Identification of SARS CoV-2 Omicron BA.1 and a novel Delta lineage by rapid methods and partial spike protein sequences in Sulaymaniyah Province, Iraq.

BACKGROUND: Five variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) have been globally recorded including Alpha, Beta, Gamma, Delta, and Omicron. The Omicron variant has outcompeted the other variants including the Delta variant. Molecular screenings of VOCs are important for surveillance, treatment, and vaccination programs. This study aimed to identify VOCs by using rapid inexpensive methods and partial sequencing of the virus's spike gene. METHODS: Mutation-specific rRT PCR probes were used for both D614G and K417N mutations to potentially discriminate between Delta and Omicron variants. These were followed by sequencing of a fragment of spike gene (748 nucleotides), which covers the most notable VOC mutations in the receptor binding domain of SARS CoV-2. RESULTS: Rapid methods showed that out of 24 SARS CoV-2 positive samples, 19 carried the N417 mutation, which is present in the Omicron variant. Furthermore, 3 samples carried K417 wildtype, which is present in the Delta variant. Additionally, 2 samples containing both K417 and N417 suggested mixed infections between the two variants. The D614G mutation was present in all samples. Among the 4 samples sequenced, 3 samples carried 13 mutations, which are present in Omicron BA.1. The fourth sample contained the two common mutations (T478K and L452R) present in Delta, in addition to two more rare mutations (F456L and F490S), which are not commonly seen in Delta. Our data suggested that both Omicron variant BA.1 and a novel Delta variant might have circulated in this region that needs further investigations.

Association of tumor necrosis factor alpha -308 single nucleotide polymorphism with SARS CoV-2 infection in an Iraqi Kurdish population.

Uncovering risk factors playing roles in the severity of Coronavirus disease 2019 (Covid-19) are important for understanding pathoimmunology of the disease caused by severe acute respiratory syndrome Coronavirus 2 (SARS CoV-2). Genetic variations in innate immune genes have been found to be associated with Covid-19 infections. A single-nucleotide polymorphism (SNP) in a promoter region of tumor necrosis factor alpha (TNF-α) gene, TNF-α -308G>A, increases expression of TNF-α protein against infectious diseases leading to immune dysregulations and organ damage. This study aims to discover associations between TNF-α -308G>A SNP and Covid-19 infection. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping a general Kurdish population and Covid-19 patients. The homozygous mutant (AA) genotype was found to be rare in the current studied population. Interestingly, the heterozygous (GA) genotype was significantly (p value = 0.0342) higher in the Covid-19 patients than the general population. This suggests that TNF-α -308G>A SNP might be associated with Covid-19 infections. Further studies with larger sample sizes focusing on different ethnic populations are recommended.

Association of Toll-like receptor-4 polymorphism with SARS CoV-2 infection in Kurdish Population.

Genetic variations are critical for understanding clinical outcomes of infections including server acute respiratory syndrome coronavirus 2 (SARS CoV-2). The immunological reactions of human immune genes with SARS CoV-2 have been under investigation. Toll-like receptors (TLRs), a group of proteins, are important for microbial detections including bacteria and viruses. TLR4 can sense both bacterial lipopolysaccharides (LPS) and endogenous oxidized phospholipids triggered by Covid-19 infection. Two TLR4 single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile have been linked to infectious diseases. No studies have focused on these SNPs in association with Covid-19. This study aims to reveal the association between Covid-19 infection with these SNPs by comparing a group of patients and a general population. Restriction fragment length polymorphisms (RFLP) were used to identify the TLR4 SNPs in both the general population (n = 114) and Covid-19 patient groups (n = 125). The results found no association between the TLR4 polymorphisms and Covid-19 infections as the data showed no statistically significant difference between the compared groups. This suggested that these TLR4 SNPs may not be associated with Covid-19 infections.

Association of Apolipoprotein e polymorphism with SARS-CoV-2 infection.

