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Where can I find hemagglutination inhibition (HI) assay data?

Where can I find hemagglutination inhibition (HI) assay data?


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I am looking for hemagglutination inhibition assay data for type A influenza virus. I've checked in databases such as fludb.com, however it seems to only have genetic data. A lot of the time, scientific papers don't include the HI assay data they used, so I was wondering if anyone knows where to find such data. I specifically need influenza type A HI assays, not influenza type B.

HI data comes in the form of titers (the reciprocal of the last well plate concentration of antibody that was able to inhibit the virus from binding to the blood sample and forming a shield on the well plate.)

Could someone please give me a link to a database with this information?

Thanks.


I don't think there's any universal database containing ongoing, widely representative HI assay output. The Antigenic Cartography group has made available some historical datasets that were used in published articles, such as

  • Influenza A/H3N2 data published in Smith et al. 2004. Science 305:371-376
  • Data from H1N1pdm09 assays in 2009/2010 (click on the links for html/Excel/OpenDocument tables)
  • Data published in Koel et al. 2013. Science 342:976-979

Trevor Bedford et al. make their large set of historical HI data described in Integrating influenza antigenic dynamics with molecular evolution available in DataDryad.


Hemagglutination Inhibition

The hemagglutination inhibition (HI) assay is used to titrate the antibody response to a viral infection. The HI assay takes advantage of some viruses' ability to hemagglutinate (bind) red blood cells, therefore forming a “lattice” and preventing the red blood cells from clumping. In the HI assay, twofold dilutions of the sera to be tested are made in 96 well plates. A known titer of the virus is added, and the plate is incubated for 30 minutes at room temperature. Red blood cells are then added and the plate incubated for a further 30 minutes at room temperature. If antibodies are present in the sera sample that cross-react with the virus, the antibodies will bind to the virus and prevent the virus from hemagglutinating the red blood cells. In this way, the exact titer of the antibodies in the sera can be determined. This assay is the standard for equine influenza diagnostics. 63

This assay has the advantage of being quick and easy to perform, especially if there is a predominant virus subtype that is suspected. However, the HI assay may not detect antibodies in samples that are not cross-reactive to the virus being tested (i.e., are against different subtypes of influenza virus). Thus all the possible different viruses and viral subtypes should be tested, which is not always feasible. In addition, immunity to the virus may involve other aspects of the host response, including t-cell responses and antibodies against other components of the virus besides the hemagglutinating protein. The HI assay will not detect these aspects of the host immune response. In this regard, plaque reduction neutralization tests would be more comprehensive.


Antigenic Characterization

&ldquoAntigens&rdquo are molecular structures on the surface of viruses that are recognized by the immune system and are capable of triggering an immune response (antibody production). On influenza viruses, the major antigens are found on the virus&rsquo surface proteins (see Figure 1).

When someone is exposed to an influenza virus (either through infection or vaccination) their immune system makes specific antibodies against the antigens (surface proteins) on that particular influenza virus. The term &ldquoantigenic properties&rdquo is used to describe the antibody or immune response triggered by the antigens on a particular virus. &ldquoAntigenic characterization&rdquo refers to the analysis of a virus&rsquo antigenic properties to help assess how related it is to another virus.

CDC antigenically characterizes about 2,000 influenza viruses every year to compare how similar currently circulating influenza viruses are to those that were included in the influenza vaccine and to monitor for changes in circulating influenza viruses. Antigenic characterization can give an indication of the flu vaccine&rsquos ability to produce an immune response against the influenza viruses circulating in people. This information also helps experts decide what viruses should be included in the upcoming season&rsquos influenza vaccine.

Other information that determines how similar a circulating virus is to a vaccine virus or another virus are the results of serology tests and genetic sequencing.

The above image shows the different features of an influenza virus, including the surface proteins hemagglutinin (HA) and neuraminidase (NA). Following influenza infection or receipt of the influenza vaccine, the body&rsquos immune system develops antibodies that recognize and bind to &ldquoantigenic sites,&rdquo which are regions found on an influenza virus&rsquo surface proteins. By binding to these antigenic sites, antibodies neutralize flu viruses, which prevents them from causing further infection.

The Hemagglutinin Inhibition Assay (HI Test)

Scientists use a test called the hemagglutinin inhibition (HI) assay to antigenically characterize influenza viruses. The HI test works by measuring how well antibodies bind to (and thus inactivate) influenza viruses.

Scientists use the HI test to assess the antigenic similarity between influenza viruses. This test is particularly useful for helping to select the vaccine viruses used in the seasonal flu vaccine. HI test results can tell us whether antibodies developed against vaccination with one virus are antigenically similar enough to another circulating influenza virus to produce an immune response against that circulating virus. Scientists also use the HI test to compare antigenic changes in currently circulating influenza viruses with influenza viruses that have circulated in the past.

The HI test involves three main components: antibodies, influenza virus, and red blood cells that are mixed together in the wells (i.e., cups) of a microtiter plate. (See Image 1.)

A microtiter plate is used to perform the HI test. The plate contains wells (i.e., cup-like depressions that can hold a small amount of liquid) where the solution of antibodies, influenza virus and red blood cells are inserted and allowed to interact. These wells are arranged according to rows and columns (which are identified on the microtiter plate by letters and numbers, respectively). The rows of the plate can be used to test different influenza viruses against the same set of antibodies. The columns can be used to differentiate between greater dilutions of antibodies, like a scale from low to high going from left to right (see Figures 3 and 4 for an example).

The antibodies used in the HI test are obtained by infecting an animal (usually a ferret) that is immunologically naïve (i.e., it has not been exposed to any influenza virus or vaccine previously in its lifetime). The animal&rsquos immune system creates antibodies in response to the antigens on the surface of the specific flu virus that was used to infect that animal. To study these antibodies, a sample of blood (serum) is drawn from the animal. The HI test measures how well these antibodies recognize and bind to other influenza viruses (for example, influenza viruses that have been isolated from flu patients). If the ferret antibodies (that resulted from exposure to the vaccine virus) recognize and bind to the influenza virus from a sick patient, this indicates that the vaccine virus is antigenically similar to the influenza virus obtained from the sick patient. This finding has implications for how well the vaccine might work in people. See Flu Vaccine Effectiveness: Questions and Answers for Health Professionals for more information.

As previously mentioned, the influenza viruses used in the HI test are taken from samples from sick people. CDC and other WHO collaborating centers collect specimens from people all over the world to track which influenza viruses are infecting humans and to monitor how these viruses are changing.

For the HI test, red blood cells (RBCs) are taken from animals (usually turkeys or guinea pigs). They are used in the HI test because influenza viruses bind to them. Normally, RBCs in a solution will sink to the bottom of the assay well and form a red dot at the bottom (Figure 2A). However, when an influenza virus is added to the RBC solution, the virus&rsquo hemagglutinin (HA) surface proteins will bind to multiple RBCs. When influenza viruses bind to the RBCs, the red cells form a lattice structure (Figure 2B). This keeps the RBCs suspended in solution instead of sinking to the bottom and forming the red dot. The process of the influenza virus binding to RBCs to form the lattice structure is called &ldquohemagglutination.&rdquo

The HI test involves the interaction of red blood cells (RBCs), antibody and influenza virus. Row A shows that in the absence of virus, RBCs in a solution will sink to the bottom of a microtiter plate well and look like a red dot. Row B shows that influenza viruses will bind to red blood cells when placed in the same solution. This is called hemagglutination and is represented by the formation of the lattice structure, depicted in the far right column under &ldquoMicrotiter Results.&rdquo Row C shows how antibodies that are antigenically similar to a virus being tested will recognize and bind to that influenza virus. This prevents the virus and RBCs from binding, and therefore, hemagglutination does not occur (i.e., hemagglutination inhibition occurs instead).

When antibodies are pre-mixed with influenza virus followed by RBCs, the antibodies will bind to influenza virus antigens that they recognize, covering the virus so that its HA surface proteins can no longer bind to RBCs (Figure 2C). The reaction between the antibody and the virus inhibits (i.e., prevents) hemagglutination from occurring, which results in hemagglutination inhibition (as shown in Figure 2C). This is why the assay is called a &ldquohemagglutinin inhibition (HI) test.&rdquo Hemagglutination (as depicted in Figure 2B) occurs when antibodies do not recognize and bind to the influenza viruses in the solution, and as a result, the influenza viruses bind to the red blood cells in the solution, forming the lattice structure. When the antibodies do recognize and bind to the influenza viruses in the solution, this shows that the vaccine virus (like the one the ferrets were infected with) has produced an immune response against the influenza virus obtained from the sick patient. When this happens, the influenza virus being tested is said to be &ldquoantigenically like&rdquo the influenza virus that created the antibodies (from ferrets).

When a circulating influenza virus is antigenically different from a vaccine or reference virus, the antibodies (developed in response to the vaccine or reference virus) will not recognize and bind to the circulating influenza virus&rsquo surface antigens. In the HI test this will cause hemagglutination to occur (see Figure 2B). This indicates that the vaccine virus or reference virus has not caused an immune response (i.e., the creation of antibodies) that recognizes and targets the circulating influenza virus. Circulating influenza viruses tested via the HI test are typically obtained from respiratory samples collected from ill patients.

Assessing Antigenic Similarity Using the HI Test

The HI test assesses the degree of antigenic similarity between two viruses using a scale based on greater dilutions of antibodies. As previously mentioned, the HI test is performed using a microtiter plate. The microtiter plate contains rows and columns of wells (i.e., cups) where RBCs, influenza virus and antibodies (developed against a comparison virus, such as a vaccine virus) are mixed. Dilutions are marked across the top of the microtiter plate. These dilutions function as a scale for assessing antigenic similarity and immune response. By testing the ability of greater dilutions of antibody to prevent hemagglutination, scientists measure how well those antibodies recognize and bind to (and therefore inactivate) an influenza virus. The higher the dilution, the fewer antibodies are needed to block hemagglutination and the more antigenically similar the two viruses being compared are to each other. The highest dilution of antibody that results in hemagglutinin inhibition is considered a virus&rsquos HI titer (Figure 3). Higher HI titers are associated with greater antigenic similarity. Greater antigenic similarity suggests that vaccination would produce an immune response against the test virus.

This virus sample has an HI titer of 1280, which means that the greatest dilution of antibody that still blocked hemagglutination from occurring was at 1280 dilution. At this dilution, the antibodies were still capable of recognizing and binding to the antigens on the virus.

When CDC antigenically characterizes influenza viruses to inform decisions on the formulation of the seasonal flu vaccine, the HI test is used to compare currently circulating viruses (B&C) with vaccine viruses (A). This allows scientists to quickly determine if a virus used in the seasonal flu vaccine is antigenically similar to circulating influenza viruses and therefore capable of producing an immune response against them.

Public health experts consider influenza viruses to be antigenically similar or &ldquolike&rdquo each other if their HI titers differ by two dilutions or less. (This is equivalent to a two-well (i.e., a four-fold dilution) or less difference). Using figure 4 as an example, when circulating virus 1 is compared to a vaccine virus, circulating virus 1 differs by one dilution (a 2-fold difference) and therefore is &ldquolike&rdquo the previous season&rsquos vaccine virus. However, circulating virus 2 differs by five dilutions (a 32-fold difference) and therefore is not like the previous season&rsquos vaccine virus. Circulating viruses that are antigenically dissimilar (i.e., not &ldquolike&rdquo) the reference panel are considered &ldquolow reactors.&rdquo

Limitations

Antigenic characterization gives important information about whether a vaccine made using a specific vaccine virus will protect against circulating influenza viruses, but there are several limitations to antigenic characterization test methodology, which are described below.

Egg Adaptations

Right now, most flu vaccines are made using viruses grown in eggs. As human influenza viruses adapt to grow in eggs, genetic changes can occur in the viruses. These are called &ldquoegg-adapted&rdquo changes. Some egg-adapted changes may change the virus&rsquo antigenic (or immunogenic) properties while others may not. Egg-adapted changes have become a particular problem for selection of candidate vaccine viruses (CVVs) for the influenza A(H3N2) virus component of the flu vaccine. Influenza A(H3N2) viruses tend to grow less well in chicken eggs than influenza A(H1N1) viruses and they also are more prone to egg-adapted changes. Such changes can reduce the immune protection provided by the flu vaccine against circulating A(H3N2) viruses.


