Suboptimal Humoral Immune Response against Influenza A(H7N9) Virus Is Related to Its Internal Genes

Influenza A(H7N9) virus pneumonia is associated with a high case fatality rate in humans. Multiple viral factors have been postulated to account for the high virulence of the virus. It has been reported that patients with influenza A(H7N9) virus infection have relatively low titers of neutralizing antibodies compared to those with seasonal influenza virus infections. In this study, we compared serum hemagglutination inhibition (HI) and microneutralization (MN) antibody titers of mice challenged with wild-type A(H7N9) viruses [H7N9(Anhui) and H7N9(Zhejiang)], an A(H1N1)pdm09 virus [pH1N1(2009)], and a recombinant A(H7N9) virus with PR8/H1N1 internal genes (rg-PR8-H7-N9). All mice infected by H7N9(Anhui) and H7N9(Zhejiang) developed serum HI antibodies at 14 days postinfection (dpi) but no detectable MN antibodies, even at 28 dpi. A low level of neutralizing activity was detected in H7N9(Anhui)- and H7N9(Zhejiang)-infected mice using fluorescent focus MN assay, but convalescent-phase serum samples obtained from H7N9(Anhui)-infected mice did not reduce the mortality of naive mice after homologous virus challenge. Reinfection with homologous A(H7N9) virus induced higher HI and MN titers than first infection. In contrast, pH1N1(2009) virus infection induced robust HI and MN antibody responses, even during the first infection. Moreover, rg-PR8-H7-N9 induced significantly higher HI and MN antibody titers than H7N9(Zhejiang). In conclusion, the internal genes of A(H7N9) virus can affect the humoral immune response against homologous viral surface proteins, which may also contribute to the virulence of A(H7N9) virus.     

Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The "gold standard" for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged <60 years and 92% specificity for adults aged ≥ 60 years for detection of serologically confirmed 2009 H1N1 infections in U.S. populations during the first pandemic waves. These studies provide an approach to optimize timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.     

Serologic evidence of avian influenza virus infections among Nigerian agricultural workers

Nigeria has had multiple incursions of highly pathogenic avian influenza A (HPAI) H5N1 virus into its poultry population since 2006. This study aimed to determine if Nigerians exposed to poultry had evidence of avian influenza virus transmission to man. Between 2008 and 2010, 316 adult farmers and open market workers and 54 age‐group matched, non‐animal exposed controls were enrolled in a prospective, population‐based study of zoonotic influenza transmission in four towns in southeastern Nigeria. Questionnaire data and sera obtained at the time of enrollment were examined for evidence of previous infection with 10 avian influenza virus strains. Serologic studies on sera collected at the time of enrollment showed modest evidence of previous infection with three avian‐origin influenza viruses (H5N1, H5N2, and H11N1) and one avian‐like H9N2 influenza virus, with eight (2.4%) of animal‐exposed subjects and two (3.7%) unexposed subjects having elevated microneutralization assay antibody titer levels (ranging from 1:10 to 1:80). Statistical analyses did not identify specific risk factors associated with the elevated antibody titers observed for these zoonotic influenza viruses. These data suggested only occasional virus transmission to humans in areas thought to have been enzootic for avian influenza virus. Prospective data from this cohort will help the authors to better understand the occurrence of zoonotic infections due to avian influenza viruses in Nigeria. J. Med. Virol. 85:670–676, 2013. © 2013 Wiley Periodicals, Inc.     

The first lack of evidence of H7N9 avian influenza virus infections among pigs in Eastern China

In this study, we sought to examine whether evidence existed suggesting that pigs were being infected with the novel H7N9 avian influenza virus. From November 2012 to November 2013, blood was drawn from 1560 pigs from 100 large farms in 4 provinces of eastern China. Many of these pigs were in close proximity to wild birds or poultry. Swine sera were studied using hemagglutinin inhibition (HI) assays and enzyme-linked immunosorbent assays (ELISAs) against the H7 antigen derived from the emergent H7N9 avian influenza virus (AIV). Only 29 of the 1560 samples had HI titers of 1:20 when using the H7N9 AIV antigens, and none of the 29 (H7N9 AIV) HI-positive samples were positive when using ELISA, indicating that no samples were positive for H7N9. The negative results were also verified using a novel competitive HA-ELISA. As pigs have been shown to be infected with other avian influenza viruses and as the prevalence of novel influenza A viruses (e.g., H7N9 AIV) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be periodically conducted in the future.     

