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These books synthesize perspectives of renowned scientists from the world’s most prestigious institutions - from Fukushima Renewable Energy Institute in Japan to Stanford University in the United States, including Columbia University (US), University of Sidney (AU), University of Miami (USA), Cardiff University (UK), and many others.
\\n\\nThis collaboration embodied the true essence of Open Access by simplifying the approach to OA publishing for Academic editors and authors who contributed their research and allowed the new research to be made available free and open to anyone anywhere in the world.
\\n\\nTo celebrate the 50 books published, we have gathered them at one location - just one click away, so that you can easily browse the subjects of your interest, download the content directly, share it or read online.
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IntechOpen and Knowledge Unlatched formed a partnership to support researchers working in engineering sciences by enabling an easier approach to publishing Open Access content. Using the Knowledge Unlatched crowdfunding model to raise the publishing costs through libraries around the world, Open Access Publishing Fee (OAPF) was not required from the authors.
\n\nInitially, the partnership supported engineering research, but it soon grew to include physical and life sciences, attracting more researchers to the advantages of Open Access publishing.
\n\n\n\nThese books synthesize perspectives of renowned scientists from the world’s most prestigious institutions - from Fukushima Renewable Energy Institute in Japan to Stanford University in the United States, including Columbia University (US), University of Sidney (AU), University of Miami (USA), Cardiff University (UK), and many others.
\n\nThis collaboration embodied the true essence of Open Access by simplifying the approach to OA publishing for Academic editors and authors who contributed their research and allowed the new research to be made available free and open to anyone anywhere in the world.
\n\nTo celebrate the 50 books published, we have gathered them at one location - just one click away, so that you can easily browse the subjects of your interest, download the content directly, share it or read online.
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Sexual-cycle abnormalities in bitches may present as anestrous, shorter, or longer cycles, as well as prolonged proestrus, prolonged estrus, split estrus, or anovulatory cycles. These cycle disorders may result from abnormal ovarian functions and are a cause of infertility [1].
\nAnestrous cycles in bitches may be either primary or secondary. If a bitch does not show signs of estrus despite having reached age of puberty, anestrous cycles are primary. The age of puberty in bitches is 6–14 months. In general, the “primary anestrus” diagnosis may be used if estrus has not occurred and the cycle has not started by 24 months of age. Although some small breeds experience first estrus at 6 months of age, cycles accompanied by estrus signs may be delayed, since the first cycles may be silent. Therefore, cycle problems are not usually investigated until a bitch reaches 2 years of age. A diagnosis of “secondary estrus” is used if estrus has not occurred for 10–18 months, although first estrus had occurred. In other words, secondary estrus is defined as the presence of a period longer than 10–18 months between estruses. Normally, bitches experience estrus at 4–10 month (mean 6–7 months) intervals. Cycles shorter than 4 months or longer than 10 months are abnormal and can cause infertility [2–6].
\nFactors that lead to primary anestrus include ovariectomy or ovariohysterectomy at early age, silent heat (subestrus), abnormalities in sexual differentiation (chromosomal and genetic disorders), use of progesterone or glucocorticoids, congenital hypothyroidism, certain systemic diseases, ovarian anomalies or ovarian aplasia, progesterone-releasing ovarian cysts, and autoimmune oophoritis [2, 7].
\nSecondary anestrus may result from dysfunction of the thyroid gland or adrenal cortex, as well as nonendocrinological disorders, cachexia, obesity, and use of cycle-inhibiting drugs. Silent heat, luteal cysts, and some ovarian tumors may also lead to secondary anestrus as well as to primary anestrus [4, 5].
\nIf taken from another person, the bitch may have been sterilized, which a new owner may overlook. Presence of a tattoo in the inguinal region and palpation or inspection of a scar from operation in the ventral wall of the abdomen may be indicators of ovariohysterectomy. However, it should be kept in mind that such a scar may be present if any intra-abdominal operation has been performed, so it may be premature to conclude that the scar resulted from ovariohysterectomy. Serum LH level measurement may be used to identify bitches that have undergone ovariohysterectomy, as serum LH is continuously high in such bitches as a result of the absence of negative feedback on LH (because the ovaries have been removed). Although elevation in serum LH provides information about ovariohysterectomy, note that this indicator may also be seen in ovarian dysfunction or during the preovulatory period (i.e., the preovulatory LH peak). Therefore, repeated measurements are required to confirm and experimental laparotomy may also be performed for a definitive diagnosis [8]. Whether ovariohysterectomy has been performed may also be detected by measuring the serum estrogen level before and 60–90 min after intravenous administration of 0.02–0.03 μg/kg buserelin. If ovariohysterectomy has been performed, estrogen levels will be found above 15–20 pg/mL.
\nAnti-müllerian hormone (AMH) measurement may also show whether a bitch has undergone ovariohysterectomy. AMH levels are found to be significantly lower in bitches that have undergone ovariohysterectomy compared to nonsterilized bitches [9, 10].
\nSilent heat is defined as the maintenance of ovarian functions without the presence of vulvar edema, serosanguinous vaginal discharge, and charm for male dogs. Silent heat may be observed for several cycles before first estrus in younger bitches of smaller breeds. These animals may be evaluated as “anestrus” because the pet owner may not find the external signs of estrus, or they may be identified as healthy male dogs although ovarian functions continue normally.
\nIf silent heat is suspected, serum progesterone level should be measured once monthly in order to verify that the ovaries are functioning. A serum progesterone level above 2 ng/mL indicates functional luteal tissue. Observation of increasing superficial epithelial cells in regular vaginal cytological examinations is an indicator of functional ovaries [5].
