Parvovirus 4 (PARV4) is an emerging and intriguing virus that currently received many attentions. High prevalence of PARV4 infection in high-risk groups such as HIV infected patients highlights the potential clinical outcomes that this virus might have. Molecular techniques were used to determine both the presence and the genotype of circulating PARV4 on previously collected serum samples from 133 HIV infected patients and 120 healthy blood donors. Nested PCR was applied to assess the presence of PARV4 DNA genome in both groups. PARV4 DNA was detected in 35.3% of HIV infected patients compared to 16.6% healthy donors. To genetically characterize the PARV4 genotype in these groups, positive samples were randomly selected and subjected for sequencing and phylogenetic analysis. All PARV4 sequences were found to be genotype 1 and clustered with the reference sequences of PARV4 genotype 1. J. Med. Virol. 88:1314-1318, 2016. © 2016 Wiley Periodicals, Inc.

Introduction:Cancer immunotherapy is one of the effective treatment methods that provide a better quality of life with limited side effects for patients. Carcinoembryonic Antigen (CEA) can be an appropriate target for cancer immunotherapy. Methods: A lentivector expressing CEA antigen, pCDH-CEA, was constructed by cloning CEA cDNA downstream of the CMV promoter. The constructed plasmid was co-transfected with helper plasmids, into Lenti-X 293 T cells. The lentivector-containing supernatant was collected. Titers of the CEA-lentivector were estimated using the RT-PCR method. The CT26 cells were then infected by CEA-lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select CEA-positive cells. The ability to produce tumors in BALB/c mice was investigated. Results: The results showed that CEA expressing lentivector plasmids and the two other helper plasmids could be transfected into Lenti-X 293T cells efficiently and packaged successfully as a pseudo-lentivector. The detection of CEA mRNA and protein expression in the 6th and 14th passages of CT26-CEA cells was confirmed in the engineered stable cell line. Tumor formation was confirmed in cell inoculated mice. Conclusion: CT26-CEA cell line with stable expression of CEA can be used as a suitable tumor model to facilitate research on colorectal cancer in vitro and in mice models; therefore, it could be served as a valuable tool for cancer immunotherapy.

Introduction: Due to the role of neutralizing antibodies which can prevent human cytomegalovirus (HCMV) infection, most of the efforts have been focused on designing vaccines capable of eliciting protective humoral immunity. The aim of this study was to evaluate the antibody response of BALB/c mice to a truncated HCMV glycoprotein B produced in insect cells using Baculovirus Expression Vector System (BEVS). Methods: The ectodomain of HCMV gB coding sequence was synthesized and the recombinant protein was expressed in Spodoptera frugipedra (Sf9) insect cell line using BEVS. The expression of the recombinant HCMV gB was verified using an HRP-conjugated polyclonal antibody, specific for HCMV gB. The levels of antibody responses and characterization of the subclasses of IgG antibodies were evaluated after vaccination of the mice. Results: The expression of truncated HCMV gB protein (~ 70 kDa) in the infected insect cells was verified by Western blot analysis. Measurement of IgG subclasses showed the dominance of IgG1 subclass response among all of the IgG subclasses (P < 0.05) while the titers of IgG2a and IgG2b were approximately the same. Conclusion: This study demonstrated that BEVS could be used as an efficient approach for the expression of this truncated protein. The results also showed the use of this recombinant protein as a subunit vaccine could induce a significant antibody response, tilted toward IgG1.

Background: Taqman one-step real-time PCR (RT-PCR) has special importance due to its high sensitivity and specificity in the diagnosis of infectious diseases such as viral infections. In the recent pandemic of SARS-CoV-2, diagnostic kits based on this method are commonly used for molecular detection. One of the main systematic errors that misinterpret the results is using inaccurate internal control in RT-PCR diagnostic kits. Designing primers and probes that span exon-exon junction will avoid genomic DNA amplification and lead to obtaining high specific results. Objectives: This study aimed to evaluate the endogenous internal control of primers and probe for RNase P RNA to reduce false-negative results in respiratory samples. Methods: In this study, 30 samples of patients who were negative for SARS-CoV-2, influenza A, and influenza B were re-evaluated for SARS-CoV-2 using newly designed primers and probes for RNase P RNA (ultra-specific primers and probe). We also performed bioinformatics analysis on CDC-approved primers and probes of RNase P endogenous internal control. Results: In this analysis, we specified the location of these newly designed primers and probe on target mRNA and genomic DNA. Then, the Taqman one-step RT-PCR method was performed using both CDC-approved primers and probes along with our ultra-specific primers and probe for RNase P RNA. Based on bioinformatics analysis, the attachment sites of the CDC-approved primers and probe for endogenous internal control of RNase P are located on the first exon of this gene. In addition to identifying the target gene sequence, these primers and probe also non-specifically detect similar sequences on the genomic DNA. Conclusions: The present study showed that the use of specific primers and probes introduced by CDC for SARS-CoV-2 and influenza virus may cause false results due to non-specific binding to the genomic DNA. Therefore, choosing the right internal control for RNase P RNA can be useful in achieving very accurate results.

Background: Hemophilia is a well-known bleeding disorder with worldwide distribution. Replacement therapy, using plasma-derived or recombinant coagulation factors, comprises a gold standard regimen for the treatment. Regardless of the advancements made in viral inactivation methods in the production of plasma-derived coagulation factors, the possibility of transmission of new viral infections remained as a noticeable concern yet. The aim of the current study was to investigate the status of parvovirus 4 (PARV4) in severe hemophilia A, von Willebrand disease (vWD), and healthy control. Materials and Methods: In the current case-control study, 76 patients with hemophilia and vWD and 60 individuals from their family members entered the study. Nested PCR used to determine the presence of PARV4 in study subjects (76 cases). To characterize the PARV4 genotype, positive samples subjected to sequencing and phylogenetic analysis. Results: PARV4 genome detected in 11 (14.47%) patients with bleeding disorders. Among whom, nine patients (14.75%) were with severe hemophilia A and two (13.33%) patients with vWD. Only five healthy controls (8.33%) were positive for PARV4. All PARV4 sequences were found to be genotype 1. Conclusion: PARV4 infection in patients with hemophilia and vWD was higher than the control group. While detection of PARV4 DNA in patients with bleeding disorders may not necessarily reflect a clinical urgency, future investigations are needed to define the clinical significance of PARV4. It seems the detection of the virus immune signature of PARV4 infection, particularly in the context of acute and persistent infections, needs to focus on cellular and tissue targets.