**8. In-vivo models for epidemic and pandemic diseases**

The existing models using live bacterial infusion, feeding fungal strains and transgenic flies expressing viral proteins. Under immuno-suppressed condition would serve multiple purposes like studying host pathogen interaction and conducting preclinical trials [62, 66].

### **8.1 HIV models**

Since human viruses do not usually invade insects the use of *Drosophila melanogaster* as a model organism is critical and currently in less usage [67]. In order to establish an HIV model for drug screening in *Drosophila melanogaster* it is important to understand the structural components of human immunodeficiency virus (HIV). The envelope components are comprised of Gp120 and Gp41 encoded by *env* sequence, pol-Gag RNA material encodes for *Matrix, capcid, nuclear capsid, p6, Protease, reverse transcriptase, and Integrase), Vif, Vpr, Nef, vpu, tat,* and *Rev* [68]. Transfection of Tat (transcription activator), Vpu (helps virion budding), Nef (regulator of structural gene expression) and Rev. (Nuclear export protein) in flies (in-vitro/in-vivo) were previously shown, these transfection models could be useful due to the fact that there is no marketed drug to target these viral proteins [69].

The incapacity of Drosophila S2 cells is only associated with the expression of HIV-1 envelope proteins. It is possible to express gycosylated and cleaved Gp120 in S2 cells but fusion with CD4+ receptors of T-helper cells could not be achieved in the model expression system [70]. In another study the expression of Gp120 in drosophila was carried out in S2 cell line, the antigen Gp-120 did not exhibited T-helper cell mediated humoral immune system activation and IgG antibody generation,

when introduced in mice [71]. Due to this usual challenge in a different study they expressed HIV-1 virus like proteins in Drosophila S2 cells [72].

The nef transgenic flies exhibited JNK mediated apoptosis further nef inhibits NF-kB necessary for Relish gene activation similarly decreased immune response is common in AIDS patients [73]. In a study transfected viral protein Vpu was shown to cause immune suppression in fat body of flies via toll dependent pathway, in wings the Vpu expression caused apoptosis and hindered wing development, in mammals Vpu is known for causing T-cell lymphocyte death in infected patients [74, 75]. Active microbial invasion in nef flies should be further confirmed before targeting with potent anti-nef drug candidate. The *Rev* transfected S2 cells revealed that expression of Rev. protein directed the translocation of viral mRNA sequence into the cytoplasm, blocked by leptomycin B a secondary metabolite of *Streptomyces* species [76]. Leptomycin B remained unapproved in clinical trials due to high toxicity in cancer patients [77]. The ART drugs like zidovudine, lamivudine, stavudine, didanosine and abacavir were introduced in *D. melanogaster* to study genotoxicity profile [78, 79]. In Drosophila oocytes Tat a nuclear shuttling protein, displayed interaction with tubulin causing dorso-ventral axis mislocalization resulting in delayed microtubule polymerization, similarly tubulin dysfunction causes neurological symptoms observed in HIV+ individuals [80]. The transfected viral proteins in live Drosophila could be used to target drug in a thoughtfully designed model.

Cryptococcosis, Candidiasis and Aspergillosis are common types of fungal infections observed as clinical challenge in HIV-positive patients [81]. Under immunosuppressed condition the invasion of fungi in flies causes fatality. In Drosophila fungal infection could be difficult to achieve as the innate immune system mediates anti-fungal peptide production by haemocyte causing decrease of fungal load and increases fly survival rate [82]. Hence Toll mutant flies were generated and used to induce fungal infection.

Fluconazole and voriconazole showed anti-fungal activity against *Candida albicans* and *Aspergilus fumigatus* respectively in flies [64, 83]. Among *Cryptococcus* species only *Cryptococcus neoformans* is capable of killing flies with mutated toll receptors in drosophila, susceptible to infection acquired from *Cryptococcus* species like *Cryptococcus kuetzingii* or *Cryptococcus laurentii* [83]. Although, the toll mutant flies do not demonstrate an HIV model, it is used to induce fungal infection which can serve as a model for fungal infection in Drosophila for drug screening purpose.

