Microbiology Independent Research Journal (MIR Journal)

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Vol 8, No 1 (2021)
1-9 1027

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus.  
The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

10-17 906

In recent years, the number of infectious diseases caused by Clostridium difficile in the world has grown with a significant increase in relapses and mortality in patients, particularly among the cancer patients in hospitals. There is also observed an increase in the resistance of Clostridium difficile to the first-line drugs, namely metronidazole and vancomycin, which makes the search for new methods of treatment and prevention of this infection even more urgent. In this review, we analyze the recent data on the methods of cultivation and isolation of the pure bacterial culture of Clostridium difficile and other anaerobic enteropathogens over the course of enterocolitis treatment with antimicrobial drugs in pediatric patients with oncopathology. Novel approaches to the therapy of this infection are discussed. 

18-26 937

The production environment in pharmaceutical industry should not be a source of microbial contamination of the product. The purpose of this study was to examine the patterns of changes in the microbial contamination level in clean rooms. The study was conducted at the educational and scientific clean room module of the Biotechnology Department of the Obninsk Institute of Nuclear Power Engineering (IATE NRNU MEPhI). It was shown that the level of contamination of surfaces in production facility increases with the presence of personnel on the premises. The degree of microbial contamination of the air varies slightly depending on the type of activity and the number of personnel on premises. The compliance of the investigated clean room module with class D for clean rooms was confirmed. It was concluded that it is necessary to monitor microbial contamination of premises more often, as well as to implement the sanitary treatment in order to eliminate the sources of spore-forming microflora.

27-37 427

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

38-40 340

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease (COVID-19) and ongoing pandemic that has devastated humankind. During the COVID-19 pandemic, it was noticed that the mortality rate in men is higher than that in women. The membrane (M) protein of SARS-CoV-2 plays a pivotal role in the viral life cycle regulating intracellular trafficking and processing of spike (S) protein. In infected individuals, M protein inhibits the conversion of active testosterone to its inactive form through its interaction with Aldo-keto reductase family 1 member C2 (AKR1C2) protein. This leads to the high availability of active testosterone and boosts the formation of its complex with an androgen receptor that in turn promotes the transcription of the transmembrane protease serine 2 (TMPRSS2) gene. TMPRSS2 is known to play a pivotal role in the priming of S protein that is necessary for the SARS-CoV-2 entry into the host cell. Therefore, the interaction of the M protein of SARSCoV-2 with AKR1C2 eventually leads to the upregulation of the transcription of the TMPRSS2 gene that results in an enhanced viral infection and in turn higher mortality in men. The interaction of M protein with AKR1C2 could be a possible target for SARSCoV-2 antiviral drug design.

41-49 210

Osteomyelitis is a rare disease that is often caused by an infection. In case of microbiology analyses failure, molecular assay seems appropriate for the identification of the pathogen. Broad-range PCR is a popular tool to amplify the gene of 16S ribosomal RNA – the component of the 30S subunit of the bacterial ribosome present in various species. The subsequent sequencing of the amplified gene enables scientists to determine the bacteria species. In this review, we discuss studies and case reports where the osteomyelitis causative agent was revealed by means of broad-range PCR. The purpose of the analysis is to assess the relevance and significance of this method for the diagnosis of osteomyelitis in patients. Numerous successful applications of wide-range PCR followed by sequencing in order to identify the causative agent of osteomyelitis have proven that this method is a useful tool in cases where the culture analysеs showed negative results.

50-61 266

Avian influenza viruses of H1 and H5 subtypes were involved in the formation of highly pathogenic viruses that caused pandemics and panzootics in the 20th–21st centuries. In order to assess the zoonotic potential of viruses of these subtypes, two viruses of H1N1 and H5N3 have been isolated from wild ducks in Moscow and adapted to growth in mouse lungs. Their phenotypic properties were studied, and the genetic changes that occurred during adaptation were identified. The original A/duck/Moscow/4970/2013 (H1N1) and A/duck/Moscow/4182-C/2010 (H5N3) viruses were apathogenic for mice but became pathogenic after 7–10 passages in mouse lungs. Complete genome sequencing revealed 2 amino acid substitutions in the proteins of the H1N1 mouse-adapted variant (Glu627Lys in PB2 and Asp35Asn in hemagglutinin (HA) – numbering according to H3) and 6 mutations in the proteins of H5N3 virus (Glu627lys in PB2, Val113Ala in PB1, Ser82Pro in PB1-F2, Lys52Arg in HA2, Arg65Lys in NP, and Ser59Ile in NA). The increase in virulence is most likely due to a common substitution in the protein PB2 Glu627Lys as revealed in both viruses. The replacement of Asp35Asn in HA of the mouse-adapted H1N1 virus is associated with an increase in the pH value of the HA transition from 5.0 for 5.5 in comparison to the HA of parent virus. The found mutations in HA, NA, and PB1-F2 proteins of the adapted H5N3 variant are unique. The mutations Glu627Lys in PB2, Arg65Lys in NP, and Val113Ala in PB1 are most likely host adaptive.

ISSN 2500-2236 (Online)