Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

The Sero-Prevalence of Hepatitis B and C Viruses in Municipal Waste Collectors in Southwest of Iran.

Aims: The present study aimed to investigate the frequency of hepatitis B virus (HBV) and hepatitis C virus (HCV) serological markers among waste collectors in the municipality of Shiraz city, southwest of Iran, 2018. Settings and Design: In this cross-sectional study, a total of 385 waste collectors from all 10 districts of Shiraz city, southwest of Iran were enrolled. A questionnaire was used to gather occupational and demographic information as well as awareness about viral hepatitis. Methods and Material: Their blood samples were collected, and the sera were investigated for the presence of hepatitis B surface antigen (HBsAg), anti-HBs antibody, and anti-HCV antibody using enzyme-linked immunosorbent assay (ELISA). Results: All the participants were men with a mean age of 41 ± 8 years. Out of 385 participants, 6 (1.5%) subjects were positive for HBsAg, indicative of HBV infection. Moreover, 38 (9.9%) had a protective level of anti-HBs antibodies, while more than 90% had a low level of anti-HBs antibodies. All participants were negative for HCV antibodies. Conclusion: According to these findings, evaluation of HBsAg and anti-HBs levels is recommended in the healthy program of waste collector workers (WCWs). However, this occupation might not be a risk factor for the acquisition of HBV and especially HCV infections.

The molecular epidemiology, genotyping, and clinical manifestation of prevalent adenovirus infection during the epidemic keratoconjunctivitis, South of Iran.

PURPOSE: Adenoviral-mediated keratoconjunctivitis is among the emergency diseases of ophthalmology with long-term sequels. The role of adenovirus infection, ocular-related genotypes, and association with ocular symptoms need to be investigated for epidemiological as well as clinical purposes. METHODS: The affected patients from two close keratoconjunctivitis epidemics were included in the study. The swab samples were taken from patients; the total DNA was extracted and then used as a template for in-house Real-time PCR. Besides, partial Hexon genes of 11 adenovirus positive samples were amplified and submitted to sanger sequencing. Moreover, they were finally evaluated by phylogenetic analysis. RESULTS: Of 153 patients, 92 (60.1%) were males and 47 cases (30.7%) had a history of eye infection in the family or colleagues. Real-time PCR tests of 126 samples (82.4%) were positive for adenovirus, and all eleven cases that underwent sequencing analysis were determined to be group 8 (HAdV-D8). Adenovirus infection has a significant relationship with infection among family or colleagues (p = 0.048), membrane formation (p = 0.047), conjunctival bleeding (p = 0.046), tearing, and pain(p < 0.05). CONCLUSIONS: The results indicated that Adenovirus is the major cause of keratoconjunctivitis, and HAdV-D8 was the most common genotype in the area. There were some clinical manifestations associated with Adenovirus infection of the conjunctiva.

Serosurvey of hepatitis A virus and E virus infection among municipal sweepers working in the largest city in the south of Iran.

This study was conducted to determine the exposure rate of Hepatitis A and Hepatitis E viruses in urban solid waste collectors/sweepers in the south of Iran. The 385 samples (serums) were collected from Shiraz Municipality waste sweepers.. A questionnaire was used to gather data on their demographic and occupational characteristics, as well as their awareness of viral hepatitis disease. The viral seroprevalence was determined by commercial IgG ELISA kit. All participants were male, mean age of 41 ± 8 years. ELISA assay showed that all of them were positive for anti-HAV IgG. Also, 62 out of 385 individuals were positive for anti-HEV IgG. The statistical analysis showed that the frequency of HEV IgG antibody among age groups 20-30, 31-40, 41-50 and >50 years old had an increasing trend, 4.5%, 10.1%, 17.4%, and 36.7%, respectively, indicating age factor significance (p = .001). Based on some investigated factors including the duration of work experience, current and previous jobs, habitation, personal hygiene status, and knowledge on viral hepatitis diseasees/their transmission, there was no statistically significant difference between anti-HEV IgG positive versus negative sweepers. The results indicated a slighty higher frequency of anti-HAV and anti-HEV IgG among sweepers compared to other pre-investigated population. It doesn't seem that garbage collecting/sweeping could be a significant risk factor for HAV and HEV infection.

