Virus Hemagglutination - an overview (2023)

Hemagglutination-Inhibition Assay

Immune Response

Infection with a TBE complex virus stimulates antibodies against each of the recognized structural and nonstructural proteins. The predominant protective antibodies are those stimulated by the E and NS1 proteins. Antibodies against the E protein neutralize virus infectivity, inhibit virus hemagglutination and are generally crossreactive with TBE complex and other flaviviruses in nonfunctional tests such as ELISA or immunofluorescence microscopy. NS1-specific antibodies are less broadly crossreactive with non-TBE complex flaviviruses. Nonfatal infections by TBE complex viruses induce long-lasting immunity to reinfection with TBE complex-related viruses but there is insufficient information to predict the extent to which crossprotection occurs amongst more distantly related mosquito-transmitted flaviviruses. Conventionally, immune animals are not thought to be hosts for virus transmission by infected vectors; however, there is experimental evidence of TBE virus transmission by ticks cofeeding on immune rodents.

Diagnosis

Clinical signs, hematological changes, and postmortem findings are characteristic and sufficient for presumptive diagnosis of feline panleukopenia. The usual confirmatory tests include either antigen-capture enzyme immunoassay or immunofluorescence for the detection of antigen in tissues, or PCR assay for the detection of viral DNA in feces or tissues. Virus isolation or hemagglutination assays also can be used. Serologic diagnosis is by hemagglutination-inhibition assays, ELISA, or indirect immunofluorescence. Positive PCR results should be carefully interpreted in the absence of other confirmation, and quantitative PCR showing high levels of viral DNA can be useful to confirm an active infection. The viral DNA may persist at low levels in tissues for months (or perhaps years) in the absence of active viral replication. Truly persistent shedding of DNA in feces is not generally seen.

Diagnosis of Acute Infection by Demonstrating a Rise in Antibody

Following the traditional approach, “paired sera” are taken from the patient, the “acute-phase” serum sample as early as possible in the illness and the “convalescent-phase” sample at least 10 to 14 days later. Blood is collected in the absence of anticoagulants and given time to clot, and then the serum is separated. Acute and convalescent serum samples should be tested simultaneously. For certain tests that measure inhibition of some biological function of a virus, for example, virus neutralization or hemagglutination-inhibition, the sera must first be inactivated by heating at 56°C for 30 minutes and sometimes treated by additional methods, in order to destroy various types of non-specific inhibitors of infectivity or hemagglutination, respectively. Prior treatment of the serum is not generally required for assays that simply measure antigen–antibody binding, such as EIA, RIA, Western blot, latex agglutination, immunofluorescence, or immunodiffusion.

The paired sera are then titrated for antibodies using any of a wide range of available serological techniques. Demonstration of a significant rise in antibody titer (conventionally, a four-fold rise when titrating the test serum as two-fold dilutions) is taken as proof of “seroconversion,” that is, acute infection with the agent in question. In practice, there are many subtleties in the interpretation of results. For example, the timing of the collection of the paired sera in relation to the date of onset of illness must be carefully assessed to allow for inappropriate sample timing. Moreover, an individual with immunity due to prior infection or immunization may undergo a second or reinfection, often of a subclinical nature, but showing a rise in antibody titer. It is obvious that in reality, two well-timed paired sera to allow demonstration of a four-fold rise in titer, are often not available. Attempts are sometimes made to infer that a recent infection has occurred, on the basis of one serum sample containing a relatively high antibody titer; indeed laboratories may issue guidelines about what titer of antibody in a single serum may imply recent infection. In practice this is often unreliable, as the peak antibody titer seen in the convalescent stage of an infection can vary significantly from person to person.

a. Hemagglutination-Inhibition Test

The procedure adopted for microtitration (Sever, 1962) of the original protocol (Clarke and Casals, 1958) has been widely used for a variety of objectives, ranging from case diagnosis to serosurvey.

The principle of this test is based on the propensity of most arboviruses to aggregate erythrocytes of certain animals. If, however, virus is mixed with a serum specimen containing an antibody against the virus, hemagglutination is abrogated, the highest serum dilution causing the inhibition corresponding to the antibody concentration in the specimen. Because hemagglutination is pH dependent, selection of an optimal pH is critical.