\nNormal sexual development occurs in three stages: (1) chromosomal (genetic) sex development, (2) gonadal sex development, and (3) phenotypic sex development. Therefore, disorders of sex development and differentiation are classified into three matching groups: (1) sex chromosome disorders, (2) gonadal sex development disorders, and (3) phenotypic sex development disorders. All three groups of disorders result in abnormal sex differentiation and may vary in presentation between genital structure of normal and obscure appearance, and all three groups of disorders may lead to sterility or infertility [11–13].
\nChromosomal analysis, anatomical and histopathological definitions of the gonads, and examination of the internal and external genitalia are required for the definite diagnosis of dogs suspected to have disorders of sexual development [11, 14].
\nLong-term use of some drugs, such as androgen and progestogens, causes anestrus by inhibiting cycles. Exogenous glucocorticoid administration is also reported to affect serum LH level and the normal cycle. Puberty is inhibited due to suppression of genital-canal development and ovarian activity in bitches that have undergone administration of long-acting GnRH agonists like deslorelin [15].
\nSimilarly, cyclic activity may not be observed for a long time in prepubertal bitches that have been actively immunized against GnRH [16].
\nA comprehensive anamnesis should be obtained from the pet owner if drug-related anestrus is suspected; sufficient data must be obtained regarding the medical history of the bitch. If a pet owner has recently acquired the bitch and if he or she has no or insufficient information about previous vaccinations and medications, drug-related anestrus should always be considered. In such situations, the only treatment is to wait until the effects of the drug(s) disappear or until the antibody titration decreases, if immunized against GnRH.
\nThere is an indirect and strict association between thyroid dysfunction and reproductivity. Hypothyroidism leads to reproductive disorders such as prolonged anestrus, silent heat, prolonged proestrus, and ovulatory problems. The prolactin level increases, which leads to impair or no development of ovarian follicles by inhibiting GnRH in bitches that have insufficient thyroid hormone release [17].
\nIn bitches, hypothyroidism usually manifests as primary hypothyroidism, resulting from destruction of the thyroid gland. The serum total and free thyroxin levels are low in bitches with thyroid dysfunction. Thyrotropin-releasing hormone (TRH) secretion from the hypothalamus increases due to low levels of thyroid hormones; consequently, thyroid-stimulating hormone (TSH) secretion from the pituitary gland increases. As a result of this totally physiological process, low serum total and free thyroxin levels and elevated serum TSH levels are observed in bitches with primary hypothyroidism [7].
\nHypothyroidism should be kept in mind in the presence of numbness or mental fatigue, hair loss, weight increase or obesity, dryness or loss of body hair, hyperpigmentation, cold intolerance, bradycardia, high plasma cholesterol level, or anemia in a bitch with an anestrous problem. Measurement of only thyroid hormones may yield misleading or conflicting results. Therefore, a full thyroid profile should be obtained and a definitive diagnosis made in bitches suspected of hypothyroidism. For this purpose, the serum-free thyroid hormone level (particularly T4 measurement) and the response of the thyroid gland to TSH administrations should be investigated. Furthermore, autoantibodies against thyroxin (T4) and triiodothyronine (T3) or thyroglobulin should be investigated in serum, as they are produced in most bitches with lymphocytic thyroiditis [7, 18, 19].
\nThe sexual cycle may return to normal with hormone replacement therapy within 3–6 months in bitches diagnosed with hypothyroidism. For this purpose, synthetic thyroid hormone (levothyroxin) should be administered through the peroral route. A dose of 22 μg/kg b.i.d. is usually sufficient.
\nAlthough hyperthyroidism with consequent primary anestrus is a rare condition in bitches, a case has been reported in a Pinscher dog with diet-related hyperthyroidism in which primary anestrus developed; the bitch reached proestrus 13 days later after dietary regulation, with estrus then induced by cabergoline [20].
\nThe pituitary gland is important for the endocrinological functions of the adrenal glands, thyroid gland, and the ovaries. Abnormalities of the pituitary gland also negatively affect these organs. Prolonged anestrus is unusually seen in bitches with dwarfism caused by a congenital anomaly. Ovariohysterectomy is recommended in bitches that show prolonged anestrus related to pituitary gland insufficiency [6].
\nSuch diseases may negatively affect reproductive function. Cycles probably will not develop if an animal is unhealthy.
\nProgesterone-releasing ovarian cysts, ovarian aplasia, and oophoritis may lead to primary anestrus. Definitive diagnosis can be made by histopathological examination of the ovarian tissue [21].
\nFirst, it should be determined whether the pet owner has correct and sufficient knowledge of the cycle and estrous signs of bitches. The age of the bitch and whether it has experienced ovariectomy, ovariohysterectomy, or administration of any drug or vaccine (especially GnRH vaccine) should be determined. Serum progesterone level should be monitored monthly for 6–8 months before any intervention in anestrous bitches. In normal bitches, the serum progesterone level rises above 2 ng/mL within 2 months after estrus; progesterone level below 2 ng/mL for 6–8 months definitely indicates prolonged anestrus. In addition, vaginal epithelial cells should be monitored for any alterations with weekly vaginal smears.
\nRoutine blood and urine tests (complete blood count, biochemical analysis) and thyroid function tests should be performed following general examination in anestrous bitches. Progesterone measurement and ultrasonographic examination of the ovaries should be carried out whenever luteal cyst or tumor of the ovaries is suspected. Karyotype analysis should be performed if developmental anomalies of the genital organs are suspected (such as hermaphroditism) [5].
