Dengue Fever (DF登革热) is due to mild infection caused by one of the four serotypes of Dengue virus that may progress to a severe Dengue Hemorrhagic Fever (DHF) and subsequently to a fatal Dengue Shock Syndrome (DSS), if treatment not initiated in right time (WHO 1999). Dengue viruses belong to the family Flaviviridae, genus Flavivirus. Dengue virus (DENV) an enveloped RNA virus, is an arthropod-borne human viral pathogen that causes dengue fever epidemics in tropical and subtropical areas every year. DENV is categorized into four distinct antigenic serotypes (DENV1–4)(血清型1-4), which are further divided into different genotypes（基因型）. Within each DENV antigenic type, genetic and antigenic diversity exists which results in multiple distinct genotypes.
Primary infection with one DENV serotype has been shown to provide life-long immunity against a similar serotype and shortterm immunity against other serotypes. A longitudinal survey of DENV clinical strains over a 20-year time span from Bangkok, where all four DENV serotypes have co-circulated suggests the extinction of one clade and the replacement with another. These shifts have been shown to be linked to the epidemic cycle in the region.
Dengue epidemics in Philippines and in Thailand in the 50′s of the last century raised attention to the virus occurring in 4 serologic types. A successful cure of the infection caused by one of the serotypes does not mean immunity against the other serologic types. Dengue can thus affect one individual repeatedly and the reinfections are caused by different serotypes. Moreover, the course of the infection is predetermined by the sequence of the individual serotypes, which induce the re-infection in one individual. Both older children and adults are involved.
Serotype dynamics play a crucial role in the persistent burden of dengue worldwide. Immune response after infection with one serotype confers temporary cross-immunity against other serotypes of the same pathogen as evidenced from laboratory and cohort studies, but in the longer term when cross-immunity wanes, secondary infection may be possibly more severe.
Anti-envelope human antibodies are primarily directed against two glycoproteins on the surface of the viral envelope, the precursor membrane (pre-M) and envelope (E) proteins. Genetic diversity of pre-M and E proteins can significantly modulate antibody neutralization activity against DENV2 strains. Antigenic variation in the E protein has also been shown to impact the neutralization efficiency of monoclonal antibodies, antisera from DENV-infected patients, and immune sera from DENV-vaccinated individuals. Additionally, DENV serotypes contain antigenic heterogeneity. Mapping of E protein amino acid differences from different DENV antigenic serotypes revealed four distinct clusters whereby each serotype was clustered closely together on the antigenic map. However, cross-reactivity of antisera with different serotypes can lead to discrepancies in the DENV antigenic cluster and certain DENV strains have shown more antigenic variance to inter-typic viruses than intra-typic viruses.
Antigenic characterization of DENV would greatly aid in DENV vaccine development. In order to promptly identify DENV antigenic variants, an in-silico model for DENV has been devised based on possible antigenicity-dominant positions of the E protein providing a convenient way to calculate the difference in viral antigenicity.
Although the four dengue serotypes are antigenically distinct, there is evidence that serologic subcomplexes may exist within the group. For example, a close genetic relationship has been demonstrated between DENV-1 and DENV-3 and between DENV-2 and DENV-4. The sizes of the genomic open reading frames of DENV-1, DENV-2, DENV-3, and DENV-4 are 3392, 3391, 3390, and 3387 amino acids, respectively, the shortest among the mosquito-borne flaviviruses. An amino acid sequence positional homology of 63%–68% is observed among the DENV serotypes compared to 44%–51% between DENVs and other flaviviruses such as yellow fever and West Nile.
Serotypes within the dengue virus complex are most accurately and easily identified with an indirect immunofluorescent antibody (IFA) assay using serotype-specific monoclonal antibodies which react with epitopes on the structural proteins, or by a polymerase chain reaction (PCR) using serotype specific primers.
Both antigenic and biological variation among dengue viruses has been documented. As mentioned above, DENV-3 viruses isolated in the Caribbean and the South Pacific in the 1960s were found to be antigenically distinct from the prototype and Asian strains of DENV-3 using PRNT. They were also biologically unique in that they did not grow as well in baby mice and mosquitoes as did the Asian strains. DENV-4 viruses isolated in the Caribbean after the introduction of this serotype into that region in 1981 were antigenically distinct from DENV-4 viruses from Asia.