In order to study HPV and EBV there are two model systems to study the effect in flies. In the study with HPV co-expression of viral oncoprotein E6 and human UBE3A did not resulted in tumorigenesis requires Ras or Notch pathway in flies, E6-UBE3A requires insulin receptors for cancer to develop [84]. Upon introducing the BZLF1 gene of EBV led to interaction with shaven gene in flies a homolog of pax gene family of humans responsible for B-cell development [85]. Expression of BRLF1 and BZLF1 genes using *GMR-R* model in Drosophila showed BRLF1 caused overproliferation of cells in flies whereas, BZLF1 resulted in interaction with several tumor suppressor genes and both viral genes showed interactions with core tumor suppressor genes like *reaper, p53, Rab5*, and *Tor* [86]. EVB DNA injection in flies caused Imd mediated pathway to increase diptericin production at the same time hemocyte proliferation and remarkable increase in numbers of hemocyte cells [87]. Human cytomegalovirus derived immediate early gene transfection in flies resulted in embryonic lethality similar to humans [88].

#### **8.2 Influenza infection models**

Influenza virus like most other viruses fails to infect the *Drosophila melanogaster*. To construct a suitable model for drug screening was also an important aspect due

**115**

Drosophila melanogaster: *A Robust Tool to Study Candidate Drug against Epidemic…*

to high mortality rate caused by flu virus. The influenza virus coat protein consists of hemagglutinin (HA) and neuraminidase (NA). Matrix protein 2 (M2) plays a vital role in maintaining the pH level through proton transport enabling viral uncoating. Expression of M2 protein in flies is achieved through insertion of M2 cDNA sequence in upstream activation sequence (UAS) of pCaSpeR3 p-element insertion vector gave rise to UAS-M2 flies. The crossover between UAS-M2 and C135-Gal4 flies resulted in death at the pupal stage. Therefore the larvae were exposed and not the adult flies to anti-influenza drug amantidine which is a M2 antagonist. Amantadine and several other drugs of its class are not capable of acting against the flu virus due to varying viral strain types. Moving further the flu-fly model of UAS-M2 could be used to study potential anti-influenza drug [56].

There are several pneumococcal pneumonia infection model studied in drosophila using *Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa* and *Klebsiella pneumonia.* The biofilm formation is widely observed during *Streptococcus pneumoniae* infection in humans [89]. The nasopharyngeal tract is colonized primarily by these gram positive rods bacteria prior to infecting the lower respiratory tract. Similarly the *Pseudomonas aeruginosa* exhibit the biofilm formation during nasocomial infection in humans, a common culprit causing community acquired pneumonia hospitalized in patients [57, 90]. *Pseudomonas aeruginosa* was shown to infect *Drosophila melanogaster* causing its gut epithelium inflammation [57]. *Staphylococcus aureus* causes osteomyelitis, endocarditis, septicaemia and pneumoniae in humans, it can be selected for mimicking pneumoniae infection in Drosophila [91]. *Staphylococcus aureus* caused rapid death of flies within 48 h due to inoculation of high lethal dosage, extended survival seen upon exposure to antibiotic (tetracycline) [92]. The teichoic acid of peptidoglycan layer in *Staphylococcus aureus* was found to suppress the toll receptors of flies similar to *Streptococcus pneumoniae* toxins autolysin and pneumolysin interacts with toll receptors of macrophages in human [93–95]. *Klebsiella pneumonia* the gram positive bacteria are capable of killing drosophila at higher dose [96]. *Streptococcus pneumoniae* causes the maximum deaths in human causing pneumoniae which could be used as a suit-

There are at present two bacterial models for studying mycobacterium infection in flies, induced by *Mycobacterium marinum* and *Mycobacterium abscessus*. *Mycobacterium marinum* is a non spore forming, non motile, gram positive acid-fast bacillus, which is genetically 99.3% similar *to Mycobacterium tuberculosis* [97, 98]. Vacuole acidification is inhibited by *M. marinum* in drosophila phagocytic cells has been previously identified to be similar with tuberculosis pathogenesis in humans [99, 100]. Tigecycline plus linezolid was shown to have extended fly survival during the *Mycobacterium abscessus* infection. Rifampicin a very potential wide range antibiotics effective to inhibit multi drug resistance tuberculosis (MDRTb), it showed antimycobacterial efficacy in Drosophila infected with *Mycobacterium marinum* [101]. Any potential drug candidate

able model for antibiotic screening in *Drosophila melanogaster*.

capable of anti-mycobacterial activity can be studied in these models.

