of this information to FluNet, the global dataset managed by GIP (16). This integrated surveillance has been enhanced by the development, validation, and procurement of multiplex assays to test samples for influenza and SARS-CoV-2 simultaneously. In response to the recommendations of the 6th International Health Regulations (2005) (IHR) Emergency Committee for COVID-19, WHO encourages GISRS laboratories to sequence SARS-CoV-2-positive samples from its sentinel surveillance in a timely manner and share genetic sequence data with accompanying metadata through publicly accessible databases such as the GISAID EpiCoV database, with a focus on data quality over quantity (17).

CURRENT CHALLENGES AND THREATS
Much progress has been made on building capacities globally, regionally, and nationally to protect the world from influenza. However, in addition to the disruptions caused by the COVID-19 pandemic to livelihoods and health care systems, and the threat of misinformation, there are some challenges unique to influenza surveillance worth including here.
Although in most parts of the world, influenza activity has been lower than usual during the COVID-19 pandemic, an unusual epidemic can break out anytime. On the other hand, animal influenza viruses continue to sporadically infect humans, highlighting the continuing threat of zoonotic transmission and the need to remain vigilant for the unexpected and work with the One Health concept in mind (18). We also need to remain open to the possibility of another respiratory virus emerging and causing the next pandemic.
It is not clear when seasonal influenza activity will resume, and at what levels globally, but it is clear the surveillance systems need to be in place and functioning to quickly detect the return of influenza activity, alongside monitoring the trends post-COVID-19. In many countries, the current priority is understandably the response to the COVID-19 pandemic and exhaustive case finding and management efforts to control transmission. Contextspecific solutions to achieving the pandemic response objectives and the ongoing influenza surveillance objectives need to be put in place. The collecting and sharing of influenza data and viruses need to continue to ensure the world is as prepared as possible against influenza epidemic and pandemic threats.