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Human adenoviruses (HAdV): Molecular diagnosis (PCR); IgM and IgG antibodies; Identification of species and genotype (PCR and sequencing)

 

Information 22-07-2018.

Human Adenoviruses (HAdV) are non-enveloped icosahedral double-stranded DNA viruses classified in the genus Mastadenovirus. These viruses have a worldwide distribution, causing infections throughout the year and are considered responsible for 5 to 10% of all febrile diseases in newborns and young children, causing more important infections in children and immunocompromised people. Most people have serologic evidence of an adenoviral infection prior to the age of 10 years.

Initially, antigenic differences were differentiated into serotypes. Subsequently, the application of molecular techniques have differentiated human adenoviruses in 7 species (A to G: HAdV-A, HAdV-B, ...,). The differentiation of species depends on characteristics such as: phylogenetic distance, genome organization (mainly in the E3 region), nucleotide composition, oncogenicity in rodents, range of hosts, cross-neutralization, recombination capacity, number of RNA genes associated with virus and haemagglutination tests.

In addition to the species, Adenoviruses are differentiated into types (genotypes), of which up to 84 have now been described. This is due to the fact that the genome of this virus is very unstable since coinfections can give rise to the homologous recombination that generates its molecular evolution with the appearance of new types, some of them considered emerging pathogens. Most new types of HAdV arise by homologous genomic recombination.

Some of the species have been subdivided into subspecies. For example, the species HAdV-B is divided into two subspecies, B1 and B2, each of them, including several genotypes: B1 (HAdV-B3, -B7, -B16, -B21 and -B50) and B2 (HAdV- B11, -B14, -B34, -B35 and -B55).

Human Adenoviruses (HAdV) are responsible for outbreaks of infections, particularly those related to day-care centers, schools, children's camps, hospitals, military environments and other centers. Of all the types of known adenoviruses, a third of them are associated with human diseases such as gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, meningoencephalitis and hepatitis, while the others produce asymtomatic infections.

Different species and some types of each species have been linked to different preferred types of infections. Thus, some of the types included in the species HAdV-B, -C and -E are mainly associated with respiratory infections; those of HAdV-A, -D, -F and -G with gastroenteritis; those of HAdV-B, -D and -E with eye infections, including epidemic keratoconjunctivitis; HAdV-B with acute hemorrhagic cystitis; HAdV-A, -B and -D with meningoencephalitis and those of HAdV-C with hepatitis. Of the subspecies, subspecies B1 causes respiratory infections with the exception of HAdV-B50 and those included in subspecies B2 cause kidney and urinary tract disease.

The genotypes of the HAdV-A species have historical importance as oncogenic in certain rodent model organisms, for example HAdV type 12, it is highly oncogenic, with the capacity to induce tumors in 4-months old newborn rodents. In immunocompetent individuals, HAdV infections are usually self-limited and are relatively infrequent to produce lethal infections, with some exceptions such as epidemics due to certain historically important strains and genotypes, for example, "Ad-7h", recently classified as type 66. In immunocompromised individuals, HAdV infections are of great concern because they can cause lethal infections. Specifically, HAdV-A, -B and -C are most frequently associated with infections of allogeneic transplant recipients such as serotypes HAdV-A31, -B3, -B7, -C1, -C2, -C5, while many new types of the species HAdV-D have been identified, for the first time, in patients with AIDS and other immunocompromised patients as possible opportunistic pathogens.

All this means that HAdV is considered an important public health problem for both immunocompetent and immunocompromised individuals.

These viruses have been found in waters around the world, including sewage, river water, drinking water, ocean water and swimming pools, even surpassing Enteroviruses, when both are detected in surface waters. Evidence has shown that Adenoviruses survive longer in water than various Enterovirus species or as the Enterovirus that causes hepatitis A, perhaps because of its double-stranded DNA, compared to Enteroviruses (RNA). Adenovirus infections can be caused by the inhalation of aerosolized droplets, by direct conjunctival inoculation, by fecal-oral spread, through infected tissues, airflow filters or environmental surfaces.

Clinical Tropism of Adenovirus according to species and type (genotype)

In recent years, multiple cellular receptors for Adenovirus have been identified, which determine the affinities of HAdV species for specific tissues. The human adenoviruses included in the species D (HAdV-D) are the most frequent, and show a great variability in their tropisms, with growth in ocular, gastrointestinal and respiratory tissues.