The major advantages of the this test are (i) it does not require expensive equipment or instruments and (ii) it is highly useful to initially screen etiologic agents at the major group level because of its extensive and exclusive cross-reaction to all members of one virus group (antigenic complex, genus, or family) and excellent ability to segregate that group from others.

Although it has been sometimes erroneously believed to be an IgG assay, actually it measures other immunoglobulins, such as IgM and IgA, as well. Kaolin treatment of serum specimens for removal of nonspecific inhibitors still leaves a considerable amount of HI-reactive IgM (Granström et al., 1978; Wiemers and Stallman, 1975), although it was once thought to remove it (Mann et al., 1967).

For the visualization of agglutination, goose erythrocytes have been used in most laboratories. Other investigators have found trypsinized human type “O” blood cells or goose cells preserved with formalin treatment useful in laboratories where fresh goose blood cells are difficult to procure (Ahandrik et al., 1986). As for antigen, sucrose-acetone extracts of infected suckling mouse brains were popularly used in the past, but some of them have been replaced with antigens prepared from infected cell cultures. More recently, recombinant antigens, such as Japanese encephalitis (JE) viral antigen expressed as extracellular subviral particles, became available. However, although some recombinants had a hemagglutination activity (Heinz et al., 1995; Hunt et al., 2001; Konishi et al., 1996), other recombinant antigens either have not been evaluated for utility in the HI test or were found to be nonreactive (Davis et al., 2001; Konishi et al., 2001). Availability of a good HI-reactive recombinant antigen for the diagnoses of West Nile fever (WNF) and other viral infections in wildlife is important, because an HI test with such a safe antigen obviates development of antispecies antibodies necessary in the popular ELISA but currently unavailable commercially.

Although HI antibodies in neurotropic flaviviral infections, compared with those of non-neurotropic infections, are sometimes detectable within 3 to 5 days after the onset of illness because of longer intervals between infection and development of symptoms; generally, a disadvantage of this test is that for case diagnosis it is essentially a retrospective diagnostic test because both acute phase and convalescent phase specimens must be obtained to determine a significant change in antibody titer. Many recovered former patients do not feel a strong need to return to clinics for second blood samples; thus, unless convalescent phase specimens are actively sought by physicians or diagnostic laboratories, many cases with only acute phase specimens would remain inconclusive. Furthermore, it is one of the most cross-reactive tests to flavivirus. It should also be remembered that in the microHI test, which is the standard today, titers obtained are often lower compared with those by the macroHI test (Akov, 1976).

C Epizootiology

12.5.1 Plant and Other Lectins, and Virus Hemagglutinins

Lectins were first found almost exclusively in plants.^{398,1084–1086} Since they were detected by the agglutination of erythrocytes, they were named “hemagglutinins” or “phytohemagglutinins.” As plant lectins are able to distinguish between erythrocytes of different blood types, as early as 1954 Boyd and Shapleigh coined the name “lectin,” from the Latin legere, “to select.”¹⁰⁸⁷ This definition was later used for all carbohydrate-binding proteins, irrespective of their occurrence.

To go back in history, before 1860, S. Weir Mitchell in Pennsylvania had observed agglutination of pigeon blood by the venom of a rattlesnake.¹⁰⁸⁸ The first lectin, called “ricin,” was discovered in 1888 in the beans of the castor tree (Ricinus communis) by Hermann Stillmark at the University of Dorpat (now Tartu) in Estonia.¹⁰⁸⁹ In fact, it was a mixture of weakly agglutinating protein toxins, which were later found to bind to galactose and N-acetylgalactosamine.^1084,1090 Furthermore, Simon Flexner and Hideyo Noguchi,¹⁰⁹¹ from the Rockefeller Institute (New York, USA), described in 1902 snake venom agglutinins in some detail in the horseshoe crab (L. polyphemus)¹⁰⁷⁸ and the American lobster Homarus americanus. In the first decade of the 20th century also the first bacterial agglutinins were found, which were later identified as lectins. Investigations on blood type-specific lectins stem from the early 1950s, when it was demonstrated for the first time that lectins can bind monosaccharides.¹⁰⁹²