Relative frequency of hepatitis B virus, human papilloma virus, Epstein-Barr virus, and herpes simplex viruses in the semen of fertile and infertile men in Shiraz, Iran: A cross-sectional study.

BACKGROUND: About 8-12% of couples on reproductive age suffers from infertility worldwide. Since 1993, the role of genital tract infections by microbes, including viruses that can infect the sperm, in human infertility has been proposed. OBJECTIVE: To investigate the frequency of hepatitis B virus (HBV), human papilloma virus (HPV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV) infection in the semen of fertile and infertile men referred to the Mother and Child Hospital, Shiraz, Iran. MATERIALS AND METHODS: In this cross-sectional study, 350 men including 200 infertile and 150 fertile men were included. All semen samples were allowed to liquefy, followed by the assessment of sperm parameters. DNA was extracted using a DNA extraction kit (CinaGene, Tehran, Iran) according to the manufacturer's instructions. Detection of HBV, HPV, EBV, and HSV1/2 was done by the PCR method. RESULTS: The mean age of the participants was 36 ± 7 yr. Molecular results showed that 16 samples (8%) of infertile men and 5 (3.3%) of fertile men were positive for HBV, which was not statistically significant (p = 0.069). Only one sample of the fertile participants was positive for HPV. None of the semen samples of the infertile or fertile groups was positive for the presence of EBV or HSV1/2. CONCLUSION: The results of this study indicated that HBV, HPV, EBV, and HSV might not be involved in men's infertility. Further studies are recommended for clarifying the role of these viruses in infertility.

Evidence of Hantavirus circulation among municipal street sweepers, southwest of Iran.

Hantaviruses are rodent-borne zoonosis pathogens that cause hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS) in humans. Rodents spread the virus via their excretions. The outbreak of Hantaviruses pose a significant public health problem. The epidemiology and history of Hantaviruses in Iran is not clear and regardless of the data from the few available studies, little is known about its epidemiology in this country. Herein, we discuss the prevalence of IgG antibody against Hantavirus serotypes in 385 street sweepers from southwest of Iran. Serum samples were investigated, using Hantavirus Pool 1 "Eurasia" IgG kit and Pool 2 "America" ELISA IgG kit (Euroimmun, Germany) to detect IgG antibodies against Old and New World Hantaviruses. The results showed a specific IgG antibody in two samples (0.5%). Both of seropositive cases had specific IgG antibody against Old World Hantaviruses. The data of the current study along with the previous data, indicate the circulation of Hantaviruses in Iran. Hence, the risk of Hantavirus infection in high-risk groups should be considered as a serious health issue.

Serosurvey of parvovirus B19 and cytomegalovirus infections among female university students in Shiraz, Southern Iran.

Infection with parvovirus B19 and cytomegalovirus (CMV) during pregnancy might lead to fetal infection, resulting in congenital abnormalities. This study aimed to investigate the frequency of IgM and IgG antibodies against parvovirus B19 and CMV in female university students in Shiraz, in Fars province, Southern Iran. In this cross-sectional study, 370 female university students were included. Blood samples were collected from each participant and tested for anti-parvovirus B19 and CMV IgG and IgM antibodies, using commercial ELISA kits. The mean age of the participants was 24 (±7)years. Out of 370 participants, 327 (88.4%) and 9 (2.4%) were positive for IgG and IgM antibodies against CMV. Moreover, 211 (57.0%) and 4 (1.1%) of the participants were respectively positive for IgG and IgM antibodies against parvovirus B19. The difference in CMV or parvovirus B19 seropositivity between different age groups was not statistically significant (P>0.05). The findings of our study showed that more than 50% of the female university students are seropositive to CMV and parvovirus B19 infections. It highlights the importance of health education and also the laboratory screening of females at childbearing age to reduce the risk of congenital infections resulting from these viral infections.