Factors responsible for the emergence and spread of the severe form of the disease, DHF/DSS, are not fully understood. The changing disease pattern described above provides support for the principal hypotheses regarding the pathogenesis of DHF/DSS, a secondary infection, and an increased virulence of the virus. Thus, increased transmission in urban areas and the development of hyperendemicity increases the probability of secondary infections and of genetic changes in the virus which may result in more severe disease due to antibody dependent enhancement (ADE) or to an epidemic virus strain with greater virulence. And increased spread of viruses between population centers via modern transportation increases the probability of introducing a virus strain with increased epidemic potential and virulence into new geographic areas.
Increased transmission of multiple dengue serotypes thus increases the probability that severe disease will occur, regardless of whether the underlying cause is due to increased virulence, ADE, or, more likely, a combination of both.
Globally, there is significant diversity among DENV strains. The four serotypes of DENV (DENV-1, DENV-2, DENV-3, and DENV-4) are genetically distinct but cause similar diseases and share epidemiologic features. All DENV strains are members of the Dengue antigenic complex; inclusion of a strain as DENV is based on antigen cross-reactivity, sequence homology, and genome organization. The four serotypes of DENV were historically distinguished by limited cross-neutralization or hemagglutination inhibition using serum from infected individuals. Subsequent sequencing analysis revealed that individual serotypes of DENV can differ from one another at the amino acid level significantly, with 30% to 40% variation in the viral envelope proteins. However, within a given serotype, amino acid homology is much greater, with conservation levels at approximately 90% or higher. Therefore, individual DENV serotypes (e.g., DENV-1 versus DENV-4) vary far more than distinct viruses in Japanese encephalitis serocomplex (e.g., WNVs and JEVs vary by 10% to 15% at the amino acid level), which has led some to consider DENV as a group of four different viruses that are linked by serology, epidemiology, and disease pathogenesis. Differences in severity associated with individual serotypes or particular sequences of serotypes in sequential infection have been observed, and it still is unclear whether some serotypes are inherently more pathogenic than others.
In some species, the Spike（S，刺突蛋白） protein is cleaved into two subunits, the N-terminal S1 fragment being slightly smaller than the C-terminal S2 sequence. The S protein is anchored in the envelope by a transmembrane region near the C-terminus of S2. The functional S protein is highly glycosylated and exists as a trimer. The bulbous outer part of the mature S protein is formed largely by S1 while the stalk is formed largely by S2, having a coiled-coil structure. S1 is the most variable part of the S protein; some serotypes of the avian coronavirus, infectious bronchitis virus (IBV) differ from one another by 40% of S1 amino acids. S1 is the major inducer of protective immune responses. Variation in the S1 protein enables one strain of virus to avoid immunity induced by another strain of the same species. Conventional diagnostic methods to differentiate IBV serotypes include virus isolation in specific pathogen–free (SPF) embryonated eggs followed by virus neutralization (VN) tests, hemagglutination inhibition (HI) tests, or antigen-capture enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. However, genetic-based tests to identify IBV types have become the test of choice since the discovery that sequences in the S1 gene are correlated with different serotypes of IBV. Reverse transcription polymerase chain reaction (RT-PCR), targeting the S1 portion of the spike protein, followed by sequencing of the RT-PCR product, restriction enzyme fragment length polymorphism (RFLP), or hybridization with IBV-specific probes have been developed for differentiating serotypes and variants of the virus.