The already described studies of Hirst in the 1940s on the agglutination of human erythrocytes by influenza virus (see Sections 2.1 and 11.7) can be considered as the first example of the involvement of a lectin/hemagglutinin, a sialic acid-binding agent. Influenza virus hemagglutination was found to be inhibited by a brain lipid fraction, known to contain gangliosides.¹⁰⁹³ In the following decades the structure and pathophysiological significance of the viral hemagglutinin (designated “H” antigen) was most intensively studied, until today, by the group of Hans-Dieter Klenk at Philipps-Universität Marburg (Germany).^958,959,1094 Influenza viruses demonstrate pronounced receptor binding specificity. Human influenza viruses preferentially bind to Neu5Ac(α2→6)Gal sequences, whereas avian influenza viruses prefer Neu5Ac(α2→3)Gal as ligand (or receptor in the terminology of virologists).¹⁰⁹⁵ Competitive inhibitors of the binding of avian or human influenza viruses to sialylated glycans are soluble glycoconjugates carrying (α2→3)- or (α2→6)-linked sialic acids, respectively. The earlier discussed mucin from the nests of the Chinese swiftlet, genus Aerodramus collocalia (collocalia mucoid, edible bird's nest substance),¹⁰⁹⁶ rich in sialic acids, is a potent inhibitor of the agglutination of myxoviruses.¹⁰⁹⁷ This may have been the reason that it has been used in Chinese medicine for hundreds of years in order to prevent and cure influenza and its following bronchial diseases. The substance, which is a good substrate for sialidase, has additional health benefits and is therefore highly esteemed in Asia.¹⁰⁹⁸

Sialic acid-recognizing proteins are manifold in Nature. The first sialic acid-binding protein from vertebrates reported was Complement Factor H, an early response factor of the innate immune system.^1100,1101 In a thorough search about 230 sialic acid-specific lectins in viruses (being the largest group), bacteria, toxins, protozoa, fungi, plants, invertebrates, and vertebrates were described.¹¹⁰² In the intervening time, more sialic acid receptors have been detected, especially in the field of virus research, e.g., of the human Noro virus,¹¹⁰³ the Middle East Respiratory Syndrome Coronavirus (MERS-CoV),¹¹⁰⁴ and the Infectious Salmon Anemia Virus (ISAV), the latter binding to Neu4,5Ac₂.¹¹⁰⁵ Most of these sialic acid lectins bind to Neu5Ac, but a few only to Neu5Gc or to both sialic acid types. An example of Neu5Gc as virus receptor is the transmissible porcine gastroenteritis coronavirus.¹¹⁰⁶ The various lectins may discriminate between (α2→3) and (α2→6) linkages and rarely prefer (α2→8) linkages, like e.g., siglec-7 and -11. Some invertebrate and viral lectins bind to 4- or 9-O-acetylated sialic acids, the best known example being influenza C virus.^826,1102

Not much is known about how and where influenza C viruses enter the human organism, but the human respiratory tract is the most likely place. In the nasal secreted mucin of only one person studied so far, about 10% of the sialic acids were 9-O-acetylated. In a pool of nasal mucosa probes collected after surgery in 1994, a 9-O-acetylated sialoglycoprotein fraction of 100–130kDa was detected by Western Blot analysis. This could be the influenza C virus receptor. Since also the ganglioside fraction was found to be O-acetylated, the influenza C virus receptor must be studied further.¹¹⁰⁷ Influenza C virus hemagglutinins, specific for (α2→3) and (α2→6) linkages, can differentiate between receptor determinants bearing N-acetyl-, N-glycolyl-, and O-acetylated sialic acids. This was shown by the agglutination of Neu5Ac, Neu5Gc, or Neu5,9Ac₂ covering erythrocytes.¹¹⁰⁸

Very recently, probes from viral sialic acid-binding proteins were developed to detect 4-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl sialic acids on cells and tissues of humans and a great variety of animals.⁴⁰⁷ It is astonishing how widely distributed these esterified sialic acids are. The HE proteins were expressed in insect cells and fused to the Fc region of human IgG1 and to a hexahistidine sequence for histochemical or microarray use (see also Section 7.4). Binding of influenza A virus and Siglec-2 (CD22) is inhibited by Neu5Ac O-acetylation.⁸²⁶