Combating H7N9: an interview with Lanjuan Li and Yi Shi

On March 31, 2013, the Chinese government reported that three persons from Shanghai and Anhui had died from a novel avian influenza virus, known as H7N9. This avian flu is unique in that it has a high human mortality rate, while fatality in poultry remains low. In other words, this virus can circulate ‘silently’ among poultry, but be a very possible cause of the next flu pandemic. Since 2013, human cases of H7N9 infection have been reported continuously, posing a potential threat to public health in China and attracting global attention. Will H7N9 be the next flu pandemic? With this question, NSR spoke to clinical expert, Professor Lanjuan Li from Zhejiang University, and the virologist, Dr. Shi Yi, from the Beijing Institutes of Life Science, Chinese Academy of Sciences and Shenzhen Third People’s Hospital. In this interview, they share with us the biological information of H7N9, its clinical manifestation and treatment, and its future pandemic potential.

Lanjuan Li, Chief physician and infectious disease specialist from the First Affiliated Hospital of Zhejiang University, China. She participated in the treatment of H7N9 patients and is an expert in infectious diseases. (Courtesy of Lanjuan Li) Jiangxi, Jilin, Guangdong, Hunan, Guangxi, Guizhou, Hebei, Taiwan and Hong Kong regions. Imported cases were reported in Canada and Malaysia. Several clusters of cases with H7N9 infection have been reported, and the recessive infection was also found among some people with occupational exposure to live poultry. Over the past two years, the H7N9 virus epidemic was typically seasonal, occurring during winter and spring.
Exposure to infected poultry is a key risk factor for human infection with H7N9. In order to cut off the transmission route, authorities closed live poultry markets in the central districts of major cities affected. The number of human cases has significantly decreased this year in Shanghai and Hangzhou. However, we should be aware that the H7N9 virus is still circulating and undergoing dynamic gene re-assortment in domestic poultry. This is why sporadic human cases continued during 2014-2015 and will likely persist for many years. We are still on alert.

ORIGIN AND DYNAMIC CHANGING OF H7N9 VIRUS
NSR: After the emergence of the novel virus H7N9 in China, many scientists, including your team, immediately commenced a search for this virus. What are the key findings about the H7N9 virus? Li: Let's start with the influenza virus family. It is a big virus family classified into type A, B and C on the basis of their core proteins, which is essential for virus replication in host cells.
Gene reassortment is a way in which a virus changes its genome, keeps evolving, and possibly gains the potential to jump easily between different species hosts.
-Lanjuan Li INTERVIEW Xu 315 Among these three types, influenza A viruses are common, and responsible for most epidemic outbreaks and all the pandemics, for example the 1918 Spanish flu. Influenza A viruses can be further divided into different subtypes based on the nature of the virus surface glycoprotein haemagglutinin (HA) and neuraminidase (NA). Each subtype is labeled with a combination of an H number (for HA) and an N number (for NA). HA helps invading flu particles latch on to cells. HA is also the virus' most important surface antigen, against which virus-neutralizing antibodies are directed. NA helps the progeny virus release. NA is also the target for the current antiviral drugs zanamivir and oseltamivir. Influenza A viruses infect a range of mammalian and avian species, but the subtypes differ in their host range. Normally, H7 subtype family viruses circulate amongst avian populations, with some variants known to occasionally infect humans such as H7N7 and H7N2. H7N9 has previously been isolated only in natural hosts (birds) with low pathogenesis. Until this 2013 outbreak in China, no human infection with H7N9 viruses has ever been reported.
According to the gene sequence analysis, the H7N9 strain in this outbreak is a reassortant virus, which consists of eight gene sections from several different parent viruses: the H7N3 wild bird virus, at least two chicken H9N2 viruses and the duck H11N9 virus. Gene reassortment is a way in which a virus changes its genome, keeps evolving, and possibly gains the potential to jump easily between different species hosts. (The Spanish flu is a case of reassortment). In this H7N9 reassortment, the segments from H9N2 should be of concern. The six internal gene segments of H7N9 were recruited from different H9N2 stains. However, in recent decades, H9N2 viruses are known to widely and diversely spread in poultry, and also cause infection in swine and humans. Thus, the contribution of H9N2 segments to the host jumping from bird to human should be further monitored and investigated.
In addition to gene reassortment, site mutation is also significant in H7N9 with respect to its pathogenesis. Currently, several crucial mutations in HA (138,186,221,226) have been found in H7N9, which have been shown to promote the switch in the tropism of the cell surface receptor. This switch contributes to the acquisition of its ability to bind to human sialic-acid containing receptor (α-2,6 SA glycan receptor), consequently allowing it to infect humans. Almost all human H7N9 viruses carry the specific adaptive mutation (E627K, D701N or Q591K) in the PB2 gene, which is associated with viral replication at the lower temperature of the mammalian respiratory tract. This is not observed in patients infected by other avian flu viruses.
A novel method, named the 'four-anti and two-balance' approach was developed and used to reduce the mortality rate of H7N9 virus infection in clinical practice.
-Lanjuan Li Fortunately, no mutation that may lead to more human-tohuman transmission was found. Thus far, no apparent change in virology and transmission among human cases has been found. This greatly reduces the risk and opportunity for the occurrence of the H7N9 pandemic.