\nTreatment of primary or secondary anestrus is targeted towards its etiology. No treatment is available if a bitch has undergone ovariectomy or ovariohysterectomy or in the presence of congenital sex development or differentiation anomalies, ovarian aplasia, or autoimmune oophoritis. Ovariohysterectomy is recommended for autoimmune oophoritis. Hormone replacement therapy may be administered in bitches determined to have hypothyroidism-related anestrus [22].
\nEstrous stimulation may be performed in anestrous bitches for which the underlying cause cannot be detected. Synthetic estrogens (diethylstilbestrol), dopamine agonists (bromocriptine and cabergoline), GnRH agonists (lutrelin, buserelin, fertirelin, deslorelin, leuprolide), or exogenous gonadotropins (LH, FSH, hCG, PMSG) may be administered for this purpose [23, 24].
\nDopamine agonists are effective for stimulating fertile estrus in most bitches; however, prolonged use may be required. Bromocriptine (Parlodel, Gynodel) or cabergoline (Galastop, Dostinex) is used for this purpose. Bromocriptine is less preferred, as it causes vomiting and requires lengthy periods of administration in order to stimulate estrus. Although more expensive than bromocriptine, cabergoline may induce estruses effectively and safely with fewer side effects. Cabergoline is usually recommended at a dose of 5 μg/kg daily via the peroral route until 3–8 days after proestrus has begun [23–25].
\nUsing GnRH and its analogs may also induce estruses. However, prolonged use for 8 days or longer or long-acting analogs such as lutrelin, deslorelin, or leuprolide is needed to induce fertile estrus. It is impractical to induce estrus by using short-acting natural GnRH or GnRH agonists, because GnRH should be given as a pulsatile continuous infusion at a dose of 0.2–0.4 μg/kg every 90 min via the intravenous or the subcutaneous route for 3–9 days. This requires 3–9 days of hospitalization and the availability of a pulsatile infusion pump. Long-acting formulations of GnRH analogs such as lutrelin, deslorelin, and leuprolide have been quite successful when implanted subcutaneously or submucosally. Deslorelin-containing implants (Suprelorin®, 4.7 mg deslorelin) are used most frequently for this purpose [23–27].
\nHypophyseal (FSH and LH) and chorionic (PMSG and hCG) gonadotropins are also used for inducing estruses. It has been found that chorionic gonadotropins are more successful than hypophyseal gonadotropins in bitches. Although various protocols have been attempted, successful results have been reported with 20 IU/kg/d PMSG applied subcutaneously for 5 days, with 500 IU intramuscular hCG on day 5. PG600, a preparation containing PMSG and hCG first produced for pigs (80 IU PMSG and 40 IU hCG/mL), is quite effective for inducing estruses [8, 28].
\nThe mean duration between estruses is 7 months (4–13 months) in bitches, and the long part of the cycle (2–10 months) comprises a mandatory anestrus phase following diestrus. In the anestrous phase, the uterus enters the involution process and the endometrium is regenerated. Anestrus shorter than 2 months naturally results in repetition of estrus at 4-month or shorter intervals, which is defined as “recurrent estrous.” It should be kept in mind that the estrous period is shorter in breeds such as the
The period between estruses may be prolonged up to 6 months by using a weak androgen, mibolerone (Cheque Drops), recommended at a dose of 30–180 μg per day to suppress estruses [1, 30].
\nFurthermore, if infertility resulted from short estrous period, bitches are reported to return to normal fertility in the following cycles when estrus is suppressed with synthetic progestin administration. For this purpose, 2 mg/kg per day megestrol acetate or 0.5 mg/kg per day of chlormadinone acetate may be administered for 8 days, so administration should begin within a maximum of 3 days following the beginning of proestrus [29].
\nProlonged interestrous interval is defined as an interestrous period longer than 12 months. While estrus repeats 12–13 months after the previous estrus (prolonged interestrous interval) in some adult bitches, some are not observed to experience estrus again for a long time (secondary anestrus). It should be kept in mind that the interestrous period is longer in breeds such as the
The mean duration of estrus is 9 days in an adult dog, which may sometimes be prolonged up to 3 weeks. Estrus of longer than 21 days with the absence of ovulation at the end of this long period is defined as prolonged estrus. Prolonged estrus is related to persistent and elevated estrogen levels, which remain continuously high during the estrous phase of the cycle. This disorder is encountered frequently in younger bitches, especially during the second cycle [1].
\nThe most important clinical signs of the continuation of estrus include cornification in vaginal epithelial cells, continuation of the desire for copulation, vulvar edema and swelling, and hyperemia in vaginal mucosa for longer than 21 days. The serum progesterone level is low, while estrogen level is high.
\nPersistent estruses are usually related to an estrogen-releasing source, which may be an anovulatory follicle, follicular cysts, or functional ovarian tumors (granulose cell tumors). The follicles that develop in bitches receiving exogenous gonadotropins in order to experience estrus may sometimes lead to prolonged estruses. Exogenous estrogen administration for the treatment of urinary incontinence or vaginitis, hormone replacement therapy, and prevention of undesired pregnancy may also cause persistent estruses. Persistent estrus may also develop alongside tumors of the hypophysis or the hypothalamus or in a hepatic disease defined as portosystemic shunting in which an abnormal vascular junction is formed between the hepatic portal vein and the systemic circulation. In these cases, metabolism of estrogen in the liver is impaired.
\nDetermination that 90% or more of the cells in a vaginal smear specimen are permanently cornified on cytological examination and nondetection of the normal increase in serum progesterone levels (remaining within the preovulatory range <2 ng/mL) indicate prolonged estrus. Detection of serum estrogen level is not a reliable method for diagnosis [1].