1 Introduction

Accurate measurement of individuals' pathogen exposure history is an essential tool for understanding risk factors of infection and population-scale patterns of transmission. Determined through a variety of methods, the concentration of antibodies in sera is considered the gold standard method to estimate past exposure to pathogens. Two of the most common methods for measuring serum antibody to influenza are the hemagglutination inhibition (HI) and virus neutralization (NT) assays. 1 Although both tests serve as measures of antibody concentration in sera, they have important differences in how they are conducted and how they measure immunity. The HI test, which is fast and relatively easy to perform, is considered to be easily standardized and reproducible across laboratories. However, only the effect of antibodies on the hemagglutination process, by which a virus binds to red blood cells, is measured with HI, and the endpoint is only a correlate of the ability of antibodies to inhibit virus infection of host cells. 2, 3 In contrast, NT assays, also known as microneutralization assays, measure the titer needed to block the cytopathic effects of the virus, by measuring antibodies that block entry of the virus into the cell, internalization of the virus, and fusion of the HA. Although NT is intuitively more appealing because it more closely mirrors the disease process in vivo, it is more time-consuming and expensive and considered harder to standardize across laboratories. 2, 3

Despite the widespread usage of these two methods, there have been few formal comparative studies of these measures. In a 2007 study by Stephenson et al., HI and NT tests were performed in 11 laboratories to investigate reproducibility of each assay for detection of anti-H3N2 influenza antibodies. They found significantly higher variation in NT results between laboratories than in HI results, yet better discrimination among NT and generally limited correlation between the tests. 2 In a follow-up study of anti-H1N1pdm antibodies, significant correlation between HI and NT was found, yet the conversion factors between laboratories varied significantly. Furthermore, NT titers were both significantly higher and significantly more variable than HI titers. 3

The difference in reliability between laboratories with these two assays is a direct result of how they are measured. Hemagglutination inhibition and viral neutralization assays assess the level of functional immunity to a virus in a similar manner, both using serial dilution of sera applied to a fixed amount of virus to determine at which titer of sera the virus is effectively inhibited. The difference is in the biological mechanism used as an indicator for inhibition. The HI assay utilizes the natural process of viral hemagglutination, a process in which a lattice forms by binding of viruses to red blood cells this process is blocked when sufficient antibody with affinity to the virus is present. A serum HI titer of ≥40 is assumed to indicate a 50% reduction in susceptibility compared with an individual with undetectable titer. 4-6 The NT assay, in contrast, measures cytopathic effects of the virus, the invading and killing of cells, through plaque formation. Again, the antibodies in the sample serum are tested for their ability to block this activity. Results are expressed as reciprocal of the highest dilution at which virus infection is blocked. 7

The viral neutralization test is valued for its high sensitivity and specificity, which have been found to be higher than for microneutralization fluorescent antibody test (MFA) and HI, although some have indicated similar sensitivity and specificity between HI and NT tests for certain viruses, including influenza A H1N1 2009. 2, 5, 8, 9 Additionally, it has been found to be more strain-specific than HI for seasonal and H5N1 viruses, and HI tests have been found to be insensitive for the detection of human antibody responses to avian hemagglutinin, especially when intact virus is present. 2, 6 According to Gross and Davis, the neutralization test “appears to detect lower levels of viral antibody than does the HI test,” a difference that “may be related to the high serum concentrations and the additional viral antigens detected by NT.” 10 It is, however, a laborious and time-consuming procedure, making it less suitable for testing large numbers of samples. 11 Disadvantages of NT include its difficulty level and time required to perform, the need for live virus, and that technical aspects of the assay can affect titers. 3, 8 However, the major disadvantage of it has been poor reproducibility between laboratories. 2, 3

In addition to low sensitivity, in particular as compared with radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), disadvantages of HI include subjectivity of result interpretation and reliability issues in relation to freshness of reagents. For both tests, immunity measurement is not exact, but rather based on titer cut points, and the endpoint of both assays requires visual inspection. Other than cost, ease, and reduced variability, a major advantage of HI over NT for measurement of seasonal influenza immunity is that HI does not require cytopathy, which does not always occur for each influenza virus in this assay. 7

Hemagglutination inhibition and NT assays have been utilized for years to investigate influenza immunity, although only a few studies have directly compared the assays' influenza antibody detection capabilities, and most of these studies evaluated vaccine-derived immunity. 2, 3, 12 Here, we compare HI and NT antibody titers from a sample of individuals from Guangdong Province, China, in order to formally compare the performance of HI and NT titers for measuring naturally derived immunity to twelve historic and recently circulating strains of influenza A. These twelve strains are seasonal influenza strains of both H3N2 and H1N1 subtypes that have been or are in broad circulation since 1968. Additionally, we will attempt to determine an equivalence factor between HI and NT titers for direct translation and comparison of results from both assays.


インフルエンザ特異抗体価を定量化する最適化された赤血球凝集抑制 (HI) 試験

抗体は、インフルエンザや他の病原体に対する血清学的保護のサロゲート マーカーとして使用されます。ワクチンによる免疫を理解する前と後のワクチン接種の抗体の生産の詳細な知識が必要です。この資料では、特定のインフルエンザ抗体価を決定するための信頼性の高いポイントによってプロトコルについて説明します。最初のプロトコルでは、赤血球凝集は、2 番目のプロトコル (赤血球凝集アッセイ、HA アッセイ) その後の使用のための集中を標準化するのに必要な抗原量を指定する方法について説明します。2 番目のプロトコルでは、ひと血清・細胞培養上清 (赤血球凝集抑制法、HI 試験) のシリアル希薄を使用して異なるウイルス株インフルエンザ特異抗体価の定量について説明します。

応用例として 3 価不活化インフルエンザ ワクチンを受けた健康コホートの抗体応答を示す.また、インフルエンザ ウイルスと交差反応を示す、さまざまな種類の動物の赤い血球 (赤血球) を使用して、交差反応を最小限に抑える方法を説明しました。議論は、長所と短所提示法の特定のインフルエンザ抗体価の定量免疫のワクチン関連の理解を改善する方法を強調表示します。

Introduction

インフルエンザ ウイルスによる感染症は、かなりの罹患率、死亡率と高い医療費 1 , 2 , 3 , 4 に関連付けられます。特に、高齢者、新生児、妊婦、慢性疾患患者より重篤な臨床転帰のリスクです。したがって、循環のインフルエンザ ウイルスに対するワクチン接種ハイリスク集団のこれらの疾患の負担を減少する主な尺度であります。予防接種、例えば、保護しきい値を超えるインフルエンザ特異的抗体の後個々 の免疫応答の増加人口 内のウイルスの伝達の可能性感染症と一般的に個々 のリスクが軽減されます。5 . ワクチン誘発される体液性免疫応答の別の集団と様々 な年齢グループ間で詳細な理解は重要な臨床質問 6 , 7 、 8 に答えるための重要な要素 , 9 、よう: 一部の高齢患者はなぜ感染症前のワクチン接種にもかかわらずを持って?「良い」と「十分な」ワクチン誘発される保護とは何ですか。どのくらいの頻度ワクチンは保護抗体に到達する免疫抑制患者に適用されるべきですか。最も効果的な投与量とは何ですか。ワクチン接種後の抗体価と新規アジュバントの影響は?ワクチンの抗体産生の測定は、これらの重要な質問に答えるし、予防接種の成果を向上させるに役立つことがあります。

ウイルス特異抗体価の定量は、様々 な免疫学的方法で実行できます。これには、固相 10 や中和の試金 13 ビーズに基づく ELISA 11 試金こんにちは試金 12 が含まれます。ELISA 法は、様々 な抗原に対する血清の比較的大量のスクリーニングを許可します。また、病原体特定免疫グロブリン (Ig) M および IgG 個別に検討します。抗原、例えば、直線のアミノ酸シーケンスまたはウイルスのような粒子の特性は、抗体の結合に影響を与える可能性があります潜在的なエピトープのスペクトラムは非常に広いと抗体かどうかに関する情報を提供しませんレスポンスは、機能的関連性です。

この資料は、特定のインフルエンザ抗体価を定量化するこんにちは世界保健機構 WHO ベース プロトコル 12 ステップバイ ステップの説明します。赤血球凝集は赤血球の凝集につながるいくつかのウイルスの特徴的な効果です。患者血清をこの効果の抑制には、中和効果を反映する抑制性抗体濃度測定が可能します。

同時に複数のサンプルのより効率的な処理と必要な時間を短縮できるように WHO プロトコルのワークフローを変更しました。最初のプロトコルでは、特定のインフルエンザ抗原の凝集反応電位の決定について説明します。そうすることで、正しいインフルエンザ抗原濃度は 2 番目のプロトコルの決定されます。この部分は、血液の各バッチと同様、すべての新しいウイルスの抗原で繰り返す必要があります。

2 番目のプロトコルでは、特定のインフルエンザ抗体価の定量について説明します。示されたプロトコルはしかしインフルエンザ ウイルスとひと血清サンプルの調査用に最適化された、それはも刺激免疫細胞、例えば、ウイルス特異 B 細胞からマウス血清や細胞培養上清中の適用することができます。結果は、絶対測定抗体として算定できます。多くのワクチン研究の幾何平均抗体価と 95% 信頼区間はそれぞれの特定の人口のため表示されます。解釈、seroprotection またはセロコン バージョンは、特定のウイルス集団の感受性を記述するよく使用されます。Seroprotection は、4 倍以上の抗体価を高める 2 つの時間ポイント (ほとんどの一般的予防接種前および 30 日後予防接種を使用) の間の seroprotective 抗体の達成と、≥1:40、および陽転の力価として定義されます。

両方のプロトコルが使いやすいし、彼らの研究の質問の広い範囲に適応することができます。特に、確実かつ迅速を決定する使用できます赤血球凝集反応、麻疹や polyomaviruses、流行性耳下腺炎、風疹 14 , 15 , 16 などの容量を持つ他の各種のウイルスに対する抗体価 .

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Protocol

研究プロトコルはローカル倫理審査委員会 (www.EKNZ.ch) によって承認された、すべての参加者から書面によるインフォームド コンセントを得た。

  1. 興味の時点で人間から血清サンプルを収集します。この研究のため、ワクチン接種後 0 (インフルエンザの予防接種の時間)、+7、+30、+60、+180 日で血清を集めました。
  2. 血清を得るためには、部屋の温度 (20-25 ° C) で 10 分の 1,200 x g でサンプル チューブを遠心します。
    メモ: 4 ° c と 24 h 以内、非遠心血液サンプルを保存する必要があります。
  3. 因数に異なる管 (低温瓶) と使用するまで-80 ° C で凍結血清。
  4. ポイントを含むすべての時間 - 患者内での変動を減らすために一人の batch-wise、その後の試金を実行します。

注意: 5 つの異なる抗原を使用 (材料の表を参照してください)。バイオ セーフティ レベル 2 (BSL-2) 研究室では、抗原を準備します。

  1. 製造元の指示に従って進む前に室温で 5 分間の最小値のために立つ溶存抗原が 1.0 mL の蒸留水と 1 つの凍結乾燥インフルエンザ抗原アンプルの合計内容を再構成でき。
  2. 分注 1.5 mL に抗原ソリューションはチューブし、さらに使用まで-80 ° C で凍結します。

注: コレラの濾液は、WHO プロトコル 12 によると酵素 (RDE) を破壊する受容体として使用されます。これは血清アッセイ 17 と邪魔になるから生得的な阻害剤を削除します。

注:、こんにちの試金、いくつかのプレートの間で比較できるように、同じウイルス粒子量は各プレートの使用する必要があります。赤血球凝集反応に必要なウイルス粒子を定量化に HA の試金 (HA 滴定とも呼ばれます) は、HA 単位に記録されます。赤血球凝集反応の「単位」HA の滴定で使用される方法に依存して運用ユニットし、ウイルスの絶対的な量の測定ではないです。したがって、HA ユニットは、標準化された赤血球懸濁液の等量に凝集するために必要なウイルス量として定義されます。WHO によるとこんにちは試金のための標準量は 4 HA 単位 25 μ L です。HA アッセイの原理図は、図 1を参照してください。


図 1: 凝集性と赤血球凝集抑制の原則です。ウイルスや抗体 (左列)、なしのネガティブ コントロール状況で凝集が発生せず、赤血球は、インフルエンザ ウイルス (中欄) の存在下で hemagglutinate。しかし、インフルエンザ ウイルスのヘマグルチニンがウイルス特異的抗体ない凝集によってブロックされた場合 (右側の列) に発生します。この図の拡大版を表示するのにはここをクリックしてください。

注: 使用される赤血球 (表 1) アッセイにおけるインフルエンザ ウイルスの型に依存しています。さらに、96 ウェル マイクロタイター プレートのさまざまな種類の非分離細胞の出現と同様、インキュベーション時間異なります (表 2)。

インフルエンザ抗原 A/カリフォルニア/7/09 (H1N1) A/スイス/9715293/2013 (H3N2) A/テキサス/50/2012 (H3N2) B/ブリスベン/60/08 B/マサチューセッツ/02/2012
RBC 種 チキン モルモット モルモット トルコ トルコ