Information on the complexity of CoV serotypes is crucial for predictions on whether antibodies against a previous CoV infection or a specific vaccine may protect from reinfection, which has important implications for vaccine design and neutralizing antibody therapy. Antigenic variability in the S protein, the major target of neutralizing antibodies, is extremely low between different MERS-CoV strains (Drosten et al., 2015). A recent serological study using infectious MERS-CoV isolates collected from patients in Saudi Arabia in 2014 showed no significant differences in serum neutralization, indicating that all these isolates belong to the same serotype (Muth et al., 2015). Based on these data, it seems likely that the S genes of all currently circulating MERS-CoVs are interchangeable in candidate vaccine formulations. The potential relevance of neutralizing antibodies directed against other envelope proteins remains to be studied. （by L. Enjuanes, S. Zuñiga, C. Castaño-Rodriguez, J. Gutierrez-Alvarez, J. Canton, I. Sola）
Amongst group 2 coronaviruses sequence variation is also greater in S1 than S2, a C-terminal region of S1 being hypervariable. Indeed, this region is deleted in variants of MHV. Avian infectious bronchitis virus (IBV), a chicken Gammacoronavirus, is a major poultry pathogen, and is probably endemic in all regions with intensive poultry production. Since IBV was fi rst described in 1936, many serotypes and variants of IBV have been isolated worldwide. Most IBV serotypes differ from each other by 20 to 25% of S1 amino acids. However, some serotypes differ by 50% of S1 amino acids. The differences between the S1 proteins undoubtedly have a selective advantage; generally speaking, the immunity induced by inoculation with one serotype protects poorly against infection with heterologous serotypes. Differences of as few as 2 to 3% of S1 amino acid residues can result in a change in serotype, defined as lack of cross-neutralization using convalescent sera. These few differences may contribute to diminished cross-protection in challenge experiments in chickens. Crossprotection between them often being poor. Consequently IBV vaccines have been developed with several serotypes (by Dave Cavanagh).
Recombination of coronaviruses appears to be a process of significant importance in the wild. Its occurrence has been shown to contribute to the natural evolution of IBV. This highly contagious virus comprises many different serotypes, and new ones emerge regularly, with the result that these viruses escape from host immunity and cause new outbreaks. Although many of the new variants arise by genetic drift as a result of subtle mutations in the spike protein (S) gene, similar to the changes that lead to antigenic drift in influenza viruses, new serotypes apparently also originate from genetic exchange of S gene sequences between different viruses through homologous RNA recombination (Kusters et al. 1990; Cavanagh et al. 1990; Wang et al. 1993; Jia et al. 1995).
Feline coronavirus (FCoV) is composed of nucleocapsid (N) proteins, membrane (M) proteins, and spike (S) proteins. FCoV has been classified into serotypes I and II according to the amino acid sequence of its S protein and antibody neutralization. Both serotypes consist of two biotypes: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). FECV infection is asymptomatic in cats, whereas FIPV infection causes lethal disease: FIP (by Tomomi Takano and Tsutomu Hohdatsu). Efforts to make effective vaccines against infectious peritonitis caused by FCoV have been ongoing for many years. A phenomenon that has militated against their widespread application has been that of antibody dependent enhancement (ADE) of disease. That is, antibodies induced by a first infection or vaccination may enhance the disease caused by a subsequent infection. Infection of cats by FCoV usually results in an infection confined largely to the digestive tract. In some cases the virus disseminates to other organs, leading to fatal infectious peritonitis. This dissemination is facilitated by macrophages. It is believed that uptake of FCoV by macrophages is enhanced when the virus has immunoglobulins, induced by a prior infection or vaccination, on its surface; the Fc moiety of the immunoglobulin attaches the virus-antibody complex to the surface of the macrophage (by Dave Cavanagh).
An isolate or group of isolates that are distinguished from biologically related isolates by reaction (or lack of reaction) with key serological reagents such as defined polyclonal antisera or monoclonal antibodies.
血清型的定义：A serotype is defined as either exhibiting no cross-reaction with others or showing a homologous/heterologous titer ratio of greater than 16 (in both directions).
Hyperendemicity：the co-circulation of multiple virus serotypes in the same population.
Viral Isolates, Strains, and Serotypes
The term virus isolate refers to any particular virus culture that is being studied and it is thus simply an instance of a given virus.
A viral strain is a biological variant of a virus that is recognizable because it possesses some unique phenotypic properties that remain stable under natural conditions. Characteristics that allow strains to be recognized include (1) biological properties such as a particular disease symptom or a particular host, (2) chemical or antigenic properties, and (3) the genome sequence when it is known to be correlated with a unique phenotypic character. If the only difference between a ‘wild type’ virus taken as reference and a particular variant is a small difference in genome sequence, such a variant or mutant is not given the status of a separate strain in the absence of a distinct phenotypic characteristic.