CLINICAL SYMPTOMS AND PATHOGENESIS OF H7N9
NSR: H7N9 infection leads to only mild symptoms in poultry, but results in severe illness or death in humans. Why? Li: In humans, H7N9 virus infection causes aberrant cytokine expression, which contributes to the high pathogenic properties of H7N9 in humans. The substantially increased pro-inflammatory and anti-inflammatory serum cytokines and chemokines are compatible with the clinical severity of this novel H7N9 infection, a situation also occurring in H5N1 infection. In particular, the serum concentration of interleukin 10 is persistently increased, similar to that found in severe A H1N1 pdm09 infection. The cytokine storm may be one of the reasons behind the high fatality, but we still do not understand how the influenza virus triggers the cytokine storm. NSR: What are the key findings about the pathology of H7N9? How might it differ from other influenza viruses? Li: Common symptoms of H7N9 include fever, cough and shortness of breath. Most patients develop severe pneumonia. As far as pathology is concerned, some patients experience intra alveolar hemorrhage, diffuse alveolar damage and hyaline formation without fibro proliferative changes. These features are compatible with the acute exudative inflammatory phase when 316 Natl Sci Rev, 2015, Vol. 2, No. 3 INTERVIEW immunostaining for influenza A virus and the nucleoprotein expression is positive in the alveolar epithelium. Some patients can develop neumocyte hyperplasia and interstitial fibrosis in addition to the diffuse alveolar damage, compatible with the fibroproliferative phase.
To assess the pathogenesis of these histopathological and clinical features of H7N9 infection, we have studied the patients' serial viral loads, and found that H7N9 virus could invade on pulmonary, but not extra-pulmonary systems. In addition, some studies showed that the viral load was higher and the duration of shedding was longer in sputum specimens compared to nasopharyngeal swabs, suggesting that lower respiratory tract specimens would be preferred for the diagnosis of severe cases. This predilection of the H7N9 virus for the lower respiratory tract in humans is similar to another avian influenza H5N1 virus, but generally quite unusual for the avian influenza viruses. NSR: What does the predilection for the lower respiratory tract mean for the human H7N9 infection case? Shi: This affects the pathogenicity of human H7N9. When infected by a flu virus, the first step is for the virus to bind to and enter the cells. This process is mediated by the specific cell surface receptor. In their natural host birds, avian influenza viruses typically bind alpha-2,3 sialic acid (α-2,3 SA) glycan receptor of epithelial cells. Interestingly, humans also have α-2,3 SA glycans in the lower respiratory tract. Under normal circumstances, the bird flu viruses do not easily enter the lower respiratory tract of people.
Receptor binding is one of the key factors affecting the pathogenicity of influenza viruses. With regard to H7N9 and H5N1, they display a strong α-2,3 SA glycan receptor binding capacity, and can easily replicate in the human lower respiratory tract, including the lung, thus causing severe disease. By contrast, the seasonal flu viruses (for example H1N1 and H3N2) display human α-2,6 SA receptor binding preferences and can easily replicate in the upper respiratory tract, resulting in relatively mild disease.

