Genetic Diversity of HPV 16 and HPV 18 Based on Partial Long Control Region in Iranian Women.

BACKGROUND: Human papillomavirus (HPV) 16 and HPV 18 account for 75% of all cervical cancers. The L1 gene, encoding the major surface protein (MSP), is used to classify HPV types (lineages and sublineages), genotypes, and intratypic variants. Therefore, this study aimed to investigate the lineages, sublineages, genetic variabilities, and mutation effects on transcription factor binding sites by using partial sequences of the HPV 16 and HPV 18 long control regions (LCRs) in these samples. MATERIALS AND METHODS: After DNA isolation from 56 positive samples, the LCR of HPV 16 and HPV 18 were amplified using specific primers, and phylogenetic trees were drawn through MEGA X. Compared to the reference sequences, single nucleotide polymorphisms (SNPs) were identified. The transcription binding sites were also evaluated using the online PROMO database. RESULTS: The LCRs of 52 samples were successfully sequenced. Overall, 81.58% of all HPV 16 variants belonged to the D1 sublineage, followed by A4 (13.16%), A1 (2.63%), and C1 (2.63%) sublineages. All HPV 18 isolates belonged to A sublineage, 92.85% to A3 sublineage, and 7.15% to A4 sublineage. Out of 27 SNPs in the HPV 16 LCR, A7382T, T7384G, C7387T, C7393G, A7431G, T7448C, and C7783A were HPV 16-specific. Also, among 14 SNPs in the HPV 18 LCR, C7577A and A7943T were not previously reported. An insertion (C) between 7432 and 7433 positions was identified in all studied HPV 16 variants. Besides, most of the HPV 16 mutations were embedded in the YY1, TFIID, Oct-2, and NF-1 binding sites, while c-Fos and MBF1, as the most common binding sites, were affected by HPV 18 LCR mutations. CONCLUSION: The present results showed that D1 and A3 were the dominant sublineages of HPV 16 and HPV 18, respectively. Therefore, women infected with these variants need to be examined in further longitudinal studies to obtain more information about the oncogenic potential of these dominant variants in Iran. Besides, YY1, TFIID, Oct-2, NF-1, c-Fos, and MBF1 were the most frequent binding sites, which were influenced by the mutations.

Complete genome sequencing and molecular characterization of SARS-COV-2 from COVID-19 cases in Alborz province in Iran.

Iran was among countries which was hard hit at the early stage of the coronavirus disease 2019 (COVID-19) pandemic and dealt with the second wave of the pandemic in May and June 2020; however, there are a very limited number of complete genome sequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Iran. In this study, complete genome sequences of the virus in the samples obtained from three patients in Alborz province in May and June 2020 were generated and analyzed using bioinformatic methods. The sequenced genomes were positioned in a cluster with B.4 lineage along with the sequences from other countries namely, United Arab Emirates and Oman. There were seven single nucleotide variations (SNVs) in common in all samples and only one of the sequenced genomes showed the D614G amino acid substitution. Three SNVs, 1397 G > A, 28688T > C, 29742 G > T, which had already been reported in February, were found with high frequency in all the sequenced genomes in this study, implying that viral diversity reflected in the early stages of viral transmission in Iran were established in the second wave. Considering the importance of molecular epidemiology in response to ongoing pandemic, there is an urgent need for more complete genome sequencing and comprehensive analyses to gain insight into the transmission, adaptation and evolution of the virus in Iran.

Full-length infectious clone of an Iranian isolate of chicken anemia virus.

An Iranian field strain of chicken anemia virus (CAV), designated IR CAV, was isolated in the Marek's disease virus-transformed lymphoblastoid cell line MDCC-MSB1 (MSB1) culture for the first time. The full-length CAV DNA of this strain was cloned in the bacterial plasmid pTZ57R/T to create the molecular clone pTZ-CAV. The nucleotide and deduced amino acid sequences of viral proteins of IR CAV were compared with those of representative CAV sequences including reference and commercial vaccine strains. IR CAV was not related to vaccine strains and also found to have glutamine at positions 139 and 144 confirming previous studies in which such mutations were associated with a slow rate of virus spread in cell culture. pTZ-CAV was digested with PstI to release IR CAV DNA and then transfected into MSB1 cell by electroporation. The transfected cells showed cytopathic effect similar to virion-initiated infection. One-day old specific pathogen-free chicks were inoculated with the regenerated virus, which had been obtained from transfected MSB1 cells, and compared with the chicks inoculated with IR CAV. Gross lesions in the birds inoculated with the regenerated virus illustrated the infectious nature of the regenerated virus from the cloned IR CAV DNA.

Evidence of apoptosis induced by viral protein 2 of chicken anaemia virus.