表 1: インフルエンザ抗原と対応する種赤血球の。よると製造元の指示 (NIBSC)。

RBC 種 チキン トルコ モルモット ヒト型 O
赤血球 (v/v) 濃度 0.75% 0.75% 1% 1%
マイクロ プレートの種類 V 底 V 底 U 底 U 底
培養時間、RT 30 分 30 分 1 時間 1 時間
非凝集のセルの外観 ボタン * ボタン * ヘイロー ヘイロー

表 2: 赤血球の種を条件アッセイします。WHO のプロトコルによると。(* 傾いているときに流れる)。

  1. 赤血球懸濁液の準備
    1. 赤血球在庫懸濁液を希釈 (10%、v/v O 人間型を除く) (材料の表を参照してください) リン酸緩衝生理食塩水 (PBS) それぞれ適切な 0.75% と 1%、鳥類と哺乳類の赤血球濃度を確認します。


    図 2: HA アッセイのデザインをプレートします。重複で HA の滴定を行います。コントロールの行に抗原が追加されませんでした。また最高の抗原濃度の定量のための図 4を参照してください。この図の拡大版を表示するのにはここをクリックしてください。

    1. 96 ウェル マイクロタイター プレートの準備
      注: プレート デザインの概要については図 2を参照してください。
      1. 96 ウェル マイクロタイター プレートの使用される各行の 1 に 12 の井戸に 25 μ L の PBS を追加するには、マルチ チャンネル ピペット (図 2) を使用します。鶏と七面鳥のような鳥の赤血球を操作するときは、V 字マイクロタイター プレートを使用します。哺乳類赤血球、モルモットやヒト型 O (表 2) を操作するときは、U 字マイクロタイター プレートを使用します。
      2. 重複整理される抗原行の最初の井戸にインフルエンザ抗原の 25 μ L を追加します。コントロールの行には、抗原は追加されません。コントロールの行ない凝集効果を示す、ネガティブ コントロール (図 2) となります。
      3. シリアル 2 倍希釈を実行するには、マルチ チャンネル ピペットを使用して連続井戸に抗原行の最初の井戸から 25 μ L を転送します。各希釈段階をミックスするには、上下にゆっくり 10 回ピペッティングします。
      4. 最後のウェルスの最終的な 25 μ L を破棄します。
      5. 使用される各行の 1 に 12 の井戸に 25 μ L の PBS を追加するには、50 μ L/ウェルの容量に達するためにマルチ チャンネル ピペットを使用します。
      6. 各マルチ チャンネル ピペットを使用して、よく使用する赤血球懸濁液 50 μ L を追加します。
      7. タップ プレートを慎重に 10 回四方を混在させることにします。
      8. 蓋付きプレートをカバーし、RBC 種使用 (表 2参照) に応じて適切な時間室温で孵化させなさい。抱卵中板を移動しないでください。


      図 3: 鳥および哺乳類の赤血球の凝集パターン。V 字マイクロタイター プレートは、鳥の赤血球を操作するときに使用されます。傾斜プレート位置の読み出しを実行し、非凝集赤血球が涙のような形を形成して実行を開始します。U 字のマイクロタイター プレートは、哺乳類の赤血球を操作するときに使用されます。読み出しが非傾斜の位置に実行され、非凝集赤血球を形成する小さなハロー。この図の拡大版を表示するのにはここをクリックしてください。

      1. 版を読む
        注: 異なる形マイクロタイター井戸 (図 3) ため哺乳類赤血球と比較して鳥の赤血球を使用する場合、読み出しは若干異なります。
        1. 鳥類の赤血球の読み出し
          1. プレートを 25 の 90 ° 傾け s。
            注: は、凝集パターン (完全に付属、一部付属と非凝集) のすべての 3 種類が傾かないときボタンとして表示されるため鳥パターンの分化の重要なプレートを傾斜です。
          2. プレートの傾斜の位置、96 ウェル プレートの印刷方式の中、すぐに結果をマークします。鳥類の赤血球の凝集パターンは、図 3のとおりです。
          1. プレート (ベンチの水平位置) で傾くことなし 96 ウェル プレートの印刷方式の結果をマークします。
            注: 赤血球凝集が発生すると、付属の細胞に定着しない下、井戸の底でヘイローのような非凝集の細胞に対し。部分的に付属のセルのハローは薄くより大きい直径 (図 3)。


          図 4: 4 HA 単位の力価を決定する鳥の赤血球と HA の滴定の読み出し。赤血球凝集アッセイ (抗原滴定試金) 赤血球凝集反応に必要な最適な抗原量を測定します。完全な凝集が発生した最後の井戸、HA 滴定エンドポイントであり、1 HA ユニットが含まれています。抗原、HA 滴定終点の前の 2 つの井戸の 2 倍希釈のため、力価は 4 HA 単位に対応します。この図の拡大版を表示するのにはここをクリックしてください。

          注: プロトコルの作業フローは、同時に複数のサンプルのより効率的な処理を許可する、pcr 法を用いたチューブ ストライプと熱サイクラー (下記参照) に最適化されています。

          1. 血清試料の調製
            注: は、BSL 2 研究室で血清を準備します。
            1. 一人一人の各時点の冷凍血清サンプルを解凍 (ステップ 1.2 参照) 室温で。
            2. PCR チューブ ストリップ (1 つのストリップでの 10 管) の管に各解凍血清サンプルの 10 μ L の因数を追加します。
              注: PCR チューブ ストリップを使用しての大きな利点は、こんにちの試金 の次の手順でマルチ チャンネル ピペットを使用できることこれは大量の血清サンプルをテスト時に、異なるウイルス株に対する抗体価の同じサンプルの測定を繰り返し実行するときに多くの時間を節約できます。
            3. 使用するまで-80 ° C で PCR チューブ ストリップに検体の血清サンプルを格納します。
            4. 1 日こんにちは試金の実行前に、室温で関心の血清サンプル因数を解凍します。
            5. 空の PCR チューブに適切な抗血清の 10 μ L を追加します。
              注: 肯定的な制御として、特定のウイルスに対する抗血清は使用されるウイルスを一致しなければなりません。肯定的な制御により複数の板プレートのパフォーマンスの標準化。
            6. マルチ チャンネル ピペットを使用して各血清因数と抗血清・血清の 1 ボリュームにコレラ濾液の 3 巻) コレラ濾液ソリューションの 30 μ L を追加します。
            7. PCR チューブ PCR 96 ウェル ラックまたは空のヒント ボックスと 5 の渦をおいて s。
            8. 一晩で 37 ° C 熱 cycler を使用してサンプルをインキュベートします。
            9. 熱 cycler を使用してコレラの濾液を不活化する 30 分の 56 ° C でサンプルをインキュベートします。
              注: 熱 cycler によってこのステップはプロセスをさらに自動化するプログラムできます。
            10. PCR チューブ PCR 96 ウェル ラックまたは空のヒント ボックスと 5 の渦をおいて s。
            11. こんにちは試金のための使用するまで冷蔵庫で 4 ° C でサンプルを格納します。


            図 5: プレート デザインとこんにちはアッセイのワークフロー 。一皿に二人の 5 つの時間ポイントを測定できます。HI 抗体価は 8 から 1,024 を範囲します。肯定的な制御使用される抗原の抗血清と抗原希釈 4 HA 単位に等しいかどうかをチェックする背部滴定を行った。2 個別ワクチン接種者の血清サンプルのシリアル希薄化が表示されます。 この図の拡大版を表示するのにはここをクリックしてください。

            1. こんにちはアッセイ
              注: こんにちはアッセイの原理図は、図 1を参照してください。ウイルスによって赤血球の種、(表 1) 試金のために使用されます。96 ウェル プレートの様々 な種類で使用する赤血球の種と非分離細胞の出現と同様、インキュベーション時間が異なります (表 2)。こんにちは試金のためウイルスや抗原の 4 HA 単位はサンプルの 2 倍希釈系列に追加されます。
              1. 抗原溶液の調製
                1. 96 ウェルのプレート使用数に応じて抗原溶液の体積を計算 (96 ウェル プレートあたりも × 96 = 2,400 μ L 抗原あたり 25 μ L 抗原 100 μ L プレート マルチ チャンネル ピペット のための貯蔵の使用のために余分な/を追加合計 antige 2.5 mLプレートごと n)。
                  注: たとえば場合 100 の血清サンプルを測定、10 皿が必要 (10 サンプル プレートごと): 合計で必要な抗原溶液 2.5 mL × 10 = 25 mL。
                2. PBS を使用する計算された容積の 4 HA 単位の適切な希釈を準備します。
                  注: HA の試金のため 4 HA 単位が決定されます。抗原の適量 4 HA 単位に対応する抗体によって計算されたボリュームを分割します。たとえば、1/64 の希薄化に対応する 4 HA 単位、15,000 が必要抗原溶液の μ L が必要: 15,000/64 = 234.4 溶存凍結乾燥インフルエンザ抗原の μ L を追加。
                1. 96 ウェル マイクロタイター プレート使用数に応じて赤血球懸濁液の体積を計算 (よく × 96 = 4,800 μ L 赤血球懸濁液 96 ウェル プレートあたりあたり 50 μ L の赤血球懸濁液 200 μ L プレート マルチ チャンネル ピペット用の貯水池の使用のために余分な/を追加).
                2. 赤血球在庫懸濁液を希釈 (通常 10%、v/v O 人間型を除く) 適切な 0.75% と 1%、鳥類と哺乳類の赤血球濃度をそれぞれする PBS の。
                1. (サンプル ID、肯定的な制御、および背部滴定) 96 ウェル マイクロタイター プレートにラベルを付けます。5 プレートの向きをよく確認ください。
                2. 25 μ L の PBS を加える「滴定を戻る」行 (図 5、12 番目 の行) 最初井戸中にを除くすべてのよくマルチ チャンネル ピペットを使用しますします。
                  注: 使用される抗原希釈 4 HA 単位に等しいかどうかをチェックする背部滴定を行った。4 HA 単位の抗原抗体価を示す赤血球凝集が発生します 4 番目ではなく「戻る滴定」の行の最初の 3 つの井戸でも。
                3. 「滴定を戻る」行 (12 番目 の行) 最初井戸に (5.2.1 で説明) 準備された抗原溶液 50 μ L を追加します。
                4. マルチ チャンネル ピペットを使用して、各プレートの行 1 に 10 の最初の井戸に RDE 治療血清サンプルの 25 μ L を追加します。
                5. 肯定的な制御として 11 番目 の行の最初の井戸の中に適切な抗血清を 25 μ l 添加を追加します。
                6. シリアル 2 倍希釈を実行するには、マルチ チャンネル ピペットを使用して連続井戸に (1-12) の各行の最初の井戸から 25 μ L を転送します。各希釈段階の上下に 10 〜 15 回をピペッティングで混ぜます。サンプルごとの各希釈段階の同じヒントを使用できます。
                7. 最後のウェルスの最終的な 25 μ L を破棄します。
                8. 抗原溶液の 25 μ L を追加するには、行 1 に 11 (血清と抗血清) の各ウェルにマルチ チャンネル ピペットを使用します。彼らは井戸を触れないでください場合、同じヒントを使用できます。
                9. 「滴定を戻る」行 (12 番目 の行) の各ウェルに抗原ではなく 25 μ L の PBS を追加します。
                10. タップ プレートを慎重に 10 回四方を混在させることにします。
                11. 蓋付きプレートをカバーし、30 分間室温でインキュベートします。抱卵中板を移動しないでください。
                12. 赤血球懸濁液 50 μ L を各ウェルに追加します。
                13. プレート慎重に 10 倍をタップ 4 四方をミックスします。
                14. 蓋付きプレートをカバーし、RBC 種使用 (表 2参照) に応じて適切な時間室温で孵化させなさい。抱卵中板を移動しないでください。
                1. 鳥類の赤血球の読み出し
                  1. プレートを 25 の 90 ° 傾け s。
                    注: は、凝集パターン (完全に付属、一部付属と非凝集) のすべての 3 種類が傾かないときボタンとして表示されるため鳥パターンの分化の重要なプレートを傾斜です。
                  2. プレートの傾斜の位置、96 ウェル プレートの印刷方式の中、すぐに結果をマークします。鳥類の赤血球の凝集パターンは、図 3のとおりです。
                  1. プレートを傾けることがなく 96 ウェル プレートの印刷方式の結果をマークします。
                    注: 赤血球凝集が発生すると、付属の細胞我慢しないでください井戸の底でヘイローのような非凝集の細胞に対し。部分的に付属のセルのハローは薄くより大きい直径 (図 3)。
                  2. 各サンプルの HI を決定し、コンピューター ベースの表 (図 6) に転送
                  3. 注: 低力価として部分的に付属の井戸を求めた。たとえば、血清サンプルを完全に阻害も 4 まで赤血球凝集 (希釈 1: 64)、5 th も (1:128 希釈) は部分的に、上水、HI 抗体価は、最終的な分析 (図 6、低価 64 に設定されています。4 番目 の行)。