Human Adenoviruses of the species D (HAdV-D), including the types -D8, -D37, -D53 (recombinant strain between HAdV-22, HAdV-37 and HAdV-8), -D54, -D56 and -D64 ( previously identified as -D19a), but also by the human adenoviruses HAdV-E4, -C5, -B3, -B7, -B11 and -B14 are implicated in outbreaks of adenoviral epidemic keratoconjunctivitis (EKC) which is an important cause of ocular morbidity, although they can also cause pharyngoconjunctival fever (PCF) and nonspecific follicular conjunctivitis.

The types HAdV-D8, HAdV-D64, HAdV-D37, HAdV-54 (novel serotype that has been misclassified in recent years as HAdV-D8 due to its high similarity), cause conjunctivitis more serious than the others, and among the latter the HAdV-D8 is considered the main cause of the ECK that is associated mainly with community outbreaks, military and industrial institutions. HAdV-D64 (previously identified as -D19a) arose as it is a recombinant strain between pathogenic HAdV-D37 and non-pathogenic HAdV-D19. The cases of PCF have been preferentially related to serotypes HAdV-B3, HAdV-B7 and HAdV-B11.

The types HAdV-F40, -F41, -G52 and different members of species D, including some of the most recently identified types (types 65 and 67), are the main causes of gastrointestinal manifestations. Although many types of Adenoviruses can be found in the feces of patients with diarrhea, only Adenoviruses F (species F) that include types (genotypes) 40 and 41 have been shown to be the cause of acute gastroenteritis. Other Adenoviruses considered non-enteric, such as types 1, 2, 5, 3 and 7, may persist for months and even years in the stool after infection. The number of deaths from diarrhea caused by adenovirus (HAdV-F40 and F41) was estimated in 2011 in 3-4% of all diarrhea deaths among children under 5 years of age in the world.

Acute respiratory infections (ARI) are caused worldwide mainly by HAdV of species B (types -B3, -B7, -B14, -B21, -B55), species C (-C1, -C2, -C5, -C6) and species E (-E4). Among them the HAdV-C2, -B3, -B7, -E4 and -B55, are the main pathogens of these infections in children and adults and HAdV-C1, -C2 and -C5 are the most likely observed.

Among the most frequent pathogens is HAdV-B55, followed by HAdV-E4. Human adenovirus B type 55 (HAdV-B55) has been identified with a recombinant genome between renal HAdV-B11 and respiratory HAdV-B14, being more similar to the HAdV-B14 genome than to the HadV-B11 genome. HAdV-B55 is a highly contagious pathogen, associated with pneumonia and acute respiratory distress syndrome.

HAdV-E4 is the only representative of species E, this virus is of zoonotic origin and is involved through two interspecies recombinations with lateral partial gene transfer. HAdV-E4 contains 97% similarity to the genome of SAdV-E26 (Simian Adenovirus).

These examples indicate that certain adenoviruses have tropisms for specific tissues, but that the same clinical manifestations can be caused by other types and species of human adenoviruses. In addition, the prevalent types of HAdV have changed and will change over time.

Diagnosis and typing of Adenovirus by PCR

Conventional methods for detecting HAdV infections in patient samples include immunofluorescence staining to demonstrate the presence of virus antigen in infected cells and virus isolation in cell cultures inoculated with the samples. However, these methods have largely been supplanted by diagnostic methods based on molecular methods (PCR) to detect Adenovirus genomes directly in samples received in laboratories. Molecular methods allow the detection of the presence of all species and types of Adenoviruses, with high sensitivity and specificity in any sample of infections, so these tests have become the standard method of diagnosis.

The molecular diagnostic tests of HAdV use conserved molecular targets within the HAdV genome, in general of the gene coding for the hexon (vertices capsometers of the icosahedral structure), but the inclusion of additional target regions, for example, in the gene of the fiber (which emerges from the hexons), may be necessary to ensure reliable detection of all known types. Because the newly identified HAdV types generally result from recombination processes within the same virus species or from different virus species, the target regions of the PCR assays are retained in most cases, thus allowing detection with equal sensitivity of the new recombinant types. This is the case of HAdV-D genomes that seem to recombine more frequently than other human Adenovirus species. Thus, it is admitted that more than 40 types of HAdV-D have arisen through recombination between the genomic regions that encode the hexon and fibers. Most new types of HAdV identified by genomic analysis belong to species D, and have been shown to include sequences derived from multiple other types of the same species.

The identification of Adenovirus at the level of species (before serotypes), of types and even strains is relevant for epidemiological studies and for the study the exact documentation of nosocomial outbreaks.

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