Although viral protein 3 (VP3) of chicken anaemia virus (CAV) has been well recognised as an inducer of apoptosis, viral protein 2 (VP2) of the virus has only been speculated to have apoptotic activity. This has not been verified because the open reading frame (ORF) encoding VP2 completely encompasses that encoding VP3, and thus the possibility of expression of VP3 cannot be excluded. The aim of this study was to elucidate the potential role of VP2 as an inducer of apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 by early termination of its translation with a stop codon without imposing any change in the amino acid sequence of VP2. The mutated sequence was inserted into the pCAT plasmid preceded by a favorable Kozak's consensus sequence to create pCAT-VP2(+)VP3(-). The absence of VP3 expression in MSB1 cells transfected with this plasmid was confirmed using Western blotting, and DNA strand breaks and nuclear morphological changes were assessed to detect apoptosis. There was an increased level of apoptotic death in cells transfected with pCAT-VP2(+)VP3(-) compared to those transfected with the vector alone. This provides evidence that CAV VP2 can induce apoptosis.

Molecular characterization of the surface glycoprotein genes of highly pathogenic H5N1 avian influenza viruses detected in Iran in 2011.

Highly pathogenic avian influenza (HPAI) H5N1 virus is causing the death of a large number of wild birds and poultry. HPAI H5N1 was reported in the north of Iran in 2011. In this study, two A/Chicken/Iran/271/2011 and A/Duck/Iran/178/2011 viruses were genetically characterized by sequence analysis of Hemagglutinin (HA) and Neuraminidase (NA) genes. Phylogenetic analysis revealed that these viruses were different from previous Iranian isolates (Clade 2.2) and belonged to the subclade 2.3.2.1. The results showed that the detected viruses are almost identical to each other and closely related to HPAI H5N1 strains isolated in Mongolia in 2010. Based on the amino acid sequence analysis, these viruses at their HA cleavage sites contained the multibasic amino acid motif PQRERRRK-R/GLF lacking a lysine residue compared with the previous reports of the same motif. There is also a 20-amino acid deletion (resides 49-69) in the NA stalk similar to other viruses isolated after 2000. It seems that introduction of HPAI H5N1 to Iran might have happened by wild birds from Mongolian origin virus.

Evaluation of chicken anaemia virus mutants as potential vaccine strains in 1-day-old chickens.

The aim of the work reported here was to study the potential of chicken anaemia virus (CAV) mutants as CAV vaccine strains. Seventy 1-day-old chickens were divided into seven groups of 10 birds, and at 1 day old birds were inoculated subcutaneously with RPMI medium, with mutants S77N, Q131P, D186G or R/K/K150/151/152G/A/A, or with wild-type CAV. At day 14 post inoculation (p.i.) one-half of the birds in each group were killed to assess the effect of mutants on their thymuses. At day 21 p.i., birds were inoculated subcutaneously with wild-type CAV. At day 35 p.i., the remaining birds in each group were killed to assess the protective effect of vaccination with the mutants. The thymus weight to body weight ratios were determined and the thymic cortical thickness measured using immunofluorescent staining with an anti-CD8 monoclonal antibody. There was no significant decrease in the thymic cortical thickness at day 14 p.i. in birds inoculated with mutants E186G or R/K/K150/151/152G/A/A. At 14 days after challenge with wild-type CAV, birds inoculated with these mutants and with mutant Q131P had significantly increased thymic cortical thickness compared with unvaccinated unchallenged birds. A serum neutralization assay, based on viral detection using a quantitative polymerase chain reaction assay, was used to determine the neutralizing antibody titres in blood samples collected at day 35 p.i. Mutant E186G induced the highest post-challenge neutralizing antibody titres, followed by mutants Q131P, S77N and R/K/K150/151/152G/A/A. These studies have shown that mutant E186G is an appropriate candidate mutant CAV vaccine strain.

Viral load in 1-day-old and 6-week-old chickens infected with chicken anaemia virus by the intraocular route.

Although the effects of chicken anaemia virus (CAV) infection have frequently been investigated in young chickens, there have been few studies of the pathogenesis of CAV infection in older birds. The aim of the work reported here was to study viral loads in 6-week-old chickens and to compare these with those seen in younger birds. Specific pathogen free chickens were inoculated at 1 day or at 6 weeks of age with 10(4) median tissue culture infective doses of CAV by the intraocular route. Chicks infected when 1 day old were euthanized at day 14, 18 or 22 post inoculation (p.i.), and those infected when 6 weeks old at day 16, 18 or 20 p.i. Their body and thymus weights were determined and samples were collected from their spleen, liver and thymus. A quantitative polymerase chain reaction assay was developed and used to determine the number of viral genome copies in the tissue samples. In both age groups, viral genome concentrations increased in all organs up to day 18 p.i. and reached a peak in the spleen and liver at day 18 p.i. The peak viral concentrations in the thymus were detected at day 18 in the younger birds and at day 20 p.i. in older chickens. These studies have shown that exposure to CAV in older birds leads to similar levels of active viral replication to those seen in younger birds, and may result in subclinical infections in older birds with the potential to increase susceptibility to other infectious agents.