\nThe first step in treatment should include a determination of the source of estrogen causing prolonged estrus. For this purpose, the ovaries should be examined ultrasonographically for the presence of abnormal structures (e.g., ovarian cyst, granulose cell tumor); if ovarian structures cannot be identified by ultrasonographic examination, exploratory laparotomy should be performed, followed by biopsy, if required.
\nFollicles or follicular cysts causing prolonged estrus may heal spontaneously. If estrus is determined to last longer than 3 weeks, interventions are recommended in order to prevent bone marrow hypoplasia and/or pyometra. Treatment options should match the pet owner’s expectations regarding having a puppy; if a puppy is not expected, ovariohysterectomy is the best option.
\nOvulation or luteinization may be obtained by GnRH or hCG injections into follicles if the pet owner wants a puppy. hCG administration at a dose of 22 IU/kg via the intramuscular route for 3 days and GnRH (gonadorelin) administration at a dose of 10 μg/kg via the intramuscular route for 3 days are recommended. Copulation is not recommended, as the target of these applications is not the induction of ovulation but rather the termination of the signs of prolonged estrus [1]. Ovariohysterectomy is inevitable in cases in which no response is obtained from medical applications and in prolonged estrus cases due to ovarian tumors [5].
\nMegestrol acetate (Ovaban, Ovarid) may be applied to reduce the signs of prolonged estrus. Low doses of megestrol acetate are recommended via the peroral route for 2 weeks. A dose of 0.1 mg/kg is proper for the first week, and a dose of 0.05 mg/kg is proper for the second week. Although progesterone treatment with megestrol acetate is effective in bitches with persistent estrus, there is the potential to trigger the development of cystic endometrial hyperplasia. Therefore, the treatment is contraindicated for bitches to be later considered for copulation. In general, ovariohysterectomy is performed within 3 weeks after treatment with progesterone in bitches with persistent estrus.
\nBone-marrow suppression related to long-term estrogen toxicity may develop in bitches with persistent estrus. Nonregenerative anemia and thrombocytopenia are observed in such bitches [31]. Therefore, erythropoiesis-stimulating agents, such as synthetic erythropoietin, darbepoetin, granulocyte-colony stimulating factor, and granulocyte-macrophage colony-stimulating factor, may be used beside proper antibiotic and blood products in bitches that have anemia due to bone-marrow suppression. In addition, lithium [30, 32], synthetic anabolic steroids such as nandrolone decanoate (Deca-Durabolin), or a dihydrotestosterone derivative such as stanozolol (Winstrol) can be quite useful [33, 34].
\nProlonged proestrus is defined as a proestrous phase that is not followed by an estrous phase and that lasts 3 weeks or longer. In bitches with prolonged proestrus, estrus and ovulation do not occur, as the estrogen level insufficiently increases during the proestrous phase.
\nProlonged hemorrhagic vaginal discharge, cornified cells higher than 50–90% on examination of vaginal smear, and serum progesterone level remaining below 2 ng/mL indicate prolonged proestrus. Treatment principles are similar to those for prolonged estrus.
\nSplit estrus is a disorder in which no or quite short estrous signs develop despite the presence of proestrous signs. In this situation, pregnancy usually does not develop even if copulation occurs; the bitch is observed to enter proestrus again within 3–4 weeks. In these bitches, the next cycle is usually a normal ovulatory cycle.
\nSplit estrus is usually seen in young bitches that have shown first estrus. However, continuous or frequent split estruses should suggest chronic premature luteolysis or hypothyroidism. Split estrus may be confused with recurrent estrus (short interestrous interval). Ovulation will not develop in dogs showing split estrus but without the typical progesterone elevation. The condition usually recovers spontaneously [1, 7].
\nA serum progesterone level not exceeding 2 ng/mL despite cytological estrous signs is defined as anovulation. Although the cell type in vaginal cytology is noncornified, diestrous-specific progesterone elevation does not occur, and the bitch enters anestrus.
\nThe most typical signs are low serum progesterone levels and the absence of ovulation during the days after copulation in a bitch showing proestrous and estrous signs. Its incidence is about 1%. The following ovulatory cycles were observed to be normal in 45% of bitches that had an anovulatory cycle [1, 21, 35].
\nThus, treatment is usually not required in bitches with an anovulatory cycle. hCG or GnRH may be administered, if desired; however, their application carries the potential to trigger pyometra [1, 21].
\nThere are many factors leading to abnormalities in the sexual cycle of bitches. A decent anamnesis is required to find out the causes of these abnormalities. Supporting the anamnesis information by clinical and laboratory examinations is of importance for the accuracy of the diagnosis. Vaginal cytology among the diagnostic methods should be used and interpreted efficiently. Accuracy of the diagnosis forms the first step of an effective treatment. Uses of hormones, particularly gonadotropins, come into prominence in the treatment of sexual abnormalities in these animals.
\nInfluenza viruses are RNA viruses that cause infectious respiratory diseases that are majorly characterized by fever, congestion, and myalgia, which ranges in severity from mild to life-threating, and they are estimated to cause about 250,000 to 500,000 deaths globally per year [1]. They are single-stranded, helically shaped, and belongs to the orthomyxovirus family consisting of 5 influenza virus genera, ranging averagely from 80 to 120 nm in size [2]. They often contain 8 gene segments that encodes 11 proteins (Figure 1). These segments encode viral proteins including hemagglutinin (HA), neuraminidase (NA), nonstructural 1 (NS1), NS2, matrix 1 (M1), M2, nucleoprotein (NP), nuclear export protein (NEP), polymerase acid (PA), polymerase basic 1 (PB1) and PB2 [3]. Influenza viruses are uniquely known to express spike glycoproteins such as hemagglutinin (HA) which facilitates viral recognition of host receptor binding site and neuraminidase (NA) which also aids viral release after replication within the host cells [2, 4]. HA binds Sialic acid bonded with galactose, in avian influenza (H5N1) affinity binding occurs with the α-2,3 sialic acid galactose receptor complex of birds in contrast with the α-2,6 binding in human Influenza virus A infections [1, 4, 5].