Serological survey of avian H5N2-subtype influenza virus infections in human populations

To investigate the distribution of antibodies against H5N2 influenza virus in humans living in Ibaraki prefecture, Japan, 266 single serum samples were collected to perform serological tests. Results were compared to investigate the relationship between positive results and several factors. The number of positive serum neutralization antibody titers (≥40) against avian influenza virus A/H5N2 was significantly greater (P < 0.05) among poultry workers, in comparison to a Japanese healthy population. The geometric mean titers of serum neutralization antibody against A/H5N2 were significantly higher (P < 0.05) among Ibaraki inhabitants and poultry workers (P < 0.0001) when compared to a Japanese healthy population. Seropositivity against A/H5N2 virus was significantly (P < 0.05) associated with age (≥50 years old) in poultry workers. These results suggest that seropositivity against H5N2 virus in Ibaraki specimens is significantly higher than those of a Japanese healthy population and that the surveillance of avian influenza viruses is very important to evaluate the invasion or emergence of new pandemic influenza viruses from species other than humans.     

Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.     

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (K_D) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.     

Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.     

Serological report of pandemic and seasonal human influenza virus infection in dogs in southern China

From January to July 2012, we looked for evidence of subclinical A (H1N1) pdm09 and seasonal human influenza viruses infections in healthy dogs in China. Sera from a total of 1920 dogs were collected from Guangdong, Guangxi, Fujian and Jiangxi provinces. We also examined archived sera from 66 dogs and cats that were collected during 2008 from these provinces. Using hemagglutination inhibition (HI) and microneutralization (MN) assays, we found that only the dogs sampled in 2012 had elevated antibodies (≥1:32) against A(H1N1)pdm09 virus and seasonal human influenza viruses: Of the 1920 dog sera, 20.5 % (n = 393) had elevated antibodies against influenza A(H1N1) pdm09 by the HI assay, 1.1 % (n = 22), and 4.7 % (n = 91) of the 1920 dogs sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus by the HI assay. Compared with dogs that were raised on farms, dogs that were raised as pets were more likely to have elevated antibodies against A(H1N1)pdm09 and seasonal human influenza viruses. Seropositivity was highest among pet dogs, which likely had more diverse and frequent exposures to humans than farm dogs. These findings will help us better understand which influenza A viruses are present in dogs and will contribute to the prevention and control of influenza A virus. Moreover, further in-depth study is necessary for us to understand what roles dogs play in the ecology of influenza A.     

北京市首例人感染H9N2禽流感病例的流行病学调查

目的 分析2016年12月北京市朝阳区确诊的l例人感染H9N2禽流感病例流行病学特征.方法 访谈病例发病前后的相关知情人,调查病例发病经过和可能感染来源.采集病例、密切接触者及环境标本,应用实时荧光定量PCR(real-timePCR)方法,检测甲型流感病毒及H9N2禽流感病毒特异的核酸片段.对病例咽拭子标本和l件阳性环境标本中的H9N2禽流感病毒血凝素(Hemagglutinin,HA)基因进行序列测定和分析.结果 病例表现为轻症流感样病例症状,发病前15天曾有活禽市场暴露史.该市场环境标本中H9N2禽流感病毒阳性率为18.2％ (2/11).病例咽拭子标本与市场环境标本中病毒的HA基因高度同源,氨基酸相似度为100％,在进化关系上同属于欧亚系的Y280分支.4名密切接触者在医学观察期内未出现过发热、咳嗽等呼吸道症状.结论 病例感染来源与活禽市场环境暴露有关.加强流感监测以及活禽市场的管理对于防控人感染禽流感疫情具有重要意义.     

Lack of evidence of avian-to-human transmission of avian influenza A (H5N1) virus among veterinarians,Guangdong, China, 2012

BackgroundCases of human infections with H5N1 avian influenza viruses have been reported all over the world with the reason of direct contact with sick or diseased poultry, which suggests the direct contact with poultry may be one of the major risk factors for human infection.ObjectivesIn this study, we estimated the seroprevalence of antibodies against avian influenza A (H5N1) virus in veterinarians with exposure to avians.Study designFrom May 21, 2011 through April 22, 2012, 406 veterinarians exposure to poultry in Guangdong province were interviewed a questionnaire. A serum specimen was collected from participants to test for H5N1 antibodies by HI and NT assay.ResultsNone of the 406 sera from occupationally exposed veterinarians was positive according to the HI test and the NT test with the H5N1 AIV.ConclusionOur seroepidemiologic survey suggests that the risk of avian-to-human transmission of the H5N1 AIV is very low based on the samples that we tested. However, prevention regarding the risk of H5N1 AIV transmission is essential and should be recommended as public health measures.     