                  図 6: 鳥の赤血球とこんにちはアッセイの読み出し。前と後のワクチン接種によるインフルエンザ抗体応答は、こんにちの試金によって決まります。この例では、1 つ人は 2 人よりも高い HI 抗体を持っています。両方の人はワクチン接種の後抗体応答を表示します。ワクチン接種後 180 日間両方の人の抗体価は再び減少しました。この図の拡大版を表示するのにはここをクリックしてください。

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                  Representative Results

                  前と後のワクチン接種による抗体応答をインフルエンザ A 型 H3N2
                  ワクチンによる抗体は 2014 年前インフルエンザ A/H1N1/カリフォルニア/2009、A/H3N2/テキサス/2012 B/マサチューセッツ/02/2012 を含む三価サブユニットの不活化インフルエンザ ワクチンを受けた 26 の健康なボランティアで評価された/2015 年のインフルエンザ シーズン。2 ワクチン接種者の代表例を図 6に示します。興味深いことに、その特定のインフルエンザ シーズン中にテキサス/2012 A/h3n2 型循環されなかったとシーズンが多少異なるウイルス株に含まれて代わりに: A、h3n2 型、スイス連邦共和国、2013。A、H3N2、テキサス州、2012 と 2013 年 A 月 H3N2/スイス連邦共和国のウイルスのヘマグルチニン 97% 順序のアイデンティティを示すし、は異なる唯一の 11 アミノ酸 (表 4参照)、その位置は図 7で強調表示されます。


                  図 7: A/h3n2 型インフルエンザのヘマグルチニン比較します。A/テキサス/50/2012 と A/スイス/9715293/2013 株ウイルスのヘマグルチニンを比較しました。インフルエンザ A/ビクトリア/361/2011 18 、テキサスひずみと 95% 98% 順序のアイデンティティを表わす非常に同じようなヘマグルチニンの結晶構造を用いてこれらの系統の血球凝集素結晶構造がないのでスイス連邦共和国の歪みを持つシーケンス id。テキサス州、スイスの系統が異なるアミノ酸位置が強調表示されます。この図の拡大版を表示するのにはここをクリックしてください。

                  A、h3n2 型、スイス連邦共和国、2013 とテキサス/2012 A/h3n2 型ウイルス株の交差反応性の免疫反応を観察した.こんにちは価インフルエンザ A/H3N2/スイス/2013 に対して著しく低かった幾何平均抗体価で誘導される seroprotection (図 8 a) と比較してインフルエンザ A 型 H3N2 2012//テキサス/(図 8 b)。


                  図 8: 健康なドナーの幾何平均抗体価。幾何平均-抗体 (GMTs) 25 健常者前と後の予防接種の 2 つの異なる抗原を使用して決定されます。A/H3N2/スイス/2013 (A) および A/H3N2/テキサス/2012 (B) の平均抗体価が表示されます。ワクチン接種による免疫応答は、予防接種 (d0) の前に GMTs と比較して (d7 d60)、ワクチン接種後抗体を増加として観察できます。接種後 180 日間、GMTs は再び減少します。注記のうち、唯一 A/H3N2/テキサス/2012 (ワクチンが) 保護抗体価に達する。バーは幾何平均抗体価を示し、ひげが 95% 信頼区間を示します。破線は、seroprotection のしきい値を示します。Seroprotected 人の % (力価 > 1:40) がグラフで表示されます。この図の拡大版を表示するのにはここをクリックしてください。

                  予防接種後テキサス/2012 A/h3n2 型に対する抗体価はほとんどの科目で増加A、h3n2 型、スイス連邦共和国、2013 株ワクチンに存在していなかった、A、h3n2 型、スイス連邦共和国、2013 抗体価と同様にいくつかの科目に増加しました。図 9は、線形回帰モデルの 0.745 の R 2 すべての時間ポイントの両方の抗体価との相関関係を示しています。1 つと同様、2013 年 A 月 H3N2/スイス連邦共和国に対して抗体反応の誘導だった以下の強力です。


                  図 9: A/h3n2 型インフルエンザ間の交差反応します。すべての個人と時間のポイントの A/H3N2/テキサス抗体は対応する A/H3N2/スイス抗体に対してプロットされます。線形回帰モデルの 0.745 R 2 を示しています。この図の拡大版を表示するのにはここをクリックしてください。

                  使用血液の種類をに基づいて潜在的な凝集
                  ウイルスのヘマグルチニンは、赤血球を hemagglutinate に別の種依存性を示しています。この種に依存した効果には、赤血球凝集抑制試験も影響します。測定抗ウイルス抗体の特異性を改善するために行った 5 つのウイルス抗原を赤血球の最高の適している型 (A と A/H3N2/テキサス/2012 年インフルエンザ A/H1N1/カリフォルニア/2009 B/マサチューセッツ/02/2012 インフルエンザ B/ブリスベン/60/2008/H3N2/Switzerland/2013) 最大の凝集も最低の交差反応性を達成するために。生物学的標準とコントロール (NIBSC) 各抗原に対するこれらのアッセイを実行する所から陽性コントロール血清使いました。

                  インフルエンザ b、B/マサチューセッツ/02/2012 による抗体応答が B/ブリスベン/60/2008 に対して保護を提供しないことを観察すること。対照的に、B/ブリスベン/60/2008 に対する抗体は、異なる赤血球 (表 3参照) で 4 倍低価で B/マサチューセッツ/02/2012 に対して交差反応性を示した。関心のモルモット血でしたいない正しく hemagglutinate インフルエンザ * トルコ血前述 A/H3N2/テキサスから離れて相対低交差反応性を hemagglutinate する可能性と高い抗体価を示すに最善を尽くしたと/スイス連邦共和国の系統。

                  トルコ モルモット チキン ヒト型 O
                  B/ブリスベン 1024 - 1024 1024
                  B/マサチューセッツ州 1024 384 768 1024
                  A/H3N2/スイス 1024 1024 - 1024
                  A/H3N2/テキサス 1024 1024 512 1024
                  A/H1N1/カリフォルニア 1024 1024 768 768

                  テーブル 3:肯定的な制御異なった種類を渡ってそれぞれインフルエンザ HA 抗原に対する抗体です

                  違います A/H3N2/テキサス/2012 ひずみ A、h3n2 型、スイス連邦共和国、2013 ひずみ 位置
                  1 アスパラギン (N) アラニン (A) 128
                  2 アラニン (A) セリン (S) 138
                  3 イソロイシン (I) Arginine (R) 140
                  4 Arginine (R) グリシン (G) 142
                  5 アスパラギン (N) セリン (S) 145
                  6 フェニルアラニン (F) セリン (S) 159
                  7 グリシン (G) バリン (V) 186
                  8 プロリン (P) セリン (S) 198
                  9 セリン (S) フェニルアラニン (F) 219
                  10 アスパラギン (N) アスパラギン酸 (D) 225
                  11 * リジン (K) Arginine (R) 326
                  * ヘマグルチニンは残渣 325 で刈り取らため図 8 に示した結晶構造の表示されません。

                  表 4: A/H3N2/テキサス/2012 と 2013 年 A 月 H3N2/スイス系統ヘマグルチニンのアミノ酸のリスト

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                  Discussion

                  前と後のワクチン接種インフルエンザ ウイルス抗体価の定量化は、ワクチン研究に必要な重要なツールです。Seroprotection などのウイルス感染症に対する保護のサロゲートのメジャーに基づいて (> 1:40) またはセロコン バージョン (力価の 4 倍増)、予防接種戦略をすることができます最適化された 9 。指定されたプロトコルを使用して判断できます: (i) (ii) 興味のウイルス抗体価と特定のウイルスの赤血球凝集可能性。

                  変更とトラブルシューティング:

                  このプロトコルは、WHO 標準 12 に基づいています。(手順 5. を参照) 血清の準備のため PCR チューブ ストリップを使用して、プロトコルを変更しました。この変更は、作業負荷を大幅に削減し、アッセイのスループットを向上させる助けた。さらに、我々 は効果的なコスト超過が抗原の滴定手順で 4 分の 1 で抗原量を削減しました。サンプル量が限られている場合は特に役立ちます RDE の治療のため血清 (10 μ L) のより低い量を使用できます (、マウス血清)。背部滴定および肯定的な制御は、適切な内部統制として機能し、赤血球の老化を監視する抗体測定プレートに含まれます。

                  さらに、赤血球血栓良いビジュアル読み出しのための最適なサイズを設定するのに WHO 標準 12 のそれらより別の赤血球濃度を使用しました。これを保証するには、測定前に赤血球濃度をチェックをお勧めします。とはいえ、この部分を最適化プロトコル我々 していない、OD や細胞数と吸光度測定などの方法を使用できます。

                  場合は、RDE は完全に不活性化されず、赤血球は凝集反応を室温で測定する場合 desialylated と逆ハ正井戸とすることができます。我々 は決してこの問題を観察がこの場合、私達は RDE 活性は 4 ° C で有意に低いのでこんにちは 4 ° c を実行する提案します。ただし、4 ° C でこんにちは試金の実行は遅くなります。

                  こんにちは試金のいくつかの重要な側面は、次の点: 興味深いことに、血球凝集反応は赤血球 (例えば、トルコまたはモルモット赤血球) の特定の種類に強く依存します。血の最適な型は、特定のウイルス株が評価され、分析を通じて使用される同種血の前にテスト必要があります。ウイルス間の交差反応性の誘導は、わずかに新しいウイルス 19 , 20 の場合免疫学的利点を生成可能性があります、低特異性による診断の視点からいくつかの問題があります。したがって、類似のウイルス間の交差反応する必要があります慎重に対処して研究については後述します。赤血球を選択すると特定の種から、交差反応性の量をやや下げることができます。

                  こんにちは、高い再現性と信頼性の高い結果を提供する老舗のゴールド スタンダード方法です。こんにち HA 茎ループにバインドし、それにより中和と相関する抗体を検出するだけに対し、ELISA などの手法は非中和抗体を検出があります。

                  プロトコルの中で重要なステップ:

                  最も重要な手順は、RDE 非特異的阻害剤とバインディングを不活化すると血清治療ウイルスの ha。もう一つの重要なステップです、赤血球の溶血反応の制御時間をかけて彼らの年齢として。

                  将来のアプリケーション:

                  プロトコルは潜在的な凝集反応で他のウイルスの使用可能性があります。結果を示されて、ひと血清中のサンプルをここに我々 がのみ、刺激 B 細胞 (データは示されていない) と培養上清中の細胞培養、マウス血清中の抗体価を測定するアッセイを使えます。要約すると、こんにちは、ワクチンによる抗体の迅速かつ再現性のある評価をことができます。


                  Where can I find hemagglutination inhibition (HI) assay data? - Biology

                  The presence of antibodies to Newcastle disease virus in chickens is detected by serological testing. The results of these tests are used for three purposes.

                  1. To assess the efficacy of Newcastle disease vaccine in laboratory and field trials.

                  2. To assess the level of Newcastle disease virus antibodies in the field.

                  3. Serum known to contain antibodies to Newcastle disease virus is used to confirm the presence of Newcastle disease virus in a test sample of allantoic fluid. Such a sample would be obtained during the isolation of virulent Newcastle disease virus. See Section 15 .

                  There are two assays commonly used to carry out serological testing for Newcastle disease virus antibodies.

                  1. Haemagglutination inhibition (HI) test. The HI test is a convenient and commonly used assay that requires cheap reagents and is read by eye.

                  2. ELISA (Enzyme linked immunosorbent assay). This is a colourimetric assay and requires the use of a sophisticated instrument to read the optical density of the reactions. ELISA kits for Newcastle disease virus antibody detection are prepared and sold commercially. Detailed instructions are supplied with the kits. They are usually quite expensive.

                  In this manual a protocol for the HI test based on the test described by Allan and Gough will be used for serological testing. (Allan and Gough, 1974 a.)

                  Serum samples are collected for testing for the presence of antibodies to Newcastle disease virus. Blood is collected as described in Section 7. The blood forms a clot in the syringe in a few minutes. Once the blood clots, the syringes of blood can be kept with the needle upright to prevent serum filling the needle cap. The serum will separate from the clot within a few hours at room temperature or in approximately 2 hours at 37°C. Storage at 37°C will help the serum separate from the clot.