Showing all the eight gene segments and encoded proteins of influenza A virus. Influenza virus’s genome is eight-segmented and encodes for two surface glycoproteins which includes neuraminidase (NA) and hemagglutinin (HA); matrix protein 2 (M2) ion channel that are securely buried into the viral lipid envelope; matrix protein 1 (M1) which lies beneath the membrane; protein-basic protein (PB1, PB2) protein-acidic protein (PA) which makes up the RNA polymerase complex that is associated with the encapsilated genome; nucleoprotein (NP) which coats the viral genome and nonstructural proteins (NS1 and NS2) which suppresses host cell’s mRNA production and serves as interferon antagonism.
Till date, three types of influenza virus have been known to cause infection in humans: A, B, and C. Type A influenza has subtypes determined by the surface antigens hemagglutinin (HA) and neuraminidase (NA). There are 18 different H subtypes and 11 different N subtypes. Eight H subtypes (H1, H2, H3, H5, H6, H7, H9, H10) and six N subtypes (N1, N2, N6, N7, N8, and N9) have been detected in humans. Type B influenza is classified into two lineages: B/Yamagata and B/Victoria [2]. Influenza B commonly affects children while Influenza C is rarely reported as a cause of human illness, which is probably because most cases are subclinical. Influenza C has still not been associated with any epidemic disease outbreak so far. WHO currently classifies influenza A(H1N1) and A(H3N2) as circulating seasonal influenza A virus subtypes, while also classifying avian influenza virus subtypes A(H5N1) and A(H9N2) and swine influenza virus subtypes A(H1N1) and (H3N2) as zoonotic or variant influenza [2, 6].
Enormous efforts are currently aimed at preventing and treating influenza infections, including seasonal and pandemic influenza, however, outbreaks still remain a major public health challenge globally [1, 4]. This is majorly due to influenza viruses rapidly undergoing genetic mutations that restrict the long-lasting efficacy of vaccine-induced immune responses and therapeutic regimens [1]. These major viral genetic changes involve Antigenic Drift, which is caused by point mutations in genes encoding HA and N glycoproteins spikes thereby allowing for viral immune invasion against host responses and generated antibodies like vaccines. Similarly, antigenic shift which occurs in influenza virus A, caused by viral genome reassortment/swapping mechanisms among two different subtypes of influenza A which are replicating within the same host causing a jump to new species of host, and a highly diverse structure of virus able to cause the occasional pandemics seen in the world [2]. A combination of antiviral agents and vaccines remains the general prevention and treatment measures for influenza-related morbidity and mortality, however complications arising from viral genetic changes has bolstered scientific efforts on a journey to the discovery of universal vaccines.
Following respiratory transmission, human influenza virus attaches to and penetrates the respiratory epithelial cells in the trachea and bronchi. Other cell types often affected in the respiratory tract includes several immune cells, which can be infected by the virus and initiate viral protein production. However, the efficiency of replication varies among various affected cell types, and, in humans, the respiratory epithelium is the only site where the hemagglutinin (HA) molecule is effectively cleaved [5, 7]. The primary mechanism of influenza pathophysiology is a result of lung inflammation and compromise that is caused by direct viral infection of the respiratory epithelium, combined with the effects of lung inflammation also caused by immune responses recruited to handle the spread of the virus [7]. Influenza-mediated respiratory tract damage is caused by a combination of events, including: a) intrinsic viral pathogenicity due to its affinity for host airway and alveolar epithelial cells; and b) a robust host innate immune response, which, while aiding in viral clearance, can aggravate the severity of lung injury [7].
The host cell is then destroyed as a result of viral replication. Viremia, or the presence of a virus in the blood, has, on the other hand, is seldomly observed and never widely documented. Virus is released in respiratory secretions for 5 to 10 days, peaking 1 to 3 days after disease start [5, 7]. Inflammation caused by influenza pathogenic events can extend systemically and appear as multiorgan failure, the most common of which are lung compromise and severe respiratory distress [8]. Some links have also been found between influenza virus infection and cardiac complications, such as an increased risk of myocardial illness in the weeks following infection. Beyond the basic inflammatory profile, several of these processes remain uncertain [9, 10]. Researchers find it theoretically helpful to divide the progression of IAV infection into three stages, with the idea that many of these processes occur concurrently throughout the injury. The first is viral infection and replication in the airway and alveolar epithelium, during which methods that restrict viral entrance or replication might prevent or reduce the severity of the infection. The innate immune response to the virus is followed by the adaptive immunological response, which is crucial for viral clearance but may also cause severe damage to the alveolar epithelium and endothelium. The third step is the establishment of long-term immunity to the infecting virus strain, which is followed by the resolution of infiltrates and regeneration of damaged lung tissue, during which time the patient is more vulnerable to secondary bacterial infection [4, 5, 7].