A NOVEL STRATEGY TO SAVE MORE H7N9 PATIENTS
NSR: As a physician directly involved in the treatment of H7N9 patients, could you tell us the latest developments in clinical treatments? Li: As a chief physician in the First Affiliated Hospital of Zhejiang University, I have worked with colleagues to treat H7N9 patients. While researching and treating H7N9, our team developed a novel method, named the 'four-anti and two-balance' approach in clinical practice. The 'four-anti' means four crucial Early reporting and information transparency are absolutely necessary in the prevention and control of H7N9.
-Lanjuan Li Lanjuan Li checking the various body indexes of H7N9 patients with the doctor on the duty, and giving the treatment guidance during ward rounds.

(Courtesy of Lanjuan Li)
steps to treat H7N9 patients: 'anti-virus' to eliminate pathogens, 'anti-shock' to maintain effective perfusion of body organs, 'anti-hypoxemia and MODS (Multiple Organ Dysfunction Syndrome)' to maintain vital signs, and 'anti-infection' to control secondary infection. The 'two-balance' refers to maintaining the balance of water and electrolyte to keep a stable internal environment and the balance of micro-ecology to reduce the translocation of bacterium. This strategy is now widely used to reduce the mortality rate of H7N9 virus infection, and has also been adopted for the treatment of other virus infections.
In the 'anti-virus' treatment, Neuraminidase inhibitor (NAI) is normally used, however NAI is most effective at the early stage of infection and most patients only commenced the antiviral therapy at 6-7 days after infection. This delay would decrease the effect of the antiviral therapy, and may be a reason for the high mortality rates of this disease. Shock is an independent risk factor caused by H7N9 virus. The goal of 'anti-shock' therapy is to maintain effective perfusion of systemic organs and prevent the patient from further multiple organ injuries. We were the first to apply Li's Artificial Liver Support System (ALLS) in treatment of severe H7N9 cases, aiming at relieving the damage caused by shock and maintain the balance of circulation (see N Engl J Med 2013;368: 2277-85). When a patient's condition deteriorates rapidly and a cytokine storm is detected, we recommend using the ALSS. We have now treated 48 cases of H7N9 with Li's ALLS.
The H7N9 viruses destroy the diffusion function of the lung. 'Anti-hypoxemia and MODS' strategy should be considered to give timely and effective respiratory support in the treatment of severely ill patients. This includes the use of oxygen ventilation, normally in the morning, combined with the ECMO (Ex-traCorporeal Membrane Oxygenation) 3-6 hours later. ECMO can replace the gas exchange function of the lung, but it cannot replace ventilation and ALSS. Furthermore, ECMO has many side effects, including bleeding, thrombosis and infection. Thus, we apply these treatments in combination as they solve different aspects of the disease.
According to our observation, about 20% of H7N9 patients have coexisting bacterial infection acquired from the

INTERVIEW Xu 317
Although it is difficult to be certain, H7N9 does have the potential to be the next pandemic, according to our understanding of interspecies transmission of influenza viruses.
-Yi Shi community; many critically ill patients will have a secondary bacterial infection, probably acquired prior to hospital admission. For 'anti-bacterial infection', we recommend moxifloxacin administration for three days. For the secondary infection, the broad-spectrum antibiotic drug is the first choice. Finally, maintaining the fluid and electrolyte balance is important for critically ill patients. We recommend 30 mL kg −1 day −1 fluid intake to maintain the body with an appropriately negative fluid uptake balance. This keeps the body slightly dehydrated and helps to reduce the inflammatory exudation in pulmo. If necessary, ALSS can be used to maintain the fluid and electrolyte balance accurately. Maintaining the balance of intestinal microflora, by using probiotics to relieve antibiotic pressure, can reduce the rate of endogenous infection, significantly improving the survival rate.