                  • Syringes with blood samples.
                  • Sterile glass Pasteur pipettes.
                  • Microfuge tubes.
                  • Storage tubes. (1.8 mL Nunc cryotubes are ideal but microfuge tubes are a cheaper alternative.)
                  • Sharps container and discard bag for biological waste.

                  1. Remove the plunger from the syringe. Transfer the serum to a microfuge tube by pouring or using a glass Pasteur pipette.

                  2. Dispose of needles, syringes, clots and pipettes in appropriate containers.

                  3. Often the serum will contain red blood cells. Centrifuge for 30 seconds in a microfuge centrifuge or allow to settle under gravity overnight at 4°C to pellet the cells. Do not freeze the samples at this step. Freezing will lyse the red blood cells.

                  4. Transfer clear serum to a second tube.

                  5. Remember to label each tube after the transfer of the serum. This ensures the serology results can be applied to individual birds and groups.

                  Store ampoules of serum at -20°C.

                  Storage at 4°C is acceptable for a short period, up to 2 weeks.

                  Samples collected in the field may end up at room temperature overnight and often do not separate well.

                  It has been noted at the John Francis Virology Laboratory that in some samples the serum does not separate from the clot. In these cases, the whole clot is centrifuged. This usually results in a small amount of serum separating.

                  It is important that the serum samples are clear. Pink samples contain lysed red blood cells. When pink samples are tested observe both test and control samples carefully to determine effect of the colour on the results of the test.

                  Pink coloured samples can affect results of an ELISA, which is a colourimetric assay.

                  An overview of the Haemagglutination Inhibition (HI) test

                  Antibody response to the haemagglutinin protein in the Newcastle disease virus envelope can be measured by the HI test.

                  When serum containing these antibodies is mixed with Newcastle disease virus, the antibodies bind to the haemagglutinin protein in the envelope of the virus. This blocks the haemagglutinin protein from binding with the receptor site on chicken red blood cells.

                  Thus the haemagglutination reaction between the virus and the red blood cells is inhibited.

                  By performing two-fold serial dilutions on the serum prior to testing, the concentration of the serum antibodies can be expressed as an HI titre to the log base 2.

                  Standardization of the HI test

                  It is important that there is correlation between the results of tests carried out by different technicians and in different laboratories. For this reason HI tests should be standardized both within a laboratory and between laboratories.

                  Standardization is achieved by following a standard protocol. This will include:

                  Using a standard 4 HA units of Newcastle disease virus antigen.

                  Using standard positive anti-serum and negative serum.

                  Including a serum control for each test serum to detect the presence of non-specific agglutinins.

                  Using a standard 1 percent dilution of red blood cells.

                  The use of control serum in the HI test

                  Positive and negative control sera are tested to avoid errors in the interpretation of the results of the HI test. Inconsistencies in the results of the HI test may be caused by variations in reagents and procedures.

                  Examples of possible variations include:

                  The source and exact percentage by volume of the red blood cells.

                  The exact amount of antigen used in the HI test.

                  Accuracy of dilutions.

                  Temperature.

                  Time allowed for the antibody/antigen reactions to occur.

                  Quality of test serum samples (haemolysed serum samples may be responsible for non-specific reactions)

                  Two standard sera are used.

                  1. A standard negative control serum known to contain no antibodies to the Newcastle disease virus. It has no HI titre and does not agglutinate chicken red blood cells.

                  2. A standard positive control serum also known as a standard anti-serum. The HI titre of the anti-serum will have been established by repeated titration.

                  Variability in HI titres of standard positive serum

                  It is sometimes observed that the standard HI titre of the standard anti-serum tested on the same day with the same antigen preparation and protocol may vary. A difference in HI titre of one dilution that is one log base 2 (2 1 ) can be regarded as due to random errors and an inherent variability in biological responses. However if the titre of the standard positive serum differs by more than one dilution or log base 2, then the test is invalidated. In this case, fresh antigen must be prepared and tested and the HI test repeated.

                  Three categories of standard sera

                  1. International standard reference serum. Certain laboratories prepare reference serum to a very high standard. Standard reference Anti-Newcastle disease serum is available for purchase. It is used as a reference serum in the preparation of national and laboratory standard sera.

                  The National Institute for Biological Standards and Control (NIBSC) in the United Kingdom can supply an international standard reference serum. The potency of the international reference serum has been determined by the supplier and is expressed in International Units (IU) per ampoule of freeze-dried serum.

                  Email [email protected]
                  Tel 44 (0) 1707 654753
                  Fax 44 (0) 1707 646730
                  Website http://www.nibsc.ac.uk

                  2. National standard serum is prepared by a national laboratory and distributed to collaborating laboratories as required. This serum is prepared by mixing sera with known HI titres. A series of HI tests are carried out to establish the HI titre of the national standard. This is compared with the HI titre of the international standard reference serum if available. The national standard serum is then stored in multiple aliquots and distributed to collaborating laboratories within a country.

                  3. Laboratory standard serum is prepared by some laboratories in order to conserve supplies of national standard serum. The laboratory standard is prepared by mixing serum samples with known HI titres. Comparative HI testing of the laboratory standard and the national standard is used to establish the HI titre of the laboratory standard.

                  Preparation of national and laboratory standard sera

                  Each laboratory will require a supply of negative and positive control serum in order to carry out HI tests on serum samples.

                  Collection of HI negative serum

                  Collect serum from chickens that have had no exposure to Newcastle disease virus. The serum should show no inhibition of viral haemagglutination activity when tested by the HI test. It is often difficult to find serum without any HI activity. In this case use serum with low HI titres of 2 1 .

                  Collection of HI positive serum

                  There may be stored serum that is suitable for this purpose. If not, vaccinate several chickens with I-2 Newcastle disease vaccine. Two weeks after the primary vaccination, give a booster vaccination. Two weeks later, take a serum sample from each chicken and test the HI titre. Collect extra serum from chickens with a titre of 2 5 or above. The volume of blood collected from each chicken depends on the size of the chicken. Pool all the serum samples with HI titres of 2 5 or above and test the HI titre of the pooled serum.

                  Comparative HI testing of international reference and national standard sera

                  Information supplied by NIBSC in 2001 indicated their international standard reference contained 320 IU per ampoule.

                  The serum can be reconstituted in PBS. A suitable volume of PBS would be 2 mL, which would give 4 IU in the 25 µL test sample. Refer to instructions provided by the manufacturer.

                  Use the standard HI test described in this section to determine the titre of the international reference standard and the national standard.

                  Test both samples in triplicate on the same microwell plate.

                  Carry out a series of at least three tests on different days.

                  Once the HI titre of the international standard serum and the national standard serum have been established, prepare a series of two-fold dilutions of the serum that are spread around the endpoint used to establish the HI titres. Titrate the dilutions that range from complete inhibition to no inhibition as an additional check on the HI titres. The results of testing these dilutions will confirm the HI titre of the undiluted serum.

                  Example of confirming HI titre of standard samples by testing a series of dilutions of the sample.

                  Repeated testing of the pooled serum established the HI titre at 2 5 .

                  Prepare 1/8, 1/16, 1/32 and 1/64 dilutions of the pooled serum.

                  Test each dilution for HI titre.

                  Confirmatory results: The results tabled confirm that the HI titre of the serum used to prepare the dilutions had a titre of 2 5 .

                  Table 3: Results confirming HI titre of serum = 2 5

                  The test results will give a HI titre for both the International reference serum and the national laboratory serum being tested. The HI titre for 4 IU of the International reference serum can be used to determine the number of IU in the national reference serum.

                  The standard HI test of 25 µL of the international reference serum containing 4 IU and has a HI titre of 8 (2 3 ). The national serum tested HI titre of 64 (2 6 ).

                  How many IU does the national serum contain?

                  4 IU in a sample with a HI titre of 8.

                  How many (?) IU in a sample with a HI titre of 64.

                  Not all laboratories use the same protocol for testing HI titre and IU are independent of the test system used.

                  Serological results of samples expressed in IU should give equivalent results when tested by different systems.

                  By comparing the HI titre of the standard national serum with the HI titre of the international reference serum enables you to express the results of HI tests in IU. However it is not very often that you will be required to express the results of HI testing in IU.

                  Most publications of Newcastle disease serology results describe the assay used and express HI titres to log base 2.

                  Preparing a laboratory standard serum

                  Each laboratory should receive a national standard serum from a central laboratory. The HI titre and activity in International units of the serum will have been determined by rigorous testing as described above. To conserve the supply of national standard serum some laboratories will decide to prepare a secondary laboratory standard serum.

                  Collect positive serum as described above. Do comparative testing of the laboratory and national standards. Test each sample in triplicate on the same microwell plate. Repeat several times and analyze HI titres for both samples. Record the HI titre for the laboratory standard.

                  Store 1 mL aliquots of the laboratory standard at -20°C. This standard is used every time the HI test is carried out and should always show the same HI titre when tested with the 4 HA units of antigen.

                  Preparing a national standard serum without an international reference serum

                  Many laboratories will not be able to obtain an international reference serum with which to compare their national standard serum. In this case, more rigorous HI testing of the pooled HI positive serum samples will be required to establish the HI titre of the standard serum. It is suggested that the serum is tested up to ten times. Use freshly prepared 4 HA units of antigen for each test and carry out the test on different days if possible. The results will be a range of HI titres. The most frequently occurring titre can be considered the HI titre of the standard serum. All future HI tests carried out on the standard serum should give this titre.

                  Storage of standard serum

                  Once the positive and negative standard sera have been tested, aliquots can be prepared, labeled and stored frozen. Storage at -70°C is optimal (but -20°C is adequate). Do not thaw and refreeze the samples frequently. Representative samples should be thawed and tested to confirm that there is no loss of titre in the storage process.

                  Preparation of Newcastle disease virus antigen for use in HI tests

                  Antigen is prepared by inoculating embryonated eggs with a sample of Newcastle disease virus and harvesting the allantoic fluid four days later. Part of the first batch of I-2 Newcastle disease vaccine prepared from the I-2 working seed can be set aside for storage as antigen. A volume of 50 mL to 100 mL is adequate for a large number of tests. Centrifuge the sample at 1 200 g to clarify and remove any contaminating red blood cells. Store the antigen in one mL aliquots at -20°C.

                  Preparation of 4HA units of Newcastle disease virus antigen

                  The standard amount of Newcastle disease virus used in the haemagglutination inhibition (HI) test is 4HA units. It is necessary to prepare and test a suspension of Newcastle disease virus containing 4HA units in order to carry out the HI test. This involves a series of following steps.

                  1. Titrate the stored suspension of virus to be used as the antigen in the HI test. See Section 10 Calculate the HA titre.

                  2. Calculate the dilution factor required to produce 4 HA units. A simple way is to divide the HA titre by 4.

                  3. Apply the dilution factor and dilute the original suspension of antigen in PBS to produce an adequate volume of 4HA antigen to carry out the HI test. Allow 2.5 mL for each microwell plate.

                  4. Titrate the diluted (4HA) suspension of virus. This is a back titration to check the diluted antigen contains 4 HA units.

                  5. Read HA titre. It should equal 4HA units. If not adjust the dilution and titrate again.

                  6. Use the 4HA unit dilution of antigen in an HI test to test the standard positive and negative serum. The HI titre of the laboratory standard positive serum should equal the predetermined titre.

                  The results of the back titration of the diluted antigen and the HI titre of the standard positive are both used to confirm the antigen has been diluted to a concentration equivalent to the standard 4 HA units.

                  If the HI titre of the positive control serum is less than the standard titre, the antigen is too concentrated. Prepare a new dilution and test again.

                  Conversely if the HI titre of the positive control serum is too high the antigen is too dilute. Prepare a new dilution and test again.

                  Preparation of 4HA units of antigen for HI test in 10 microwell plates:

                  The HA titre of the antigen was tested according to the protocol described above. The HA titre = 128

                  Calculation of dilution factor to prepare 4 HA units: 128/4 = 32

                  Calculation of volume of 4HA unit dilution of antigen required:

                  Allow 2.5 mL per plate total volume required = 10 × 2.5 mL = 25 mL

                  Apply dilution factor = 25 mL/32 = 0.781 mL = 781 µL

                  Preparation of the diluted antigen: mix 781 µL of original virus suspension with 24.219 mL of diluent. PBS is a suitable diluent.

                  Note: In this case it would be easiest to prepare 32 mL of the 4HA antigen. This would use 1 mL of the original suspension diluted in 31 mL of PBS.