Strains that possess unique, stable antigenic properties are called serotypes. Serotypes necessarily also possess unique structural, chemical, and genome sequence properties that are related to the differences in antigenicity. Serotypes constitute stable replicating lineages which allow them to remain distinct over time. The infectivity of individual serotypes of animal viruses can be neutralized only by their own specific antibodies and not by antibodies directed to other serotypes. This inability of serotype-specific antibodies to cross-neutralize other serotypes is important in the case of animal viruses that are submitted to the immunological pressure of their hosts.
Many acute viral infections confer lifelong immunity. Upon re-exposure after initial infection, there is often reinfection but with minimal virus replication and an anamnestic immune response. Such reinfections are usually covert and almost never severe, resulting in minimal or no shedding of infectious virus. For certain viruses, such as poliovirus or rhinovirus, immunity is type speciic and confers little protection against exposure to a different serotype. These simple facts have profound implications for viral epidemiology.
1.《Encyclopedia of Virology》by Dennis Bamford and Mark Zuckerman.
2.《Fields Virology》 6th Ed.
抗原原罪Original Antigenic Sin （OAS）
The concept of “original antigenic sin”（抗原原罪） was first proposed by Thomas Francis, Jr. in 1960. This phenomenon has the potential to rewrite what we understand about how the immune system responds to infections and its mechanistic implications on how vaccines should be designed. Antigenic sin has been demonstrated to occur in several infectious diseases in both animals and humans, including human influenza infection and dengue fever. The basis of original antigenic sin （OAS） requires immunological memory, and our immune system ability to autocorrect. In the context of viral infections, it is expected that if we are exposed to a native strain of a pathogen, we should be able to mount a secondary immune response on subsequent exposure to the same pathogen. “Original antigenic sin” will not contradict this well-established immunological process, as long as the subsequent infectious antigen is identical to the original one. But “original antigenic sin” implies that when the epitope varies slightly, then the immune system relies on memory of the earlier infection, rather than mount another primary or secondary response to the new epitope which would allow faster and stronger responses. The result is that the immunological response may be inadequate against the new strain, because the immune system does not adapt and instead relies on its memory to mount a response. In the case of vaccines, if we only immunize to a single strain or epitope, and if that strain/epitope changes over time, then the immune system is unable to mount an accurate secondary response. In addition, depending of the first viral exposure the secondary immune response can result in an antibody-dependent enhancement of the disease or at the opposite, it could induce anergy. Both of them triggering loss of pathogen control and inducing aberrant clinical consequences.
•Original antigenic sin explains the failure of the immune system to generate an immune response against related antigens.
•In the original antigenic sin a prior exposure to an antigen leads to an ineffective to no response to a related antigen.
•The pathophysiologic mechanism of “original antigenic sin” includes the innate and adaptive immune systems.
•Further studies of original antigenic sin implications aimed to improve versions of already available vaccines are warranted.
Original antigenic sin (OAS) An immunological phenomenon (long-known from influenza and dengue virus infections) observed when a prior virus infection of an individual fully or partly hinders the induction of antibodies to the currently infecting related virus, and instead the original virus-specific IgG response is enhanced.
In the phenomenon called original antigenic sin, immune response against a virus can profoundly affect subsequent responses elicited against other viruses. e.g., infection by human bocavirus 1 produces a classical primary immune response. Subsequent infection by human bocavirus 2 can lead to activation of cross-reacting anti-bocavirus 1 memory B cells instead of primary immune response against bocavirus 2. Original antigenic sin not only hampers serodiagnostics but can also lead to weaker immune protection and more severe disease.
The immune response to the first infection with a virus can have a dominating influence on subsequent immune responses to antigenically related viruses, in that the second virus often induces a response that is directed mainly against the antigens of the original viral strain. For example, the antibody response to sequential infections with different strains of influenza A virus is largely directed to antigenic determinants of the particular strain of virus with which that individual was previously infected. This phenomenon, irreverently called “original antigenic sin,” is also seen in infections with enteroviruses, reoviruses, paramyxoviruses, and togaviruses. Original antigenic sin has important implications for interpretation of seroepidemiology, for understanding immunopathological phenomena and particularly for the development of vaccination strategies.