Coronavirus 2019 (COVID-19) is a viral disease caused by severe acute respiratory syndrome coronavirus-2 (SARS CoV-2). The disease resulted in global morbidity and mortality that led to considering as pandemic. The human body response to COVID-19 infection was massively different from being asymptomatic to developing severe symptoms. Host genetic factors are thought to be one of the reasons for these disparities in body responses. Few studies have suggested that Apolipoprotein Epsilon (Apo E) is a candidate gene for playing roles in the development of the disease symptoms. This work aims to find an association between different Apo E genotypes and alleles to COVID-19 infection comparing a general population and a group of COVID-19 patients. For the first time, the results found that Apo E4 is associated with COVID-19 disease in a Kurdish population of Iraq. Further study is required to reveal this association in different ethnic backgrounds all over the world.

Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation.

A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in the impact of this mutation on the virus's infectivity. Also, the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutants indicated by the presence of HEX probe and 176 bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of the G614 mutants. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.

First molecular identification of Leishmania major in Phlebotomus papatasi in an outbreak cutaneous leishmaniasis area in Iraq.

Cutaneous leishmaniasis (CL) is transmitted by Phlebotomine sand fly vectors, among which Phlebotomus papatasi is prevalent in Western Asia, Northern Africa and Southern Europe, and it is known as a vector for Leishmania major parasite in the world. However, in Iraq, morphological studies showed that P. papatasi is a predominant sand fly species and hypothesised to transmit CL causing Leishmania species including L. major and L. tropica. Few studies have found Leishmania species in sand flies in mixed pools of samples in this country. Accurate identification of sand flies as vectors of Leishmania species is required in Iraq. The current study aims to identify sand fly species, using both morphological and molecular phylogenetic analyses, in a region where CL tends to be endemic. Furthermore, molecular phylogenetic analysis has also used to confirm Leishmania species in the sand fly samples collected in 11 villages between Diyala and Sulaymaniyah Provinces. For the first time, we have found L. major in three individual sand flies, one engorged (with fresh blood meal) and two non-engorged (without visible fresh blood meal) P. papatasi females in an area of CL outbreaks since 2014-till now due to civil wars and internal conflicts happen in the region. Further study should be performed on sand fly population and Leishmania reservoirs in this region.

Gene expression of AvBD6-10 in broiler chickens is independent of AvBD6, 9, and 10 peptide potency.

The Avian ß-defensin (AvBD) gene cluster contains fourteen genes; within this, two groups (AvBD6/7 and AvBD8 -10) encode charged peptides of >+5 (AvBD6/7), indicative of potent microbial killing activities, and ≤+4 (AvBD8-10), suggestive of reduced antimicrobial activities. Chicken broiler gut tissues are constantly exposed to microbes in the form of commensal bacteria. This study examined whether tissue expression patterns of AvBD6-10 reflected microbial exposure and the encoded peptides a functional antimicrobial hierarchy. Gut AvBD6-10 gene expression was observed in hatch to day 21 birds, although the AvBD8-10 profiles were eclipsed by those detected in the liver and kidney tissues. In vitro challenges of chicken CHCC-OU2 cells using the gut commensal Lactobacillus johnsonii (104 CFU) did not significantly affect AvBD8-10 gene expression patterns, although upregulation (P < 0.05) of IL-Iß gene expression was observed. Similarly, in response to Bacteriodes doreii, IL-Iß and IL-6 gene upregulation were detected (P < 0.05), but AvBD10 gene expression remained unaffected. These data suggested that AvBD8-10 gene expression was not induced by commensal gut bacteria. Bacterial time-kill assays employing recombinant (r)AvBD6, 9 and 10 peptides (0.5µM - 12µM), indicated an antimicrobial hierarchy, linked to charge, of AvBD6 > AvBD9 > AvBD10 against Escherichia coli, but AvBD10 > AvBD9 > AvBD6 using Enterococcus faecalis. rAvBD10, selected due to its reduced cationic charge was, using CHCC-OU2 cells, investigated for cell proliferation and wound healing properties, but none were observed. These data suggest that in healthy broiler chicken tissues AvBD6/7 and AvBD8-10 gene expression profiles are independent of the in vitro antimicrobial hierarchies of the encoded AvBD6, 9 and 10 peptides.