                  • Thawed serum samples in racks
                  • V-bottom microwell plates and covers
                  • PBS
                  • 1 percent washed red blood cells
                  • V-bottom reagent trough
                  • 25 µL single and multichannel pipettes and tips
                  • Microwell plate recording sheet.
                  • Newcastle disease virus antigen diluted to 4 HA units per 25 µL
                  • Standard positive and negative serum

                  1. Fill in recording sheets to record how samples will be dispensed into microwell plates.

                  2. Calculate the number of plates required and number each plate.

                  3. Dispense 25 µL of PBS into each well of the plates.

                  4. Shake each serum sample and dispense 25 µL into the first well and the last (control) well of a row of a microwell plate.

                  5. Use a multichannel pipette to make two-fold serial dilutions along the row until the second last well from the end. The last well is the serum control. Do not dilute this well. See Appendix 4 for instructions on carrying out two-fold serial dilutions.

                  6. Add 25 µL of the 4HA dilution of antigen to each well excluding the control wells in the last column. See Section 10 for preparation of 4HA units of antigen

                  7. Gently tap the sides of the microwell plates to mix the reagents. Cover plates with a lid. Allow to stand for 30 minutes at room temperature.

                  8. Add 25 µL of 1 percent washed red blood cells to each well including the control wells in the last column.

                  9. Gently tap the sides of the microwell plates to mix the reagents. Cover the plates with a lid. Allow to stand at room temperature for 45 minutes.

                  10. Read the settling patterns for each serum sample. Read the control serum well first then read the patterns in the other wells.

                  11. Record the pattern observed in each well on a microwell plate recording sheet. Determine the endpoint. This is the point where there is complete inhibition of haemagglutination.

                  12. Record the antibody level for each serum sample. This is expressed as a log base 2. For convenience, the titre is often recorded as just the log index. For example a titre of 2 6 would be recorded as 6.

                  Interpretation of results

                  In the wells where antibodies are present there will be haemagglutination inhibition. The red blood cells will settle as a button.

                  In the wells where antibodies are absent, the red blood cells will agglutinate.

                  The end point of the titration is the well that shows complete haemagglutination inhibition. Sometimes it is not easy to determine. Look at the size of the button as an indication of the degree of haemagglutination inhibition. Use the control well as a point of comparison. Be consistent in determining the endpoint.

                  The neuraminidase enzyme present in the virus particle will eventually break the bond between the virus and red blood cells. This process is called elution.

                  When elution occurs, the red blood cells are no longer agglutinated. They roll down the side of V-bottom microwell plates to resemble the negative settling pattern, a tight button.

                  Some Newcastle disease virus strains elute more rapidly and the test must be read before this occurs.

                  Usually elution takes longer than 45 minutes. A control well with virus and red blood cells is useful to determine elution time.

                  Some sera may contain substances other than antibodies that inhibit viral haemagglutinin. These substances are described as non-specific inhibitors and are rarely observed with chicken serum and Newcastle disease.

                  Some chicken sera contain substances that will agglutinate chicken red blood cells. The settling pattern of the agglutinated cells is similar to that produced by Newcastle disease virus. These natural agglutinins are present in low concentration. The control serum well will indicate the presence of natural agglutinins.

                  Adsorption of natural agglutinins

                  If natural agglutinins are present in a serum sample, they can be removed by adsorption with chicken red blood cells. The serum can then be retested in the HI test.

                  • Suspension of 10 percent washed chicken red blood cells.
                  • Microfuge (Eppendorf) tube
                  • Micropipette, 200 mL to 1 000 mL and tips
                  • Discard tray
                  • Microfuge centrifuge
                  • Serum samples

                  1. Place 200 mL of the 10 percent washed red blood cells into a microfuge tube.

                  2. Centrifuge for 15 seconds.

                  3. Remove the supernatant.

                  4. Shake the serum sample and remove 500 mL of serum. Some samples may contain less volume. Use a new tip for each sample.

                  5. Add the serum to the red blood cells in the microfuge tube. Mix gently.

                  6. Stand for not less than 30 minutes at 4°C.

                  7. Centrifuge for 15 seconds.

                  8. Remove the serum (supernatant) immediately and transfer to a clean microfuge tube.

                  9. Store the adsorbed sera at 4°C overnight or at -20°C for longer periods.


                  Agglutination of Bacteria and Viruses

                  The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefield working with her colleagues A.R. Dochez and Oswald Avery. [1] She used antibodies to identify M protein, a virulence factor on streptococci that is necessary for the bacteria’s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.

                  Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.

                  Figure 2. Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology)

                  The method developed by Lancefield is a direct agglutination assay, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay (or latex fixation assay), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure 2). Indirect assays can be used to detect the presence of either antibodies or specific antigens.

                  To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly this indicates the presence of the antibody (Figure 3). This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient’s own IgG. RF will agglutinate IgG-coated latex beads.

                  In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure 3).

                  Figure 3. (a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. (b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies.

                  Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies.

                  In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.

                  Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient’s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient’s immune system is responding to the pathogen.

                  Watch this video that demonstrates agglutination reactions with latex beads.

                  Think about It

                  • How is agglutination used to distinguish serovars from each other?
                  • In a latex bead assay to test for antibodies in a patient’s serum, with what are the beads coated?
                  • What has happened when a patient has undergone seroconversion?

                  Hemagglutination

                  Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.

                  The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by the mother’s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).

                  The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure 4). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.

                  There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (Figure 4). IAT can be used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.

                  Figure 4. Click for a larger image. The steps in direct and indirect Coombs’ tests are shown in the illustration.

                  Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA.

                  Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure 5).

                  Figure 5. A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology)

                  A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination (Figure 6).

                  Figure 6. In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest concentration at which agglutination occurs is the titer of the antibodies in the patient’s serum. In the case of this test, Sample A shows a titer of 128, and Sample C shows a titer of 64. (credit: modification of work by Evan Burkala)

                  Think about It

                  • What is the mechanism by which viruses are detected in a hemagglutination assay?
                  • Which hemagglutination result tells us the titer of virus in a sample?

                  Animals in the Laboratory

                  Much of what we know today about the human immune system has been learned through research conducted using animals—primarily, mammals—as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.

                  Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.

                  We also use animals for the study of disease. The only way to grow Treponema pallidum for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch’s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.

                  While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one—indeed, one of Charles Darwin’s daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document “Guide for the Care and Use of Laboratory Animals” makes it clear that the use of animals in research is a privilege granted by society to researchers.

                  The NIH guidelines are based on the principle of the three R’s: replace, refine, and reduce. Researchers should strive to replace animal models with nonliving models, replace vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should refine husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that reduce the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.

                  At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.


                  Results

                  Optimization of the NA-MNT

                  In this study, the NA-MNT was developed using 4-MU-NANA as a substrate. Specifically, we performed assays and calculated the standard deviations (SDs) using cell lysate or supernatant samples containing virus. To this end, cells were first infected with virus at a multiplicity of infection of 0.001 and incubated for up to 36 h. As shown in Fig 1A, NA was detected 6 h after incubation, with significantly higher activity consistently detected in the cell lysates compared with the supernatants at all-time points evaluated (P = 0.031). In addition, the variability (SD) was significantly lower in cell lysates than in supernatants (P = 0.031).

                  (A) NA activity in cell lysates and supernatants at different time points following infection. Cells were infected with NIBRG-14 (H5N1) virus at a multiplicity of infection of 0.001. Each data point represents the mean of three separate experiments. (B) NA activity in cell lysates and supernatants with different viral inocula. MDCK cells were infected with different dilutions of NIBRG-14, with NA activity measured in either the cell culture supernatant or cell lysates 20 h after inoculation. The total NA activity in the supernatants was calculated and compared with the activity in the cell lysates. (C) The neutralizing antibody titers of serum samples measured by the NA-MNT. The neutralizing antibody titer of NIBSC reference sheep antisera (shRef H5N1), high (huHiPos) and moderate (HuMoPos) seropositive samples, and negative (huNeg) human sera were determined by NA-MNT using either cell supernatants or lysates. Each test was repeated five times, with the CVs calculated accordingly. Note: HuHiPos, HuMoPos, and huNeg samples were obtained from a clinical study of the H5N1 candidate vaccine.

                  To confirm these observations, a wider range of viral inocula were used, with NA activity being measured 20 h post-infection. As shown in Fig 1B, NA activity was consistently higher in the lysates, with lower SDs, than in the supernatants, irrespective of the amount of virus used for infection. Next, NA-MNT was performed to compare NA activities between cell lysates and supernatants by testing sheep or human antisera against H5N1 vaccines. As shown in Fig 1C, The antibody titers based on the measurements of the NA activities in the cell lysates were slightly higher than those in the supernatants (three-fold higher), but no statistically significant differences were found when logarithmic titers were compared (P>0.05 Wilcoxon signed rank test). However, the variability observed in the supernatants was larger than that in the cell lysates, as revealed by a higher coefficient of variation (CV) for the antibody titers specifically, the CV for the supernatants and lysates were 11.3–17.7% (mean, 13.7%) and 0–5.9% (mean, 3.4%), respectively.

                  Reproducibility of the NA-MNT in comparison with the ELISA-MNT

                  To determine the reproducibility of NA-MNT, three individual samples were measured by NA-MNT in comparison with ELISA-MNT. Intra-assay precision was determined by conducting eight measurements on the same day while the inter-assay precision was determined by carrying out nine measurements from each sample over 3 days. The GMT values obtained from the two assays were found to be similar. As presented in Table 1, the mean intra-assay CV% in the NA-MNT was 0, which was lower than that in the ELISA-MNT. The inter-assay CV% range was 2.52–6.40 for NA-MNT and 4.92–11.06 for ELISA-MNT (Table 2). Although the titers of the tested samples were equal between the two assays, there was always less variability with the NA-MNT, revealing this assay has greater reproducibility.

                  Correlations among the HI assay, ELISA-MNT, and NA-MNT for detection of anti-H5N1 antibodies

                  The correlations in the results obtained from the HI assay, ELISA-MNT, and NA-MNT were analyzed using serum samples collected from 40 healthy volunteers who received two doses of inactivated H5N1 influenza vaccine. All pre-immune sera tested negative for anti-H5N1 antibodies and the negative samples were assigned a value of 5 for subsequent calculations. For post-vaccination sera, lowest GMT values with the narrowest titer range were obtained using HI. Similar same range of titers was obtained with NA-MNT and ELISA-MNT, with the mean ratio of the logarithmic titers being 1.67 (Table 3). Seroconversion, defined as an antibody titer ≥1:40 after vaccination, was 75% by NA-MNT, slightly (but not significantly) lower than that of the ELISA-MNT (P = 0.152). In addition, both the ELISA-MNT and NA-MNT exhibited good correlations with the HI assay, with Spearman’s correlation coefficients being 0.781 (Fig 2A) and 0.689 (P<0.001), respectively (Fig 2B). However, a better correlation was found between ELISA-MNT and NA-MNT, with a Spearman’s correlation coefficient of 0.814 (P<0.001) (Fig 2C).

                  Antibodies were measured in 40 serum samples from healthy volunteers immunized with two doses of inactivated H5N1 influenza vaccine and compared between the (A) HI assay and ELISA-MNT (B) NA-MNT and HI assay, and (C) NA-MNT and ELISA-MNT. Linear regression equations and correlation coefficients were calculated by linear regression analysis of the log transformed data.

                  Correlations among the HI assay, ELISA-MNT, and NA-MNT for the detection of anti-H7N9 antibodies

                  We next investigated the correlations between the three assays for detection of anti-H7N9-specific antibodies. Serum samples from 92 human subjects received candidate H7N9 influenza vaccines were analyzed using the three assays. As observed with the H5N1 serum samples, all pre-immune sera were tested negative for H7N9 antibodies using all three assays. The antibody titers of serum samples harvested 21 days post-immunization were found to be slightly lower by NA-MNT than that by ELISA-MNT whereas significantly higher titers were found by NA-MNT than HI assay. As shown in Fig 3A, the seroconversion rates in the NA-MNT and ELISA-MNT were 100%, which was higher than that in the HI assay (87%).

                  92 serum samples from human subjects immunized with either (A) one dose or (B) two doses of candidate H7N9 influenza vaccine were analyzed using either HI assay, NA-MNT, or ELISA-MNT. The correlations among the titers in the 92 serum samples after immunization with one dose of candidate H7N9 influenza vaccines were measured by (C) HI assay versus NA-MNT or (D) ELISA-MNT versus NA-MNT. The correlations among the titers in the 92 serum samples after immunization with two doses of candidate H7N9 influenza vaccines were measured by (E) HI assay versus NA-MNT or (F) ELISA-MNT versus NA-MNT. Linear regression equations and correlation coefficients were calculated by linear regression analysis of the log transformed data.