NSR:
What are the key aspects in prevention and control? Li: Because the H7N9 virus is circulating among poultry, regulation of the live poultry market is still the most effective measure to prevent human exposure to this virus. It is noteworthy that developed countries do not have live poultry markets. However, live poultry markets are a tradition in China, and it is hard to change traditions quickly. Nevertheless, aggressive intervention of live poultry markets to block further animal-to-person transmission should be considered in certain regions, as we did in the last years.
Further to the temporary closure of live poultry markets, comprehensive programs of surveillance, culling, and segregation of different poultry species in poultry markets and farms are necessary to halt the evolution and further adaption of the virus. Regarding the vaccination, as H7N9 causes no disease in poultry, it is difficult to target the vaccination. Considering of the huge number of poultry in China, it is not clear whether vaccination is practical.
From the perspective of government management/regulation, early reporting and information transparency are absolutely necessary in the prevention and control of H7N9. We have benefited from previous experience dealing with SARS. When the first cases were identified, the authorities quickly made information available globally through the WHO, and made the effort of reach out to personal and research networks. This open and transparent approach helped other countries understand what was emerging in China and prepare for potential cross-border transmission. The identified genome sequences of H7N9 were uploaded as quickly as possible for public access. Transparent and timely sharing of information is helpful for the prevention and control of H7N9, and for scientific studies and vaccine development. Shi: Early warning is another good measure to prevent the virus infection. However, it depends on the surveillance network and our understanding of molecular mechanism on interspecies transmission of influenza viruses. NSR: Will H7N9 be the next pandemic flu? Could it be predicted? Li: We do not know. But we all agree that the prevalence of H7N9 is very unusual and this virus is more likely to cause human infection than any other known avian influenza viruses. It would be difficult to predict what subtype would cause the next pandemic. However, systematic surveillance for signs of host adaptation and reassortant in the virus would be very important. Shi: Although it is difficult to be certain, H7N9 does have the potential to be the next pandemic, according to our understanding of interspecies transmission of influenza viruses. As we know, pandemic viruses, such as H1N1, H2N2 and H3N2, have evolved to human influenza viruses from avian influenza viruses. For example, the receptor binding properties have been shifted in their preference from avian receptor to human receptor, and there is an intermediate stage with dual receptor binding properties. Now the H7N9 viruses have obtained a human receptor binding but kept strong avian receptor binding. It is important to note that once the H7N9 virus gains a human receptor preference, it has the potential to be a pandemic. NSR: With rapid globalization and increased travel, people are more likely to come into contact with infectious pathogens. How do you think China should tackle infectious diseases in general? Li: We should build up a sophisticated system for pathogen surveillance and detection, vaccine development and production, and clinical treatment. Surveillance is very important for tackling infectious diseases. An understanding of the source and mode of transmission of these infections, and appropriate counter measures are urgently required. In addition, it may be necessary to establish rapid response vaccine production capabilities and develop technologies for novel vaccine development. Shi: We should also keep an eye on the communicable pathogens in other countries, and any quarantine implications that may have for China. A recent example from a Korean case of MERS-CoV infection in Guangdong caused a panic in our public health system. In a globalized world, it has become increasingly important for us to spend more time and effort in surveilling pathogens and developing vaccines and clinical treatments for infectious diseases.
Xiuling Xu is a scientific editor in NSR editorial office based in Beijing.