Tasnia Ahmed,Syed Abdullah Ibn Asaduzzaman,Rehman Tanvir,Zobayda Nahar,Nishat Tasnim,Chandrika Saha,Rashed Noor＊

1Department of Microbiology,Stamford University Bangladesh,51 Siddeswari Road,Dhaka 1217,Bangladesh; 2Department of Life Sciences (DLS),School of Environment and Life Sciences (SELS),Independent University,Bangladesh (IUB),Plot 16,Block B,Aftabuddin Ahmed Road,Bashundhara R/A,Dhaka 1212,Bangladesh.

Key words:Chikungunya virus infection,Re-emerging diseases,Public health,Host immunity

Background

Chikungunya fever is already widely known to generate from the entry of the Chikungunya virus (CHIKV) into the hosts through the bites ofAedesmosquitoes (Aedes aegyptiandA.albopictus) [1].After the first case of Chikungunya encountered in Tanzania (in 1952); the fever re-emerged in Kenya (2004),Indian Ocean islands(2005),Cameron (2006),Gabon (2007),Sri Lanka(2008),India and Singapore (2009),Franceville (2010),and in Bangladesh (2017) [2,3].Chikungunya fever has already been well distinguished worldwide by the onset of arthralgia (the peripheral joint pains and the muscle aches) [4–6].Phylogenetic studies played an essential role in understanding the epidemiology of CHIKV [7–9].Interestingly,as stated above,the CHIKV and the Zika virus (ZIKV),mainly transmitted byA.aegyptimosquitoes,are decidedly pathogenic arthropod-borne viruses,co-circulating in the same geographical regions[10,11].However,the mechanism behind such coinfection by CHIKV and ZIKV is still to be investigated[10].In general,CHIKV has been reported to be introduced into Asia from Africa [7].The phylogenetic studies conducted by Lo Presti and colleagues in 2014 showed that the CHIKV epidemic within the Indian subcontinent was instigated by 2 distinct strains of East/Central/South African genotype of CHIKV; and the virus isolates were categorized into 3 distinct genotypes based on their geographical origins: the West Africa genotype (Senegal and Nigeria),the East/Central/South African genotype,and the Asian genotype [7].In Bangladesh,this virus was first introduced as a sporadic infection; and then re-emerged as a foremost lethal outbreak in 2017,resulting in infection within 10% of the total population of the Dhaka division alone.

Moreover,since there is no specific treatment in the developing countries as in Bangladesh,prevention is the most critical part for the protection against CHIKV infection.In the current review: 1) the genomic status of the CHIKV has been portrayed along with; 2) the disease manifestations in accordance with the host protective immunity against it.Site-specific clinical conditions with different physiological outputs have been diagrammatically elucidated for a better understanding of the health professionals.The methods to detect the CHIKV infection in the patients; and the preventive measures have also been included in the last part of this article.

CHIKV genome

The CHIKV genome consists of 11,811 nucleotides[12].The full genome contains two separate open reading frames between the 3ʹ and 5ʹ untranslated region (UTR) each consisting of 7,422 and 3,744 nucleotides,respectively.Another UTR region of 65 nucleotides separates both open reading frames.The larger open reading frame encodes the non-structural proteins (nsP1,nsP2,nsP3,and nsP4) (Figure 1),and the other open reading frame encodes the structural proteins (C,E1,E2,E3) and a small peptide (6K) [12–14].The structural proteins are involved in the replication process for making the structural components of the progeny viruses.C is the capsid protein that forms the virus coat enclosing the RNA genome [14].The small peptide 6K that aids in the release of pE2E1 complex (2 regions separated by 6K,E2 and E1 work together to form spike proteins) from the Golgi apparatus.E2 and E3 stay together as pE2.While leaving the Golgi apparatus,E3 protein dissociates from the pE2 complex,and forms the fusible spike protein together with E1E2 which finally displays on the host cell membrane.These spikes form a coat outside the capsid of the virus during exocytosis aided by cellular cytoskeletons-microtubules which in the later period of the infection get oriented in the apical end of the cell forming the typical round shape [15,16].

Figure 1 Genomic structure of CHIKV.

Replication of CHIKV within the human host cell

As stated earlier,the CHIKV enters into the human body via the infective bite of anAedesmosquito [17].After invading the dermis,the viruses adhere to fibroblasts and start replicating inside the host cell [18–20].After the release of new virus particles,they disseminate themselves and adhere to other fibroblasts,and the scenario continues until the host cells release the new virus particles into the blood vessels.Once inside the blood vessel,the virus attaches to the monocytes,and after entering the monocytes,the new replicative cycle starts.Thus,the replication continues,and the released progeny virus head towards the kidney,lymph nodes,brain,and the joints at the periphery of the blood circulation (Figure 2); and the consecutive symptoms of Chikungunya infection launch [21].At first,CHIKV particle adheres to the host cell receptors like phosphatidylserine (PtdSer)-mediated virus entryenhancing receptors,prohibitin,glycosaminoglycans,β1 integrin dimmer,integrin αV [22].The host cell then internalizes the virus attached to the receptor by clathrin-mediated endocytosis [23–25].Once inside the cytoplasm after its release from the endosome,translation occurs subsequently [4,23].The capsid and the 49S RNA assemble,making a new virus particle.This particle is released from the host cell by exocytosis,where the spike protein fuses with the host cell membrane coat,thereby making the viral envelop(Figure 3) [21,26].