                  For serum samples harvested 42 days after immunization, higher GMT values were obtained by both ELISA-MNT and NA-MNT compared to that by the HI assay (Fig 3B). Linear regression analysis was then performed to assess the correlation among the assays. There was good correlation between the NA-MNT and HI assay for sera obtained on day 21 (r = 0.837, P<0.01) (Fig 3C), similar to that for sera from subjects who received two vaccinations (r = 0.887, P<0.01) (Fig 3E). Moreover, there was a good correlation between the NA-MNT and ELISA-MNT in the measurement of anti-H7N9 antibodies from subjects who received either one or two doses of the vaccine. As shown in Fig 3D and 3F, the Spearman’s correlation coefficient was 0.875 or 0.866 for individuals receiving one or two immunizations, respectively. Taken together, these data indicate strong correlations among the results obtained from the three assays.


                  Results

                  Inhibition of influenza a virus multiplication in vitro by patchouli alcohol

                  The cytotoxicity of Patchouli alcohol (PA) was firstly evaluated by MTT assay in MDCK, A549 and 293FT cells [19]. The results showed that PA exhibited no significant cytotoxicity at the concentrations from 6.25 to 100 μg/ml (Fig. 1a). The CC50 (50% Cytotoxicity Concentration) values for PA in MDCK, 293FT and A549 cells were about 550.8, 914.8, and 454.5 μg/ml, respectively. These results were used to determine the dose range of PA for the subsequent experiments.

                  Patchouli alcohol inhibited replication of IAV in vitro with low toxicity. a After 24 h exposure to Patchouli alcohol (100, 50, 25, 12.5, 6.25 μg/ml) in MDCK, A549 and 293FT cells, the cell viability of Vero cells was measured by MTT assay. Values are means ± S.D. (n = 3). b IAV (MOI = 1.0) infected MDCK cells were treated with PA at the indicated concentrations for 24 h, then the antiviral activity was determined by plaque assay. Values are means±S.D. (n = 3). c Infectious virus titers from single-cycle high-moi assays performed on MDCK cells infected with PR8, Vir09 and NWS and treated with the indicated concentrations of PA. Mean percentage infectious virus titers were calculated as a percentage of infectious virus titers from untreated cells for each drug treatment condition in an experiment. Values are means ± S.D. (n = 3). d Approximately 50–100 PFU/well of Vir09 virus was pre-incubated with different concentrations of PA for 60 min at 37 °C before infection. Then the virus-PA mixture was transferred to confluent cell monolayers in 6-well plates, incubated at 37 °C for 1 h and subjected to plaque assay. e Plaque number from plaque reduction assays performed on MDCK cells infected with Vir09 and treated with the indicated concentrations of PA. Values are means ± S.D. (n = 4). f Plaque number from plaque reduction assays performed on MDCK cells infected with PR8, Vir09 and NWS and treated with the indicated concentrations of PA. Mean percentage plaque numbers were calculated as a percentage of plaque numbers from untreated cells for each drug treatment condition in an experiment. Values are means ± S.D. (n = 3)

                  PA was then assayed for its ability to inhibit IAV multiplication in vitro using plaque assay [20]. Firstly, the inhibition of PA on the virus yields from MDCK cells infected with Vir09 (A/Virginia/ATCC1/2009), NWS (A/NWS/33) or PR8 (A/Puerto Rico/8/34) at high moi (≈1.0 PFU/cell) were examined by plaque assay. As shown in Fig. 1b and c, PA treatment reduced the virus titers of Vir09, NWS, and PR8 in a dose-dependent manner when used at the concentrations of 6.25–50 μg/mL. The 50% inhibitory concentration (IC50 value) of PA for Vir09, NWS, and PR8 was about 6.3 ± 1.3, 3.5 ± 1.4, and 6.1 ± 1.7 μg/mL, respectively (Table 1). At the concentration of 12.5 μg/ml, the virus titers reduced about 30 fold of that in the untreated control group for Vir09, 3.0 fold of that for NWS, and 2.5 fold of that for PR8 virus (Fig. 1b and c).

                  To further explore whether PA had direct inhibition actions on viral particles, the plaque reduction assay was performed as previously described [21]. In brief, Vir09 virus (50–100 PFU/well) was pre-incubated with or without PA for 60 min at 37 °C before infection. Ten the virus-PA mixture was transferred to confluent cell monolayers in 6-well plates incubated at 37 °C for 1 h and subjected to plaque assay. As shown in Fig. 1d and e, pre-incubation of PR8 with PA at the concentrations of 12.5 and 25 μg/ml markedly reduced the number of plaques and protected MDCK cells, suggesting that PA may be able to inactivate viral particles directly.

                  Furthermore, the inhibition effects of PA on IAV infection was also examined over multiple cycles of infection using plaque reduction assay [17]. Briefly, MDCK cells were infected with PA pretreated virus (Vir09, NWS, and PR8) at an moi of 0.001 pfu for 1 h at 37 °C, and then subjected to plaque assay. As shown in Fig. 1f, PA also significantly inhibited the plaque formation in Vir09, NWS and PR8 (MOI = 0.001) infected cells when used at the concentration > 3.125 μg/ml (Fig. 1f). The IC50 values of PA for Vir09, NWS, and PR8 was about 2.2 ± 0.2, 3.2 ± 0.2, and 2.9 ± 0.4 μg/ml, respectively (Table 1). However, ribavirin could not significantly inhibit the plaque formation of Vir09 with IC50 value > 120 μg/ml (Table 1). Thus, PA possessed anti-IAV effects in vitro, and the pandemic H1N1 virus (Vir09) was most susceptible to PA treatment.

                  Influence of different treatment conditions of PA on IAV infection

                  Various time-points were assessed to determine the stage(s) at which PA exerted its inhibitory effects in vitro. Briefly, MDCK cells were infected with Vir09 virus (H1N1) (MOI = 1.0) under four different treatment conditions: pre-treatment of viruses, pre-treatment of cells, during adsorption, or after adsorption. At 24 h p.i., the antiviral activity was determined by plaque assay. As shown in Fig. 2a, pretreatment of Vir09 virus with 25 μg/ml PA for 1 h before infection markedly reduced virus titers, suggesting that PA may have direct interaction with IAV particles. However, either the addition of PA during adsorption or pretreatment of cells only weakly inhibited virus multiplication (Fig. 2a), suggesting that PA may not interact with MDCK cells directly. Interestingly, treatment of PA after adsorption also significantly reduced virus titers as compared to the non-treated virus control group (Fig. 2a). Thus, PA may be able to inactivate virus particles directly and block some stages after virus adsorption.

                  Influence of different treatment conditions of Patchouli alcohol on IAV infection. a MDCK cells were infected with H1N1 (Vir09, MOI =1.0) by four different treatment conditions. i) Pretreatment of virus: IAV was pretreated with 25 μg/ml of PA at 37 °C for 1 h before infection. ii) Pretreatment of cells: MDCK cells were pretreated with 25 μg/ml of PA at 37 °C for 1 h before infection. iii) Adsorption: MDCK cells were infected in media containing 25 μg/ml of PA and, after 1 h adsorption at 37 °C, were overlaid with compound-free media. iv) After adsorption: after removal of unabsorbed virus the infecting media containing 25 μg/ml of PA were added to cells. At 24 h p.i., the antiviral activity was determined by plaque assay. Values are means ± S.D. (n = 3). *P < 0.05, **P < 0.01 vs. virus control group. b PR8 (MOI = 1.0) infected MDCK cells were treated with or without 25 μg/ml of PA for the specified time period, and then the media were removed and cells were overlaid with compound-free media. Then at 24 h p.i., the cell supernatants were collected and the virus yields were determined by plaque assay. Values are means ± S.D. (n = 3). Significance: *p < 0.05 vs. virus control group. c Inactivated H1N1 (Vir09) virus and H1N1(PR8) was incubated with indicated concentrations of PA or Zanamivir (10 μg/ml), and the NA enzymatic activity was determined by a fluorescent assay. Values are means ± S.D. (n = 3). d The inhibition effects of PA and anti-HA antibody on influenza virus H1N1 (PR8) induced aggregation of chicken erythrocytes were evaluated by hemagglutination inhibition (HI) assay. e 293FT cells were transfected with pcDNA3.1 expression plasmids encoding PB2, PB1, PA, NP, and the vNS1-luc plasmid in the absence or presence of PA (3.125, 6.25, 12.5, 25 and 50 μg/ml) or Nucleozin (10 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. Significance: **p < 0.01 vs. mock control group

                  Moreover, another time course study was also performed to explore which viral stage after adsorption is inhibited by PA as described previously [17]. Briefly, Vir09 (MOI = 1.0)-infected MDCK cells were treated with 25 μg/mL of PA for different time intervals, then the virus multiplication at 24 h p.i. was evaluated by plaque assay. The results showed that PA treatment for the first 4 h (0–4 h p.i.) after adsorption resulted in a significant reduction in the virus titer (about 200-fold) (P < 0.05) (Fig. 2b). Much less inhibition was noted (less than 20-fold) when PA was added 8 h after infection (> 8 h p.i.), which suggested that PA may be able to inhibit early stages of IAV life cycle (mainly 0–4 h p.i.) (Fig. 2b).

                  Since PA may be able to inactivate virus particles directly, we then explored whether PA had direct interaction with virus surface NA and HA protein by using the neuraminidase inhibition assay and hemagglutination inhibition (HI) assay. As shown in Fig. 2c, PA could not significantly inhibit the NA activities of Vir09 virus at the concentrations of 12.5–100 μg/ml, while Zanamivir possessed high inhibition percentage (> 80%) at 10 μg/ml, suggesting that PA may have no direct interaction with virus NA protein. Moreover, the results of the HI assay showed that the anti-HA antibodies significantly inhibited the PR8 virus-induced aggregation of chicken erythrocytes at the concentrations of 0.3125–5 μg/mL (Fig. 2d), suggesting that the anti-HA antibody can block the virus attachment to red blood cells through binding to HA. However, PA did not obviously inhibit virus-induced aggregation of chicken erythrocytes even at a concentration of 50 μg/ml (Fig. 2d), suggesting that PA may have no direct interaction with viral HA protein.

                  Furthermore, we also performed mini-genome assay to evaluate the influence of PA on viral genome replication, which occur during the early stages in viral life cycle. Briefly, 293FT cells were transfected with four expression plasmids encoding PR8 virus PB2, PB1, PA, and NP proteins, and the luciferase-containing plasmid vNS1-luc/pHH21, which encodes a viral-like genome in the absence or presence of PA (3.125, 6.25, 12.5, 25 and 50 μg/ml). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h p.i. The results showed that the positive drug nucleozin caused a notable reduction in luciferase activity at 10 μM as compared with control (DMSO) treatment (Fig. 2e). By contrast, treatment with PA (3.125, 6.25, 12.5, 25 and 50 μg/ml) did not significantly inhibit luciferase activity, suggesting that NP protein may be not the direct target of PA. In summary, virus HA, NA and NP proteins may not be the main targets of PA in vitro.

                  The influence of PA on virus mRNA and protein expression

                  Since PA may inhibit some steps after virus adsorption (Fig. 2a and b), the effects of PA on viral protein synthesis and RNA replication were evaluated by using immunofluorescence assay and Real-time RT-PCR assay as described previously [19]. Firstly, Vir09 virus (MOI = 1.0) infected A549 cells were added with 10 or 20 μg/mL of PA after virus adsorption and then incubated at 37 °C for 2 h. After that, viral NP protein expression was detected by immunofluorescence assay. As shown in Fig. 3a, in virus-infected cells without drug treatment, the fluorescence of viral NP proteins could be obviously found in both the cell nucleus and cytoplasm (Fig. 3a), while nearly non fluorescence could be found in the non-infected cells (Fig. 3a). However, after treatment with PA for 2 h, the number of virus antigen-expressing cells was drastically reduced, and only very few fluorescence could be found in the cytoplasm (Fig. 3a). Quantitation of data of the fluorescence intensity in IAV infected cells showed that PA treatment (5, 10 μg/mL) significantly reduced the fluorescence intensity of NP in A549 cells, suggesting that PA may block some steps of IAV life cycle after adsorption to interfering with nuclear import and expression of NP protein (Fig. 3b).