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

中国职业暴露人群感染H6N6禽流感病毒血清学调查

目的 系统评估我国职业暴露人群感染H6N6禽流感病毒的状况.方法 本研究利用我国2009-2011年开展的高致病性H5N1禽流感病毒职业暴露人群血清学监测所采集的近15 000份血清标本,开展H6N6禽流感病毒血清学调查.结果 本研究中检测到H6N6禽流感病毒阳性血清共10份,分别来自不同的职业暴露人群,包括活禽市场、家禽规模养殖场、家禽散养户、屠宰加工场和野生候鸟栖息地.从地域上看该10份阳性血清来自8个不同的省份,分布在我国的南北方.结论 这是我国大陆地区首次报道人感染H6亚型禽流感病毒.     

Profiling of humoral immune responses to influenza viruses by using protein microarray

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.     

Experimental infection of non-human primates with avian influenza virus (H9N2)

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.     

Serological survey of antibodies to influenza A viruses in a group of people without a history of influenza vaccination

A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the haemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than those for seasonal H1N1. Samples positive for antibodies to the pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p <0.01) at 1 year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.     

Histopathological Evaluation of the Diversity of Cells Susceptible to H5N1 Virulent Avian Influenza Virus

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage.Seasonal, pandemic, and zoonotic influenza A virus infections show substantial morbidity and mortality in humans. Seasonal influenza A virus infections in humans are usually mild and cause pneumonia only in a few infected individuals. Pandemic influenza virus infections vary in their disease outcome. Zoonotic influenza virus infections in humans vary from self-limiting conjunctivitis to severe, often fatal, pneumonia. Highly pathogenic avian influenza H5N1 virus (H5N1 HPAIV), implicated in poultry outbreaks,^1,2 can be transmitted zoonotically to humans, as has been observed in areas of Asia and Africa.^3–5 Fatal outcomes have been reported at approximately 60% in the sporadic transmission of this avian influenza H5N1 virus to humans.^5–7 There is no evidence that the avian influenza virus has become efficiently transmissible among humans, a change that could result in a new pandemic.⁸The outcome after infection with influenza virus can range from slight to severe illness, depending on the kinds of cells that are affected during lung tissue infection.^9–11 Events occurring early in infection determine the extent of damage, which can range from bronchitis to pneumonia. In the most severe cases, diffuse alveolar damage (DAD) may be induced during the early stages, and healing and/or scarring may ensue, depending on the persistence of disease. Occasionally, bacterial infection also may occur, with associated effects expressed mainly in the later stages of the disease. Pathological damage caused by influenza viruses in humans and in animal models depends on the virulence of the infective agent and on the host response. All influenza viruses infect the respiratory tract epithelium from the nasal passages to the bronchioles; however, highly virulent viruses (eg, H1N1 1918 and H5N1 HPAIV) tend to infect pneumocytes and resident macrophages in the alveoli. In susceptible individuals, inflammation of the alveolar walls results in DAD. In contrast, low-virulence viruses (seasonal H1N1) primarily cause inflammation, congestion, and epithelial necrosis of the trachea, bronchi, and bronchioles. Tissue tropism is an important factor, and depends largely on the ability of the virus to attach to the host cell.^12–14 We investigated virus replication and histopathological progression of lung tissue in mice infected with H5N1 HPAIV, particularly focusing on the lower respiratory tract and alveoli, with direct comparison to the histopathological characteristics of mice infected with H1N1 pandemic (pdm) influenza virus 2009 virus.     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Testing human sera for antibodies against avian influenza viruses: Horse RBC hemagglutination inhibition vs. microneutralization assays

BACKGROUND: The hemagglutination inhibition (HI) assay is a frequently used method to screen human sera for antibodies against influenza A viruses. Because HI has relatively poor sensitivity in detecting antibodies against avian influenza A strains, a more complicated microneutralization (MN) assay is often preferred. Recent research suggests that the sensitivity of the HI assay can be improved by switching from the traditionally used turkey, guinea pig, human, or chicken RBCs to horse RBCs. OBJECTIVE: To evaluate the performance of the horse RBC HI when screening for human antibodies against avian influenza types H3, H4, H5, H6, H7, H9, H11, and H12. STUDY DESIGN: We evaluated the reproducibility of horse RBC HI and its agreement with MN results using sera from people exposed or not exposed to wild and domestic birds. RESULTS: The horse RBC HI assay had high reliability (90%-100%) and good agreement with MN assay results (52%-100%). CONCLUSION: The horse RBC HI assay is reliable, less expensive, less complex, and faster than the MN assay. While MN will likely remain the gold standard serologic assay for avian viruses, the horse RBC HI assay may be very useful as a screening assay in large-scale epidemiologic studies.