The bacteria *Vibrio cholerae* is a gram-negative and motile bacterium causes diarrheal disease in human. The pathogenesis of *Vibrio cholera* infection in humans

**8.4 Tuberculosis models**

**8.5 Cholera models**

*DOI: http://dx.doi.org/10.5772/intechopen.90073*

**8.3 Pneumococcal pneumonia models**

Drosophila melanogaster: *A Robust Tool to Study Candidate Drug against Epidemic… DOI: http://dx.doi.org/10.5772/intechopen.90073*

to high mortality rate caused by flu virus. The influenza virus coat protein consists of hemagglutinin (HA) and neuraminidase (NA). Matrix protein 2 (M2) plays a vital role in maintaining the pH level through proton transport enabling viral uncoating. Expression of M2 protein in flies is achieved through insertion of M2 cDNA sequence in upstream activation sequence (UAS) of pCaSpeR3 p-element insertion vector gave rise to UAS-M2 flies. The crossover between UAS-M2 and C135-Gal4 flies resulted in death at the pupal stage. Therefore the larvae were exposed and not the adult flies to anti-influenza drug amantidine which is a M2 antagonist. Amantadine and several other drugs of its class are not capable of acting against the flu virus due to varying viral strain types. Moving further the flu-fly model of UAS-M2 could be used to study potential anti-influenza drug [56].

#### **8.3 Pneumococcal pneumonia models**

*Animal Models in Medicine and Biology*

induce fungal infection.

in embryonic lethality similar to humans [88].

**8.2 Influenza infection models**

when introduced in mice [71]. Due to this usual challenge in a different study they

Fluconazole and voriconazole showed anti-fungal activity against *Candida albicans* and *Aspergilus fumigatus* respectively in flies [64, 83]. Among *Cryptococcus* species only *Cryptococcus neoformans* is capable of killing flies with mutated toll receptors in drosophila, susceptible to infection acquired from *Cryptococcus* species like *Cryptococcus kuetzingii* or *Cryptococcus laurentii* [83]. Although, the toll mutant flies do not demonstrate an HIV model, it is used to induce fungal infection which can serve as a model for fungal infection in Drosophila for drug screening purpose. In order to study HPV and EBV there are two model systems to study the effect in flies. In the study with HPV co-expression of viral oncoprotein E6 and human UBE3A did not resulted in tumorigenesis requires Ras or Notch pathway in flies, E6-UBE3A requires insulin receptors for cancer to develop [84]. Upon introducing the BZLF1 gene of EBV led to interaction with shaven gene in flies a homolog of pax gene family of humans responsible for B-cell development [85]. Expression of BRLF1 and BZLF1 genes using *GMR-R* model in Drosophila showed BRLF1 caused overproliferation of cells in flies whereas, BZLF1 resulted in interaction with several tumor suppressor genes and both viral genes showed interactions with core tumor suppressor genes like *reaper, p53, Rab5*, and *Tor* [86]. EVB DNA injection in flies caused Imd mediated pathway to increase diptericin production at the same time hemocyte proliferation and remarkable increase in numbers of hemocyte cells [87]. Human cytomegalovirus derived immediate early gene transfection in flies resulted

Influenza virus like most other viruses fails to infect the *Drosophila melanogaster*. To construct a suitable model for drug screening was also an important aspect due

The nef transgenic flies exhibited JNK mediated apoptosis further nef inhibits NF-kB necessary for Relish gene activation similarly decreased immune response is common in AIDS patients [73]. In a study transfected viral protein Vpu was shown to cause immune suppression in fat body of flies via toll dependent pathway, in wings the Vpu expression caused apoptosis and hindered wing development, in mammals Vpu is known for causing T-cell lymphocyte death in infected patients [74, 75]. Active microbial invasion in nef flies should be further confirmed before targeting with potent anti-nef drug candidate. The *Rev* transfected S2 cells revealed that expression of Rev. protein directed the translocation of viral mRNA sequence into the cytoplasm, blocked by leptomycin B a secondary metabolite of *Streptomyces* species [76]. Leptomycin B remained unapproved in clinical trials due to high toxicity in cancer patients [77]. The ART drugs like zidovudine, lamivudine, stavudine, didanosine and abacavir were introduced in *D. melanogaster* to study genotoxicity profile [78, 79]. In Drosophila oocytes Tat a nuclear shuttling protein, displayed interaction with tubulin causing dorso-ventral axis mislocalization resulting in delayed microtubule polymerization, similarly tubulin dysfunction causes neurological symptoms observed in HIV+ individuals [80]. The transfected viral proteins in live Drosophila could be used to target drug in a thoughtfully designed model. Cryptococcosis, Candidiasis and Aspergillosis are common types of fungal infections observed as clinical challenge in HIV-positive patients [81]. Under immunosuppressed condition the invasion of fungi in flies causes fatality. In Drosophila fungal infection could be difficult to achieve as the innate immune system mediates anti-fungal peptide production by haemocyte causing decrease of fungal load and increases fly survival rate [82]. Hence Toll mutant flies were generated and used to