The influenza viruses are significantly important human pathogens. In humans, infection of the lower respiratory tract of can result in flooding of the alveolar compartment, development of acute respiratory distress syndrome and death from respiratory failure. The extent to which the virus infiltrates the lower respiratory tract is an important factor in determining the degree of associated disease complications [8]. Infection of alveolar epithelial cells appears to cause the development of severe illness by damaging important mediators of gas exchange and permitting viral exposure to endothelial cells. Early interactions between the influenza virus, alveolar macrophages in the lung airways, and the epithelial lining are significant determinants of alveolar disease development [9]. Once this delicate barrier is penetrated, cytokine and viral antigen exposure to the endothelium layer can exacerbate inflammation, with endothelial cells being a primary source of pro-inflammatory cytokines that influence the amount and nature of future innate and adaptive immune responses [10].
In the final pathological stages, just like in the SARS-CoV-2 infection, where reports from Lancet on COVID-19 pathogenesis reveals that acute respiratory distress syndrome (ARDS) is the main cause of death in most patients [11, 12, 13, 14], influenza virus infection also initiates hypoxia and progression to ARDS [15]. ARDS is majorly experienced as shortness of breath and it’s also a common immunopathological event in SARS-CoV and MERS-CoV infections [11]. Clinically, severe Influenza A Virus infection can cause bilateral lung infiltrates and hypoxaemia, which are symptoms of acute respiratory distress syndrome (ARDS), and death from hypoxaemic respiratory failure is a major contributor to mortality [16, 17, 18, 19, 20, 21]. The cumulative incidence of ARDS related to seasonal IAV infection has been estimated to be 2.7 cases per 100,000 person-years, accounting for 4% of all respiratory failure hospitalizations throughout the influenza season [22].
In most cases, influenza produces a simple respiratory illness with a cough, fever, myalgias, chills or sweats, and malaise that lasts two to eight days. The onset is usually quick. Children might have unusual gastrointestinal symptoms such as vomiting and diarrhea. A small percentage of patients, particularly elderly individuals, young children, and those with medical comorbidities, will develop severe illness from viral or secondary bacterial pneumonia, resulting in respiratory and multiorgan failure. Extrapulmonary events are extremely uncommon [23, 24].
Common symptoms such as running nose, sore throat, muscle pains, fever, headaches and fatigue can trigger the release of pro-inflammatory cytokines and chemokines such as tumor necrosis factor or interferon from infected cells might be capable of producing a life-threatening cytokine storm [25]. Influenza does cause tissue damage compared to common cold that is caused by rhinovirus and as such symptoms might not entirely depend on inflammatory response. Also, the large amounts of cytokines have been observed to be dependent on the levels of viral replication produced by the strains [26]. Flu epidemics are difficult to control due to their rapid spread. However, influenza virus has a short generation time of two days (the time from being infected and then to infect the next person). Individuals can become infectious before being symptomatic thus quarantines following noticeable sign and symptom of the infection is not an effective public health intervention [27]. The virus shedding in an average person peak on day two while symptoms becoming apparent on day three [28].
Early anti-influenza drugs were synthesized by large scale screening methods without the knowledge of their chemical structures and mechanisms of action [29], whereas, the recent antivirals have been discovered based on the structure of influenza virus protein as drug targets using X-ray crystallography method. This is structure based, involving the use of organic compounds that are able to bind to viral target protein receptors [30]. These structures have high binding affinity to the viral target following chemical synthesis and effective antiviral screening using standard in vitro assays such as cell based antiviral screening [31] and biochemical evaluation [32]. Some cell based antiviral screening includes plaque assays for studying replication in virus, yield-reduction assays for quantifying specific viral antigens and dye uptake for measuring cytopathic effect. The application of bioinformatics, robotics, miniaturization strategies have led to an advanced and high-throughput drug screening of large drug libraries with unique chemical structures [33] and computational screening [34]. In vivo drug screening using various animal models such as chicken, mouse, ferret have been used to evaluate new drugs [35] this is followed by clinical trials to study its bio-safety, kinetics and tolerance in human [36].
Advances has since then seen the treatment of influenza virus infection basically through vaccines, monoclonal antibodies and antivirals drugs. Antiviral influenza drugs are mostly NA inhibitors; however, they generally have short therapeutic window and current show emerging drug resistance [37]. Till date, four (4) antiviral drugs have been approved for the treatment of influenza: the NA inhibitors oseltamivir (Tamifu), peramivir (Rapivab), zanamivir (Relenza), and the cap-dependent endonuclease inhibitor baloxavir (Xofuza) [23, 37]. Oseltamivir is the preferred treatment for patients with severe influenza. Intravenous peramivir is an option for these patients if there are contraindications to or concerns about reduced bioavailability of oral oseltamivir [24]. Baloxavir is preferred for the treatment of uncomplicated influenza in patients of age 12 years and older. A study was conducted to compare baloxavir with oseltamivir and placebo in 1436 healthy people between 12 to 65 years of age who had influenza, baloxavir and oseltamivir reduced symptom duration by approximately one day compared with placebo. Adamantanes (amantadine and rimantadine [Flumadine]) are also approved for influenza treatment but are not currently recommended because these medications are not active against influenza B, and most influenza A strains have shown resistance to adamantane for the past 10 years [24].
Vaccines remain extremely essential to the prevention of the infection. Vaccination is the most preferred method for prevention, and routine chemoprophylaxis within the community is not recommended. The first influenza vaccine was developed in 1945, and since seen several others produced. Multiple formulations of the influenza vaccine are available, including inactivated influenza vaccines (IIV); a recombinant inactivated vaccine (RIV); and a live, attenuated influenza vaccine (LAIV). LAIV shows one of the best efficacies at around 70% and tends to be more effective in children. It delivers more NA and M2 antigens, triggers mucosal responses including IgA, and has the potential for inducing CD8 T cell responses [38].