                  The influence of Patchouli alcohol on virus protein and mRNA expression. a Immunofluorescence assay of virus NP protein in H1N1 (Vir09) infected A549 cells at 2 h p.i. Scale bar represents 50 μm. b The average fluorescence intensity of NP proteins in (a) was measured by ImageJ (NIH) version 1.33u (USA) to calculate the average intensity per unit area of cells of different images (n = 30). Significance: ∗ P < 0.05, ∗ ∗ P < 0.01 vs virus control group. c Vir09 (MOI = 1.0) infected MDCK cells were treated with different concentrations of PA (10–40 μg/ml), and incubated at 37 °C for 8 h. After that, total RNA was extracted for real-time RT-PCR assay of IAV HA mRNA and cellular β-actin mRNA. The relative amounts of virus HA mRNA were determined using the comparative ( 2-ΔΔCT ) method. RNA levels for non-drug treated cells (virus control) were assigned values of 1. Values are means ± SD (n = 3). Significance: *P < 0.05 vs. virus control group. d MDCK cells were firstly infected with IAV (MOI = 1.0), and then treated with or without PA at indicated concentrations after adsorption. At 8 h p.i., the virus NP protein expression was evaluated by Western blotting. Blots were also probed for β-actin protein as loading controls. e Quantification of immunoblot for the ratio of IAV NP protein to actin. The ratio for non-treated virus control group (PR8) were assigned values of 1 and the data presented as mean ± SD (n = 3). Significance: **P < 0.01 vs. virus control group (PR8)

                  Moreover, the inhibition effect of PA on virus mRNA expression was then evaluated by Real-time RT-PCR assay. IAV (MOI = 1.0) infected cells were added with PA (10, 20, 40 μg/mL) after virus adsorption and then incubated at 37 °C for 8 h. After that, total RNA was extracted for real-time RT-PCR. As shown in Fig. 3c, after treatment with PA (20, 40 μg/mL) for 8 h, the IAV NP mRNA levels decreased to about 36.6 and 14.2% of that of untreated cells after PA treatment, respectively, consistent to the results of immunofluorescence assay.

                  Furthermore, Western blot assay was also performed to verify the inhibition of PA on viral protein production. MDCK cells were firstly infected with IAV (MOI = 1.0), and then treated with or without PA at indicated concentrations after adsorption. After incubation for 8 h, viral NP protein production was detected by western blot assay. As shown in Fig. 3d and e, the level of viral NP protein was significantly reduced by PA in a dose-dependent manner as compared to that of the non-treated virus control group (PR8) (P < 0.01). The treatment with PA at 40 μg/mL reduced the production of IAV NP protein by more than 80% (Fig. 3e). Therefore, PA may also be able to inhibit IAV protein and mRNA expression through interfering with some early steps of virus life cycle.

                  The cellular PI3K/Akt and ERK/MAPK signaling pathways may be involved in the anti-IAV actions of PA

                  Since PA may inhibit some steps after virus adsorption to reduce IAV mRNA and protein expression in vitro, so we further explored if PA could influence some cellular signaling pathways required for IAV infection. The cellular PI3K/Akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of PI3K/Akt signaling could inhibit both entry and replication of virus [21, 22]. In this study, after IAV infection for 2 h, the levels of phosphorylated PI3K proteins were significantly increased to about 1.3 fold higher than normal control group in IAV infected cells (P < 0.01) (Fig. 4a and e). However, after treatment with PA (6.25, 12.5, 25, 50 μg/ml) for 2 h, the expression level of phosphorylated PI3K significantly decreased from about 1.3 to about 1.1, 0.9, 0.6, and 0.3-fold of normal control group, respectively (P < 0.05) (Fig. 4a and e). Moreover, the activation of PI3K can induce the activation of some downstream signals such as Akt, and the level of phosphorylated Akt was truly significantly increased in virus-control group to about 4.5 fold higher than normal control group at 2 h p.i. (P < 0.01) (Fig. 4b and f). But treatment with PA (25, 50 μg/ml) for 2 h could significantly reduce the activation of Akt from about 4.5 to about 3.4 and 2.8-fold of normal control group, respectively (Fig. 4b and f). Thus, the PI3K/Akt signaling pathway may be involved in the anti-IAV mechanisms of PA in vitro.

                  Involvement of PI3K/Akt and ERK/MAPK signaling pathways in the anti-IAV actions of Patchouli alcohol. a-d Vir09 virus (MOI = 1.0) infected cells were treated with or without PA (6.25, 12.5, 25, 50 μg/ml) for 2 h, and then the phosphorylation of PI3K (a), Akt (b), ERK1/2 (c), and NF-κB (d) was evaluated by western blot. Blots were also probed for GAPDH, and α-tubulin proteins as loading controls. The result shown is a representative of three separate experiments. e-h Quantification of immunoblot for the ratio of p-PI3K (e), p-Akt (f), p-ERK1/2 (g), and p-NF-κB (h) protein to GAPDH or tubulin, respectively. The ratio for non-infected cells (M) was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). Significance: ## P < 0.01 vs. normal control group (Mock) *P < 0.05, **P < 0.01 vs. virus control group (Vir09)

                  Furthermore, the MAPK signaling pathway was reported to be required for efficient vRNP export from nucleus, and the inhibitors of MAPK pathway could reduce IAV replication and inflammatory symptoms [23,24,25]. In this study, ERK1/2 protein was significantly activated in virus-control group to approximately 9.2 fold higher than normal control group at 2 h p.i. (P < 0.01) (Fig. 4c and g). However, after treatment with PA (6.25, 12.5, 25 and 50 μg/ml) for 2 h, the expression level of phosphorylated ERK1/2 protein significantly decreased from about 9.2 to about 6.0, 6.3, 5.9, and 5.3-fold of normal control group, respectively (P < 0.01) (Fig. 4c and g). However, treatment with PA (6.25, 12.5, 25 and 50 μg/ml) for 2 h could not significantly reduce the expression level of phosphorylated NF-κB protein as compared to the virus control group (Fig. 4d and h). Thus, PA may inhibit ERK/MAPK rather than NF-κB pathway to interfere with IAV replication.

                  Moreover, the PI3K/Akt pathway was reported to be associated with host antiviral response [26, 27], so we further explored the influence of PA on immune response by using western blot and ELISA assay. We first evaluate the direct actions of PA on cellular PI3K/Akt pathway in non-infected A549 cells using western blotting. The results showed that PA treatment (6.25, 12.5, 25, 50 μg/ml) could not significantly influence the activation of PI3K and Akt proteins in the non-infected A549 cells (Fig. 5a and b), suggesting that the inhibition of PI3K/Akt pathway by PA may be related to its inhibition of IAV infection. Treatment of PA for different time intervals within 24 h showed no significant cytotoxicity to non-infected A549 cells (Fig. 5c). In addition, IAV infection significantly increased the production of cellular interferon-β (IFN- β) in Vir09 virus infected A549 cells, however, PA treatment (6.25, 12.5, 25, 50 μg/ml) could not significantly influence the production of IFN-β as compared to the virus control group (Fig. 5d), suggesting that PA had no direct action on cellular antiviral response. Furthermore, we also evaluated the influence of PA on the production of interferon-γ (IFN-γ) and interleukin 2 (IL-2) in mice with or without PR8 virus infection. As shown in Fig. 5e, intranasal treatment of PA (20 or 40 μg/day) for four days had no significant influence on the production of IFN-γ and IL-2 in non-infected mice. However, PA treatment could significantly reverse the reduction of IFN-γ and IL-2 in IAV infected mice (Fig. 5f), suggesting that the enhancement of PA on type-II interferon system may be related to its inhibition of IAV inhibition in vivo. Thus, the inhibition of PI3K/Akt pathway by PA may be related to its inhibition of IAV infection rather than direct actions on host antiviral response.

                  The influence of Patchouli alcohol on host antiviral response. a A549 cells were treated with or without PA (6.25, 12.5, 25, 50 μg/ml) for 2 h, and then the phosphorylation of PI3K and Akt proteins was evaluated via western blotting. Blots were also probed for β-actin and GAPDH protein as loading controls. The result shown is a representative of three separate experiments. b Plots quantifying the immunoblots (as ratios to β-actin or GAPDH) for p-PI3K and p-Akt proteins, respectively. The ratios for non-treated cells (mock) were assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). c A549 cells were treated with Patchouli alcohol (50, 25 μg/ml) for specified time period, and then the media were removed and cells were overlaid with compound-free media. Then at 24 h p.i., the cell viability of A549 cells was measured by MTT assay. Values are means ± S.D. (n = 3). d Vir09 virus (MOI = 0.1) infected cells were treated with or without PA (6.25, 12.5, 25, 50 μg/ml) for 24 h, then the content of IFN-β in the culture supernatants was detected using ELISA kits. Values are means ± S.D. (n = 3). ##P < 0.01vs. non-infected group (mock control). e and f After treatment of PA (20 or 40 μg/day) for four days in non-infected mice (e) or PR8 virus infected mice (f), the production of interferon-γ (IFN-γ) and interleukin 2 (IL-2) in lung tissues was determined by using the ELISA kits for IFN-γ and IL-2. Values are means ± S.D. (n = 3). Significance: ##P < 0.01 vs. non-infected mock control group **P < 0.01 vs. virus control group

                  Intranasal PA application supports survival of mice infected with IAV

                  The anti-IAV effects of PA in vivo were further explored using a mouse pneumonia model [18]. In brief, IAV-infected mice received intranasal administration of PA (20 or 40 μg/day) or placebo (PBS) once daily for the entire experiment, and the selected subset of treated, infected mice were then sacrificed on Day 4 and the tissue samples were removed for further analysis. Subsequently, the pulmonary viral titers were determined by plaque assay [22]. As shown in Fig. 6a, after treatment of PA (20, 40 μg/day) for 4 days, the pulmonary viral titers significantly decreased compared to that of the virus control group (P < 0.01), suggesting that intranasal therapy with PA could inhibit IAV multiplication in mice lungs. Oral therapy of oseltamivir (10 mg/kg/day) also showed significant reduction of virus titers in mice lungs (P < 0.05) (Fig. 6a).

                  The anti-IAV effects of Patchouli alcohol in vivo. a Viral titers in lungs. After treatment with Oseltamivir (10 mg/kg/day) or PA (20 or 40 μg/day) for 4 days, the pulmonary viral titers were evaluated by plaque assay. Values are the mean ± SD (n = 3). Significance: *P < 0.05, **P < 0.01 vs virus control group. b Survival rate. IAV infected mice received therapy with Oseltamivir (10 mg/kg/day) or PA (20 or 40 μg/day) for the entire experiment. Results are expressed as percentage of survival, evaluated daily for 14 days. Significance: *P < 0.05 vs. virus control group (placebo). c Histopathologic analyses of lung tissues on Day 4 p.i. by HE staining (× 10). The representative micrographs from each group were shown (n = 5 mice/group). Mock: non-infected lungs Control: IAV infected lungs without drugs Oseltamivir: IAV infected lungs with Oseltamivir (10 mg/kg/day) treatment PA 20 μg/day: IAV infected lungs with PA (20 μg/day) treatment PA 40 μg/day: IAV infected lungs with PA (40 μg/day) treatment. The red arrows indicate the presence of inflammatory cells in the alveolar walls and serocellular exudates in the lumen

                  Moreover, the survival experiments were also performed to evaluate the effects of PA on the survival of IAV-infected mice. As shown in Fig. 6b, intranasal administration with PA (40 μg/day) significantly increased survival rates as compared to the placebo-treated control group (P < 0.05). By day 14 post infection, only 30% of the individuals in the placebo group survived whereas 100% of animals in the PA (40 μg/day)-treated group survived, superior to that in Oseltamivir (10 mg/kg/day)-treated group (90%). PA treatment at 20 μg/day also increased the survival rate of IAV infected mice (50%) but without significance (Fig. 6b).

                  To further evaluate the effects of PA on viral pneumonia in mice, histopathology analysis was also performed as described previously [18]. As shown in Fig. 6c, lung tissues in virus-control group showed marked infiltration of inflammatory cells in the alveolar walls and the presence of massive serocellular exudates in the lumen. However, after treatment with PA (20 or 40 μg/day) for 7 days, the lung tissues showed intact columnar epithelium in the bronchiole even in the presence of some serocellular exudates in the lumen (Fig. 6c). Mice treated with Oseltamivir (10 mg/kg/day) also had intact columnar epithelium (Fig. 6c). Thus, PA may be able to attenuate pneumonia symptoms in IAV infected mice.


                  Affiliations

                  Influenza Centre, Department of Clinical Science, University of Bergen, Bergen, Norway

                  Anders Madsen, Åsne Jul-Larsen, Mai-Chi Trieu & Rebecca J. Cox

                  Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, USA

                  Department Microbiology, Haukeland University Hospital, Bergen, Norway

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                  Contributions

                  R.J.C., M.C.T. and Å.J.-L. designed the study. A.M., Å.J.-L. and M.-C.T. conducted laboratory analysis. F.K. provided baculovirus constructs. A.M. and R.J.C. analysed the data and wrote the manuscript. All authors have read and approved the final version of the manuscript.

                  Corresponding authors


                  Watch the video: Reaction of hemagglutination for detection of influenza virus titer (July 2022).


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