An outbreak of Leishmania major from an endemic to a non-endemic region posed a public health threat in Iraq from 2014-2017: Epidemiological, molecular and phylogenetic studies.

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected worldwide, zoonotic, vector-borne, tropical disease that is a threat to public health. This threat may spread from endemic to non-endemic areas. Current research has exploited epidemiological, molecular and phylogenetical studies to determine the danger of an outbreak of CL in the borderline area between northern and central Iraq from 2014-2017. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, using sequence analysis of the cytochrome b gene, the occurrence of CL in the borderline area between northern and central Iraq was confirmed to be due to Leishmania major. The phylogenetic analysis indicated that it was closely related to the L. major MRHO/IR/75/ER strain in Iran. CONCLUSIONS AND SIGNIFICANCE: In conclusion, the genotype confirmation of the L. major strain will improve our understanding of the epidemiology of the disease. This is important for facilitating control programs to prevent the further spread of CL. Furthermore, this area could be considered as a model for further research on the risk of global CL epidemics in other non-endemic countries where both reservoir hosts and sandfly vectors are present.

First identification of L. major in a dog in an endemic area of human cutaneous leishmaniasis in Iraq: molecular and phylogenetic studies.

Canine leishmaniasis (CanL) caused by Leishmania infantum (L. infantum) is considered as a zoonotic disease and within the last few decades, studies have identified the parasite as a major causative agent of human visceral leishmaniasis. However, in dogs, few recent studies have determined L. major as a cause of cutaneous manifestations and L. tropica as an etiological agent for cutaneous lesions involving mucosa. Interestingly, current study has found canine cutaneous lesions with mucosal involvement in a dog diagnosed with L. major, for the first time, in a focused area of human cutaneous leishmaniasis (CL) in the borderline between northern and central Iraq. Both molecular and phylogenetic studies showed that the dog L. major strain is closely related to that previously isolated from human CL in the same area. Moreover, serological study using rK39 identified IgG response against Leishmania, and the histological finding revealed the infiltration of inflammatory cells around the infection sites. These data will broaden our knowledge about CanL concerning the appearance of cutaneous clinical manifestations with mucocutaneous lesions caused by L. major. Further study on other animal reservoirs and vectors will shed the light on the epidemiology of this disease.

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut.

BACKGROUND: The importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian ß-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds. RESULTS: Time-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05). CONCLUSION: Amino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota.

Effects of rearing environment on the gut antimicrobial responses of two broiler chicken lines.

To reduce the risk of enteric disease in poultry, knowledge of how bird gut innate defences mature with age while also responding to different rearing environments is necessary. In this study the gut innate responses of two phylogenetically distinct lines of poultry raised from hatch to 35days, in conditions mimicing high hygiene (HH) and low hygiene (LH) rearing environments, were compared. Analyses focussed on the proximal gut antimicrobial activities and the duodenal and caecal AvBD1, 4 and 10 defensin profiles. Variability in microbial killing was observed between individual birds in each of the two lines at all ages, but samples from day 0 birds (hatch) of both lines exhibited marked killing properties, Line X: 19±11% (SEM) and Line Y: 8.5±12% (SEM). By day 7 a relaxation in killing was observed with bacterial survival increased from 3 (Line Y (LY)) to 11 (Line X (LX)) fold in birds reared in the HH environment. A less marked response was observed in the LH environment and delayed until day 14. At day 35 the gut antimicrobial properties of the two lines were comparable. The AvBD 1, 4 and 10 data relating to the duodenal and caecal tissues of day 0, 7 and 35 birds LX and LY birds revealed gene expression trends specific to each line and to the different rearing environments although the data were confounded by inter-individual variability. In summary elevated AvBD1 duodenal expression was detected in day 0 and day 7 LX, but not LY birds, maintained in LH environments; Line X and Y duodenal AvBD4 profiles were detected in day 7 birds reared in both environments although duodenal AvBD10 expression was less sensitive to bird age and rearing background. Caecal AvBD1 expression was particularly evident in newly hatched birds. These data suggest that proximal gut antimicrobial activity is related to the bird rearing environments although the roles of the AvBDs in such activities require further investigation.