As a primary prophylactic countermeasure, annual influenza vaccination is engaged globally with the aim of limiting influenza burden. However, the effectiveness of the current available influenza vaccines is limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The consequences of antigenic drift or shift, results in an antigenic mismatch between the current vaccines and circulating influenza isolates. Accumulation of mutations, especially at key antigenic sites in the HA globular head, due to the absence of the proofreading activity of the viral RNA polymerase and then to the selective pressure exerted by the host immune system is often responsible for the escape of influenza virus from pre-existing immunity in the case of antigenic drift [39]. There is therefore a crucial need to develop a more effective broadly-reactive (universal) influenza vaccine with the capability to confer protection against both seasonal and newly emerging pre-pandemic strains.
In influenza virus vaccine design, the major targets of the antibody response against the virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). As earlier stated, Hemagglutinin (HA) and neuraminidase (NA), are the main surface glycoproteins on influenza viral particles. NA is however less abundantly expressed on the virion in comparison HA expression, with HA to NA ratio often ranging from 4:1 to 5:1 [38]. The influenza HA is responsible for binding to sialic acid, the receptor on target host cells, and there are approximately 500 molecules of HA per virion [40]. The mature form of the HA glycoprotein exists as a homotrimer containing three HA monomers that are composed of a globular head and a stem/stalk region. The receptor binding site (RBS) is present in the globular head, which is however a hypervariable region of the protein, while the stem region is majorly involved in the pH-induced fusion event triggered by endosome acidification following viral adsorption. The stem/stalk region of the HA is more conserved among and across HA subtypes belonging to the same group [38]. Antibody response elicited against this stem/stalk region forms one of the major approaches towards developing a more responsive vaccine to both current and future strains of influenza viruses (Figure 2).
Showing the phylogenetic trees of (a) hemagglutinin (HA) and (b) neuraminidase (NA). The primary surface glycoproteins of influenza viruses, HA and NA, are divided into several categories and subtypes. During the last century, only viruses producing H1, H2, or H3 HAs and N1 or N2 NAs (such as H1N1, H2N2, or H3N2; circled in purple) have spread widely in the human population. The scale bars represent a 6% change in amino acid levels (source: [
Influenza virus infection can elicit neutralizing antibodies against both the globular head and the stem structures of the HA viral protein. Currently, ongoing strategies for more efficacious vaccine development is aimed at eliciting antibodies that target the conserved stem region of HA since previously existing influenza vaccines only show minimal induction of stem-directed humoral immunity [3]. Several studies describe ongoing approaches to elicit stem-directed antibodies including sequential immunization with heterologous influenza strains, immunization with modified proteins by removing or glycan-masking the globular head, referred to as headless HA, through minimizing epitopes of the stem region, hyperglycosylated HA head domain, Chimeric HA, and Mosaic HA [3, 38]. Self-reactivity of this antibodies may occur due to their polyreactive profile and the proximity of the HA stem region to the cell membrane which is a crucial limitation described by scientists to this approach.
Nachbagauer et al. [40] recently presented a unique concept in the stem-based approach using the context of a LAIV with a H8 head domain and an H1 stem domain (cH8/1) and a split-inactivated vaccine with an H5 head domain and an H1 stem domain (cH5/1) [40]. Using preclinical ferret investigations, the scientists assessed protection against pandemic H1N1 virus challenge using several sequential prime-boost combinations and vaccination regimens. These studies show that a sequential live-attenuated followed by split-inactivated viral vaccination strategy provides superior protection against pandemic H1N1 infection. Scientists have characterized this notion as a sequential immunization and chimeric HA proteins approach to stem-based universal vaccine design.
Furthermore, in a stem-based immunogens approach to the universal influenza vaccine design, based on the H1 subtype, Impagliazzo et al. created stable mini-HA stem antigens, where the best candidate demonstrated structural and binding characteristics with widely neutralizing antibodies equivalent to full-length HA, indicating correct folding. This immunogen totally protected mice in lethal heterologous and heterosubtypic challenge scenarios and lowered fever in cynomolgus monkeys following a sublethal challenge [42]. However, determining the effectiveness of antibodies targeting conserved epitopes in the HA stem region to offer protection remains a critical challenge [43].
Furthermore, in order to overcome the extreme variability of influenza HA, in particular at the head region, Giles and Ross, [44] described the generation of computationally optimized broadly reactive antigens (COBRAs) for the influenza HA. The COBRA-based approach is a classic reverse vaccinology approach based on multiple layering of consensus HA protein sequences, followed by the generation of a final consensus sequence capable of recapitulating, in a unique protein, amino acid changes undergone by influenza virus from the past years to the present [45]. In this approach, a phylogenetic tree is inferred using hemagglutinin (HA) amino acid sequences. Primary and secondary consensus sequences are constructed, and the secondary consensus sequences are subsequently aligned to provide the resultant consensus, known as COBRA. In multiple clinical investigations, a firm called Sanofi-Pasteur used this method with a mechanism called Elicite HAI+ antibodies [46, 47, 48, 49].
More specifically, vaccination of mice with H1N1-based COBRA candidates resulted in broad HAI activity against a panel of 17 H1N1 virus strains. Furthermore, when inoculated mice were challenged, there was little or no detectable viral replication, as found in animals immunized with a matching approved vaccine [46]. Similarly, previous studies describing the design and generation of H5N1-based COBRA found that mice, ferrets, and nonhuman primates (Cynomolgus macaques) vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had higher HAI antibody titers recognizing different isolates representing divergent subclades [44, 49].