Co-existing of HSV1/2 or EBV Infection with the Presence of High-Risk HPV DNA in Cervical Lesions in the Southwest of Iran.

OBJECTIVE: While the vast majority of the cervical lesions have been attributed to the HPVs, the role of EBV and HSV1/2 as co-factors in the progression of these abnormalities needs more investigation. In this study, we aimed to determine the co-existence of EBV or HSV in cervical lesions infected with high-risk HPVs. METHODS: Totally, 102 formaline-fixed cervical lesions with different pathological grades (LSIL, HSIL, and SCC) were enrolled in this study. DNA was extracted, and its integrity was examined by PCR assay. Two conventional PCRs were performed for the detection of EBV and HSV1/2 genomes in the tissue specimens. Besides, an in-house Real-Time PCR, as well as a nested PCR assays following sequencing, was performed to detect HPV genotypes in EBV or HSV positive samples.  Results: The mean age of the participants was 42.8±13 years. Out of 102 samples, 32% (n=33) were confirmed to be LSIL, 42.2% (n=43) were HSIL, 22.5% (n=23) were SCC and 2.9% (n=3) were adenocarcinoma.  EBV genome was detected in 13(12.7%) samples including 2 of LSIL, 8 of HSIL and 3 of SCC. All EBV positive samples harbored high risk HPV types 16,18 and/or 31 co-infections. However, the HSV genome was not found in any of the samples. CONCLUSION: Our result revealed that the frequency of EBV infection is higher in HISL than LSIL.  Moreover, the amount of HPV load showed an elevated level among co-infected patients, which indicates that EBV might be an enhancing factor of disease progression. In contrast, HSV may not has a role as a co-factor in cervical lesions pathogenesis.

Association of HPV16 and 18 genomic copies with histological grades of cervical lesions.

The possible correlation between HPV16 and HPV18 genomic copies with the grade of cervical lesions needs more investigations. The aim of this study was to quantify genomic copies of HPV16 and 18 simultaneously and to find out the correlation between genomic copies numbers and different grades of lesions. Therefore, a total of 102 formalin-fixed and paraffin-embedded tissue specimens, 33 LSILI, 43 HSIL, and 26 squamous/adenocarcinoma were subjected to DNA extraction. The ß-globin gene was selected to qualify the extracted DNA as well as normalization of viral titers using Taq-Man real-time PCR. The presence HPV16 and/or 18 were screened in tissue samples by nested PCR method, then an in- house Taq-Man Duplex real-time PCR assay was employed to quantify their genomic copies. The mean age of participants was 43 ± 13. Out of 102, 80 samples were positive for HPV16 and/or 18 DNA. There was a statistically significant association between HPV16 genomic copies and progression of cervical lesions (P < 0.001). In contrast, no such an association was found in the case of HPV18 (P = 0.51). Moreover, with 95% confidence intervals, 2.3-4 genomic copies of HPV16 genome/cell could be applicable to distinguish LSIL from HSIL and SCC. In conclusion, quantification of HPV16 genomic copy number showed a close association with progression of cervical lesion. Furthermore, HPV16 genomic copies of 4 copies/cell could be a set point to differentiate LSIL from HSIL.

No detection of EBV, BKV and JCV in breast cancer tissue samples in Iran.

OBJECTIVE: The most common cancer amongst women is breast cancer. Reports on the role of EBV, BKV, and JCV in the development of breast cancer are controversial. Hence, the aim of this study was to determine the frequency of EBV, BKV, and JCV in malignant breast tumors in comparison with benign ones. RESULTS: A total of 300 breast biopsy tissues were included, of which 150 were malignant and 150 benign. After deparaffinization, tissues were subjected to DNA extraction. ß-globin gene was amplified by PCR to evaluate the quality of extracted DNA. In house PCRs assay was performed to detect EBV, JCV, and BKV genome fragment. The mean age of malignant and benign groups was 45.0 ± 9.4 and 35.2 ± 12.1 years old. Out of 150 malignant samples, 146 were ductal, two lobular and two samples both invasive ductal and lobular carcinoma. In the benign group, 96, 52 and two samples were fibroadenoma, fibrocystic, and adenosis types, respectively. Genomic DNA fragment of EBV, BKV, and JCV was not found in any of the malignant and benign breast tissues. CONCLUSION: According to our finding, there is the possibility that EBV, BKV, and JCV are not involved in breast cancer pathogenesis.