Figure 2 Entry and dissemination of CHIKV.

Figure 3 Viral replication inside host cell (fibroblast/monocyte).

Vector and transmission

During the first encounter,Aedesaegyptimay transmit the virus in the forest areas to nonhuman primates both of which can serve as reservoirs (like bats and rodents)of the virus [27,28].However,the virus can be further propagated into the human hosts,the cycle continues,and the human-mosquito-human transmission spreads to the adjacent areas.The femaleAedesaegyptiare known as daytime (especially early morning) biting mosquitoes staying in indoor (stagnant water of flower vase,water tanks,etc.) [6,29].They have easier access to the human host to bite.On the other hand,Aedes albopictus(with higher biting frequency in late afternoon) can also take part in active transmission of CHIKV [6,30,31].These mosquitoes are the common inhabitants of the stagnant waters within the vehicle tyres left abandoned [6,32].

Pathogenesis

Bone destruction

In bones there are osteoclasts,responsible for resorption which results in the breakdown of bone tissue releasing minerals like calcium into the blood [33].The other type of cell present in the bone is osteoblast which may act by secreting the bone matrix.Firstly,CHIKV infects the osteoblast cells,resulting in the production of interleukin 6 (IL-6) and IL-1β which in turn enhance the proliferation of multinucleated monocytes,thereby acting as the progenitors to produce more osteoclasts[34].The infected cells also produce receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG) [35].In the case of CHIKV infection,OPG is produced less than RANKL,and the free RANKL binds with the monocytes enabling them to proliferate into the osteoclast (osteoclastogenesis).The more the osteoclasts form,the more resorption of the bone occurs,causing bone destruction and weakening [35,36].They also activate the CD8+T cells producing more RANKLs,and the CD4+T cells,which further differentiate into the Th17,and thus raising the RANKL blast.The Th17 type helper T cells also produce IL-17 which is an inflammatory cytokine produced during rheumatoid arthritis [36].The cartilage immune complexes are the causes of irreversible cartilage damage of the joints (Figure 4).

Involvement with eye

Recent progresses in molecular diagnostics revealed an expanded array of virus (especially from the Herpes family) mediated intraocular inflammation [37].CHIKV particles along with the ZIKV ones,sometimes pose a set of clinical complications known as the congenital ocular syndrome [38].Due to the CHIKV infection,overabundances of ocular manifestations(conjunctivitis,retinal detachment,optic neuritis,iridocyclitis,episcleritis,uveitis,blurred vision,increased intraocular pressure,subconjunctival hemorrhage,corneal edema,cotton wool spot,vascular occlusion retinal whitening,photophobia,etc.) have been reported [39].

Involvement with liver and brain

CHIKV can be disseminated throughout the body via blood circulation,and can initiate damage in distal body parts like liver,and even the brain.During energy metabolism,nitrogenous metabolites are produced in liver from the urea cycle which can initiate inflammation,resulting in arthritis.These nitrogenous metabolites can travel through the blood circulation and eventually pass the blood-brain barrier causing stress in brain which can initiate neuro-degenerative tissue damage,apoptosis,and encephalopathy (Figure 5).

Figure 4 Mechanism of bone destruction and development of arthritis after CHIKV infection.

Problems in pregnancy

CHIKV is known to be one of the responsible causes of miscarriage during the early stage of the pregnancy [40].The virus can also be transmitted to the baby during the later stages of pregnancy.During delivery,if the mother has active viremic condition,the neonate may develop CHIKV infection by vertical transmission.Otherwise,mother to fetus transmission is a rare condition [41].In recent studies,CHIKV has been detected in breast milk,and it can be transmitted to the infants via breast feeding.If the natural protective immunity is compromised,the virus may cause infection in infants[42,43].

Besides the clinical complications described above,the co-infections mediated by ZIKV and CHIKV have been reported in cases of all ages [12,44].CHIKV infection can resolve leaving the lifelong immunity for the same virus [46].However,recently the mortality has been documented in several studies [47].An exemplary occurrence of the 2006 epidemic in India is significant,in which more than 1,500,000 cases of CHIKV cases were reported [48–50].

Immunity against CHIKV

Figure 5 Incidents after CHIKV infection in liver and brain.