Aside from the COBRA-based strategy, there are several potential vaccines targeting the HA head. Song et al. [50] demonstrated the production of a fusion protein comprised of the globular HA head domains (HA1–2, spanning amino acids 62–284) from H7N9 and the Salmonella typhimurium flagellin (fliC) produced in
Internal influenza virus proteins are often highly conserved, making them viable targets for a universal vaccination. Although these proteins are rarely detected on virions or cell surfaces, making them inaccessible to antibodies, they are abundant in infected cells, where they are also processed and presented to T cells through major histocompatibility complex molecules. T cells have therefore been proven to play a significant role in influenza virus immunity. In this approach, NP and M1 have been widely studied as possible targets for universal T cell–based vaccine. Virus-based and DNA vaccination approaches have been shown in animal models to induce protective immune responses, and they are now being studied in a variety of clinical trials [41]. Over two consecutive influenza seasons, Evans et al. [51] conducted a phase 2b, randomized, placebo-controlled, double-blind trial of a recombinant viral-vectored vaccine (modified vaccinia Ankara expressing virus nucleoprotein and matrix protein 1; MVA-NP + M1), which has been shown to induce both CD4 and CD8 T cells, at eight outpatient clinical trial sites in Australia. They wanted to see if generating extra responses to conserved CD4 and CD8 T-cell antigens improves routine influenza vaccination. Based on their findings, they concluded that MVA-NP + M1 was well tolerated, with no vaccine-related major side effects. When administered within 28 days of normal QIV immunization, a vaccine intended to stimulate modest T-cell responses to cross-reactive internal proteins of influenza A did not result in an increase in incidence.
Finally, another notable vaccine technique is the epitope-based Multimeric-001 (M-001) candidate vaccine, which is now being tested in clinical trials. This vaccine, initially published by Ben-Yedidia et al. and later produced by BiondVax Pharmaceuticals Ltd., is made up of B- and T-cell epitopes taken from influenza A and B strains, containing nine conserved epitopes from the HA (including the globular head), NP, and M1 proteins [38, 52, 53]. To compensate for M-001 peptides’ poor immunogenicity and expensive cost, the epitopes are concatenated in triplicate into a single recombinant protein generated in E. coli. M-001 has been evaluated in both preclinical and clinical research, and it has been shown to protect mice against infection with various influenza strains while also being safe and generating both B- and T-cell specific immune responses [38, 53]. However, M-001 alone does not elicit HAI antibodies, which can only be generated when M-001 is followed by a boosting with seasonal or pandemic strain specific vaccinations [54].
Influenza virus infection is a continuing health and economic burden that causes epidemics with pandemic potential, impacting 5–30% of the world population each year and resulting in millions of hospitalizations and thousands of fatalities. Because of its great vulnerability to antigenic variation, influenza A is the type most responsible for pandemics. Influenza is extremely infectious, with symptoms including fever, cough, chills or sweats, myalgias, and malaise. The hypervariability of the amino acid sequences encoding HA and NA is primarily responsible for epidemic and pandemic influenza epidemics, which are the result of antigenic drift or shift. As a result, research on an effective broadly-reactive influenza vaccine capable of providing protection against both seasonal and pandemic influenza is now underway.
The authors declare no conflict of interests.
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\n\nThe Social Media Community Manager and Marketing Assistant will report to the Senior Marketing Manager. They will work alongside the Marketing and Corporate Communications team, supporting the preparation of all marketing programs, assisting in the development of scientific marketing and communication deliverables, and creating content for social media outlets, as well as managing international social communities.
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\n\nNote: This full-time position will have an immediate start. In your cover letter, please indicate when you might be available for a block of two hours. As part of the interview process, all candidates that make it to the second phase will participate in a writing exercise.
\n\n*IntechOpen is an Equal Opportunities Employer consistent with its obligations under the law and does not discriminate against any employee or applicant on the basis of disability, gender, age, colour, national origin, race, religion, sexual orientation, war veteran status, or any classification protected by state, or local law.
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A viral disease can be defined as an infectious disease that has recently appeared within a population or exists in nature with the rapid expansion of incident or geographic range. 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The combination of electronics and computer science with biology and medicine has improved patient diagnosis, reduced rehabilitation time, and helped to facilitate a better quality of life. Nowadays, all medical imaging devices, medical instruments, or new laboratory techniques result from the cooperation of specialists in various fields. The series of Biomedical Engineering books covers such areas of knowledge as chemistry, physics, electronics, medicine, and biology. 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The applications of this research cover many related fields, such as biotechnology and medicine, where, for example, Bioinformatics contributes to faster drug design, DNA analysis in forensics, and DNA sequence analysis in the field of personalized medicine. Personalized medicine is a type of medical care in which treatment is customized individually for each patient. Personalized medicine enables more effective therapy, reduces the costs of therapy and clinical trials, and also minimizes the risk of side effects. Nevertheless, advances in personalized medicine would not have been possible without bioinformatics, which can analyze the human genome and other vast amounts of biomedical data, especially in genetics. The rapid growth of information technology enabled the development of new tools to decode human genomes, large-scale studies of genetic variations and medical informatics. 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We encourage the submission of manuscripts that provide novel and mechanistic insights that report significant advances in the fields. Topics can include but are not limited to: Biotechnology such as biotechnological products and process engineering; Biotechnologically relevant enzymes and proteins; Bioenergy and biofuels; Applied genetics and molecular biotechnology; Genomics, transcriptomics, proteomics; Applied microbial and cell physiology; Environmental biotechnology; Methods and protocols. Moreover, topics in biosensor technology, like sensors that incorporate enzymes, antibodies, nucleic acids, whole cells, tissues and organelles, and other biological or biologically inspired components will be considered, and topics exploring transducers, including those based on electrochemical and optical piezoelectric, thermal, magnetic, and micromechanical elements. 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