Analytical assessment of interleukin - 23 and -27 cytokines in healthy people and patients with hepatitis C virus infection (genotypes 1 and 3a).

BACKGROUND: The immune system plays important roles in determining the outcomes of hepatitis C virus (HCV) infection. Interleukin-23 and -27 (IL-23 and IL-27) are two novel IL-12 cytokine family members known to enhance the T-lymphocyte response, but their precise involvement in HCV infection is not well known. OBJECTIVES: We investigated the serum IL-27 and IL-23 levels in patients with HCV infection and in healthy individuals. PATIENTS AND METHODS: In this case-control study, we assessed IL-23 and IL-27 levels in serum of 37 healthy individuals and 64 patients with chronic HCV using Enzyme-linked immunosorbent assay (ELISA). The relationship of cytokines level with liver enzymes (ALT, AST, and ALP), HCV genotype and viral load were analyzed. The differences of these cytokine levels in the groups of treatment and no treatment was compared. HCV genotypes were classified by HCV-specific primers methods. HCV RNA loads were determined by fluorescence quantitative PCR. RESULTS: Serum level of IL-23 was higher in HCV infected patients compared to control group (P = 0.005). However, no significant difference was seen in IL-27 serum level between patients compared to the control group (P = 0.65). There was no significant difference in IL-23 and IL-27 level between genotype 1 HCV-infected- and 3a HCV-infected- patients. Positive moderate correlation between IL-23 and IL-27 with viral load was found in type 3a and 1 HCV-infected patient. Positive relative correlation was seen between ALT and IL-23 in 1a HCV-infected patients, which was higher than 3a HCV-infected patients; but there were no significant difference between serums liver enzymes with IL-23 and IL-27 in respect to genotype 3a and 1a HCV-infected patients. CONCLUSIONS: These findings may reflect a vigorous pro-inflammatory reaction orchestrated by the host immune system against chronic HCV. Also, a better understanding of the involvement mechanism considering the correlation between other genotypes with inflammatory cytokines in various stages of disease can be obtained.

Molecular epidemiology of different hepatitis C genotypes in serum and peripheral blood mononuclear cells in jahrom city of iran.

BACKGROUND: The Hepatitis C Virus (HCV) is considered essentially hepatotropic, yet the virus compartments have also been found in important extra hepatic sites. Detection of HCV RNA in extra hepatic reservoirs such as peripheral blood mononuclear cells (PBMCs) is important for determining disease progression and treatment effectiveness. OBJECTIVES: The present study aimed to determine different HCV genotypes in patients' plasma and PBMC specimens, in Jahrom city of Iran. PATIENTS AND METHODS: Blood samples of 137 patients with established HCV were collected at the Honari clinic. These patients were anti-HCV and plasma HCV RNA positive. After plasma RNA extraction and obtaining a pellet of approximately 3-5 × 10(6) PBMCs, Real-time PCR was performed, using specific-genotype primers. Finally, data analysis was done by the Statistical Package for Social Sciences (SPSS) software. RESULTS: Subtype 3 was the most common genotype in plasma (57.7%) and PBMCs (51.1%). Subtype 1a was detected in 36.5% and 30.7% of plasma samples and PBMCs, respectively whereas subtype 4 was not detected in any of the cases. There was a genotype difference between plasma and PBMCs of 12.4% of patients. In four patients no genotype was detected in their plasma but genotype 3 was detected in the PBMCs. CONCLUSIONS: It is suggested that determination of the target genotype by plasma subtyping for choosing the proper antiviral therapy is essential but may result in therapy failure. HCV genotyping in PBMC samples, along with plasma specimens, might be more beneficial. Therefore determining the HCV genotype in PBMCs, before beginning the therapy is useful due to the possibility of occult infection detection.