No accredited vaccines or antivirals against CHIKV have been developed so far.However,a recent study employing mice delivered a bit hopeful result where the mixture of CHIKV like particle with different adjuvants were found to enhance the protective immunity [51].Moreover,some antivirals could be developed which were also found to effective against CHIKV [51,52].After getting infected with CHIKV particles,the host cell translation is actually closed down which in turn triggers the elevated production of IL-4,IL-6,IL-10 and the interferon-β [4,53].IFN type 1 is known to generate signals to the infected cells thus being the source of antiviral proteins [54,55].The natural killer (NK) cells are vastly known to sense the CHIKV infected cells with the aid of the variety of IL signals with subsequent cytotoxicity [56].Human leukocyte antigen class 2 molecule containing cells are known to be more prone to CHIKV infection; and the activated NK cells specifically bind with the human leukocyte antigen class 2 receptor containing infected host cells with their killer immunoglobulin like receptor [57].Eventually,the NK cells kill the infected cells by cellular lysis [58].Th1 cytokines activate the CD8+T cells during the early stages of the CHIKV infection.Literally,the CD8+T cells are likely to regulate the viral infection which is mediated after their attachment with the MHC class I molecule present in all antigen presenting cells.Such a process is already known to be very important for intracellular viral killing.Consequently,in the later stage of the CHIKV infection,the CD8+T cells subside,and CD4+T cells get activated after adhering to the MHC class II molecule on antigen presenting cells [59–62].

Risk factors of CHIKV infection

The prevalence of psychological distress and arbovirus symptoms has been well discussed to date [65,66].As stated above,since the first identification of CHIKV in 1950 in Tanzania,the CHIKV infection has commonly been characterized as a sudden onset of fever associated with joint pains.Such conditions can become serious if the patients already have underlying medical conditions.Several underlying factors including age,gender,immune status,genetic predisposition,bullous dermatosis,pneumonia,diabetes,hypertension,and the underlying respiratory or cardiological conditions have been reported to be associated with the extent and severity of the CHIKV infection [51,65].In a study,children below 15 years old were noticed to have a lower tendency of developing skin rash,itching and vomiting upon CHIKV exposure compared to the higher age category who have been found to develop a significantly higher frequency of headache [65].Another factor for developing Chikungunya may be based on travelling across the areas affected by an ongoing CHIKV epidemic [8].

State of CHIKV infection in Bangladesh

According to the statistical research conducted by International Centre for Diarrheal Disease Research,Bangladesh,and the Institute of Epidemiology,Disease Control and Research (IEDCR),the first encounter of CHIKV in Bangladesh was noticed in the south-western part in 2008 [63].The second outbreak occurred in Pabna in the same year [4].In 2011,IEDCR and International Centre for Diarrheal Disease Research,Bangladesh estimated more than 1110 CHIKV infected patients [64].The drastic increase of the Chikungunya attack was in 2017,resulting in nearly 3000 cases [65].IEDCR declared 23 districts of Dhaka division to be at high-risk area for CHIKV infection as well as approximately 10% people of Dhaka (18 million) to be in the risk of contracting CHIKV infection [66–68].Indeed,in perspective of Bangladesh and some other underprivileged countries with resource poor settings,the appropriate diagnosis as well as the prevention measures should be taken as discussed later.

Diagnosis of CHIKV infection

CHIKV diagnosis followed by the preventive measures is undoubtedly significant in terms of public health management as in case of the persistent flu epidemic and the emerging dengue infections [69–71].Besides CHIKV infection,the propagation of dengue virus,CHIKV and ZIKV also demands notification for the sake of confirmative epidemiological conclusions as well as to detect the co-infection [69,70].Indeed,the diagnosis of CHIKV appears to be critical when ZIKV and dengue virus are concurrently in attendance [69,70].The serum sample from the suspected patients after 5–7 days of infection is subjected to the serological test whereby the serum immunoglobulins are determined[4].Active CHIKV proliferation is understood from the presence of high titer of IgM as well as from the enzyme linked immunosorbent assay and the real time RT-PCR[69,72].Indeed,in the light of COVID-19 bioinformatics analysis and sensitive diagnosis,the possible incidence and the required advanced diagnosis methods like the nested RT-PCR,annotating the nucleotide sequences within the GenBank using BLAST program,the immunofluorescence assay of viral antigens,etc.are required to detect the CHIKV infection more precisely [4,10,70–76].Besides,as studied in case of the ongoing COVID-19 pandemic,the orchestration of host protective immunity against CHIKV infection needs to be well understood in order to design the specific counterattack against CHIKV[72–75].Besides,the repurposed or repositioned drug usage,the most effective vaccine has been found to be the TSI-GSD-218,which has already passed the Phase II clinical trial.Moreover,the ongoing phase II clinical trial vaccines VRC-CHKVLP059-00-VP and MVCHIK also appear to be effective in treating the CHIKV infection [76].

Conclusion

It’s worth to note that there are lots of comprehensive,valuable reports on CHIKV infection; and moreover,the present global situation really demands the extensive study on combating the COVID-19 pandemic.However,the current review on the molecular biological facets of CHIKV would still benefit the global public health sector due to the re-emerging trait of the virus.