First Record of HAdV-D20 Among Keratoconjunctivitis Patients in Iraq

Background: Human Adenovirus species D (HAdV-D) was common human viral pathogen especially in eye infection, consists of several types of which HAdV-D8, -D19 and –D37 were common in eye infection. This study includes detection of HAdV-D types implicated in conjunctivitis based on L2 (Penton protein) gene similarity. Methods: Conjunctival swabs were collected from Keratoconjunctivitis patients as eye infection related to adenovirus. Viral nucleic acids were extracted and specific primer pairs for HAdV-D L2 gene (encoding for penton base protein) was used to amplify the target gene and only positive samples were sent to sequencing. Results: The results revealed that only 6 samples give positive results for L2 gene amplification and then sent for sequencing for L2 (penton protein) gene-based typing. The results show that 4 local isolates (S1, S2, S3, S6) were similar to HAdV-D8 and 2 local isolates (S4, S5) were similar to HAdV-D20. Also the results display that the HAdV-37, prominent HAdV-D type of human eye infection, may be variant of HAdV-D20 due to that six variation were seen in S4and S5 local isolates nucleotide sequence in relation to HAdV-D37: T>C at position 14364, A>C at position 14411, T>C at position 14427, C>A at position 14448, G>A at position 14540 and T>C at position 14617, leading to only 2 amino acid change in resulted penton protein: T (Threonine) instead of K (Lysine) at position 204 and N (Asparagine) instead of D (Aspartic acid) at position 247. Conclusions: The current study concludes the possibility of implication of HAdV-D20 in eye infections especially conjunctivitis.

Penton base is one of the outer surfaces of viral major capsid proteins (penton-hexonfiber) contributed to antigenicity and utilized in descriptions and categorization of new recombinant strains of the HAdV (8,9). Phylogenetic analysis also can be achieved using penton base for type discrimination and assigning (10). The penton base play a vital role in adenovirus cell entry via loops of RGD that extend from penton base bind to α-vβ3 or α-vβ5 integrins. So, the tissue tropism and infection selectivity may mainly depend upon penton base variant and so the HAdV-D typespecificity will determine the type of infection (11,12). The objective of current study for investigating Human adenovirus species D type based on Penton base (L2) gene sequence variation.

Materials and Methods
One hundred patients referred to AL-Hilla teaching hospital /Babylon, Al-Imam AL-Hussein medical city hospital/Kerbela, and AL-Hakeem teaching hospital/AL-Najef from December 2018 to June 2019 were recruited for this study. All were examined by experienced ophthalmologists and diagnosed with kerato-conjunctivitis. Conjunctival swabs were collected, were inserted to viral transport media, and stored at -80 ºC. the number of cells increases logarithmically.

Ethical Approval
Informed consent was obtained from all adult participants or parents or legal guardians of minors.

Polymerase chain reaction (PCR)
Viral nucleic acid extraction has been accomplished with the use of the (FavorPrep Viral Nucleic Acid Extraction Kit II) (Cat. No.: FAVNK 002 (50 Preps) according to instructions of manufacturer (Favorgen/Tiwan). Viral nucleic acids were electrophoresed by 0.7% agarose gel electrophoresis and visualized by Gel Documentation system QUANTUM-ST5 (Vilber/France) to check the extracted nucleic acid (13). Conventional PCR for L2 gene (Penton protein gene) was accomplished using Forward: TTCGCAAGAAGCAACCTTT and Reverse: TCTTGCATGAGGTCCGG (14).
Concern implication of HAdV-D20 in Human infection we did not find any research whose document it accepts one study examine virus-spread ability in cell line which find that the HAdV-D20 can propagate successfully in HEK293 cells (Human embryonic kidney 293 cells) (25). Also, HAdV-D20 was showed to be associated with HIV/AIDS and was detected in the samples of stool from the patients who have AIDS with diarrhea, pneumonia, or both (26).
The current study concludes the possibility of implication of HAdV-D20 in eye infections especially conjunctivitis.