expressed HIV-1 virus like proteins in Drosophila S2 cells [72].

**114**

There are several pneumococcal pneumonia infection model studied in drosophila using *Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa* and *Klebsiella pneumonia.* The biofilm formation is widely observed during *Streptococcus pneumoniae* infection in humans [89]. The nasopharyngeal tract is colonized primarily by these gram positive rods bacteria prior to infecting the lower respiratory tract. Similarly the *Pseudomonas aeruginosa* exhibit the biofilm formation during nasocomial infection in humans, a common culprit causing community acquired pneumonia hospitalized in patients [57, 90]. *Pseudomonas aeruginosa* was shown to infect *Drosophila melanogaster* causing its gut epithelium inflammation [57].

*Staphylococcus aureus* causes osteomyelitis, endocarditis, septicaemia and pneumoniae in humans, it can be selected for mimicking pneumoniae infection in Drosophila [91]. *Staphylococcus aureus* caused rapid death of flies within 48 h due to inoculation of high lethal dosage, extended survival seen upon exposure to antibiotic (tetracycline) [92]. The teichoic acid of peptidoglycan layer in *Staphylococcus aureus* was found to suppress the toll receptors of flies similar to *Streptococcus pneumoniae* toxins autolysin and pneumolysin interacts with toll receptors of macrophages in human [93–95]. *Klebsiella pneumonia* the gram positive bacteria are capable of killing drosophila at higher dose [96]. *Streptococcus pneumoniae* causes the maximum deaths in human causing pneumoniae which could be used as a suitable model for antibiotic screening in *Drosophila melanogaster*.

#### **8.4 Tuberculosis models**

There are at present two bacterial models for studying mycobacterium infection in flies, induced by *Mycobacterium marinum* and *Mycobacterium abscessus*. *Mycobacterium marinum* is a non spore forming, non motile, gram positive acid-fast bacillus, which is genetically 99.3% similar *to Mycobacterium tuberculosis* [97, 98]. Vacuole acidification is inhibited by *M. marinum* in drosophila phagocytic cells has been previously identified to be similar with tuberculosis pathogenesis in humans [99, 100]. Tigecycline plus linezolid was shown to have extended fly survival during the *Mycobacterium abscessus* infection. Rifampicin a very potential wide range antibiotics effective to inhibit multi drug resistance tuberculosis (MDRTb), it showed antimycobacterial efficacy in Drosophila infected with *Mycobacterium marinum* [101]. Any potential drug candidate capable of anti-mycobacterial activity can be studied in these models.

#### **8.5 Cholera models**

The bacteria *Vibrio cholerae* is a gram-negative and motile bacterium causes diarrheal disease in human. The pathogenesis of *Vibrio cholera* infection in humans was previously reported to be symbolized as comparable disease progression in *Drosophila melanogaster*. Ingestion of cholera bacterium results in lethal infection induced by the toxins in the intestinal cells of the flies. The toxins ingestion could not cause equivalent lethal effect on flies was explained previously. The *V. cholera* infection results in inhibition of adenylyl cyclase, Gsα, or the Gardos K+ channel causing death due to oral ingestion in flies. Clotrimazole a Potassium Calcium-Activated Channel Subfamily N Member 4 (KCNN4) inhibitor exposure increased flies susceptibility to *V. cholera* infection [61]. Quorum sensing is the ability to detect and to respond to a specific density of cell population through gene regulation [102]. *Drosophila melanogaster* initiates quorum sensing during vibrio cholera infection by suppressing succinate (substrate of KEBS cycle) uptake in flies intestine, limiting the wasting process [62]. Quorum sensing enables the bacterium to remain sessile in the flies gut and Vibrio polysaccharide (VPS) gene expression was shown to have increased during *V. cholera* infection of flies [103].
