Molecular Virology (Biology)

We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.

Here, we present the complete genome sequence of Enterobacter phage vB_EcRAM-01, isolated from waters of the Río Abajo river, in Panama City, Panama. This phage has deployed lytic activity against the Enterobacter cloacae complex, a pathogen of clinical importance in intensive care units. It belongs to the Myoviridae family and has a double-stranded DNA genome that is 178,477 bp long and contains 293 open reading frames (ORFs).

We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.

This extract presents 80 pages of content from the newly revised and enlarged Fourth Edition of 'AVD: The Anti-Viral Diet', which shares the results from the most recent, cutting-edge scientific research into the anti-viral properties of widely available foods - as well as of some rarer ingredients. Most significantly, that report confirms - among other conclusions - that good nutritional status increases vaccine efficacy and decreases the probability of suffering the dangerous side effects of some vaccines.

This extract includes copious evidence regarding eight dietary ingredients from the 'Anti-Viral Diet' and a key extract on the project's proposal for an 'Anti-Viral Five-a-Day'. This extract includes 16 full-colour photographs.

The 'AVD' publication presents the general theory of an 'Anti-Viral Diet' and also the evidence from over 2000 scientists and medical researchers confirming the anti-viral and immunomodulatory properties of many dietary ingredients. At the core of 'AVD' is a database of 52 Anti-Viral Ingredients  with detailed specifications and 104 colour photographs. Key info is provided on each of the identified ingredients, including against which viruses they have exhibited potent effects, how they 'boost' the Immune System, as well as key data about Recommended Daily Allowances. Citations and extracts from over 1000 research articles enables a broad range of scientists to speak in their own words about key discoveries in nutritional immunology and anti-viral dietetics. Every section includes research findings from at least two dozen articles (mainly from peer-reviewed journals) corroborating anti-viral qualities exhibited by that ingredient.

With kind permission of the publishers LIVING TIME™ Books. Copyright © LTB™ 2020-22.

Purpose: This study was focused on (i) detection of BVDV specific antibodies in selected cattle farms, (ii) identification of persistently infected (PI) animals, and (iii) genetic typing of selected BVDV isolates. Methods: BVDV antibodies were detected in 1023 blood samples collected from three cattle farms in the Kharkiv region during 2011-2012. The total number of animals was 815 on the first farm, 900 and 5500 animals on the second and third farms, respectively. PI animals were identified by the RT-PCR method utilizing the pan-pestivirus 324/326 primers for BVDV detection in samples from antibody-negative cattle. Selected PCR amplicons were sequenced. Phylogenetic analysis of 5’-UTR (245 bp fragment) was used for the genetic typing of BVDV isolates into subgenotypes. Results: BVDV specific antibodies were detected in 694 out of 1023 samples analyzed (67.8%). This number is in agreement with findings in many cattle herds around the world. However, the number of positive samples differed in the herds. While 43 samples out of 250 (17.2%) were identified in the first herd, 398 out of 473 (84.1%), and 253 out of 300 (84.3%) animals were seropositive in the second and third herds, respectively. The BVDV's RNA was detected in a high number of animals in all herds. The RT-PCR assay detected 143 out of 1023 samples analyzed (14.1%). 32 samples out of 250 (12.8%) were detected in the first herd, 79 out of 473 (16.7%), and 32 out of 300 (10.7%) were found in the second and third herds, respectively. The total number of PI animals detected in the cattle herds was in accordance with our previous serological findings. Genetic typing of 12 isolates indicated that all viruses belonged to the BVDV-1b subgenotype and all of them contained an identical 5’-UTR region. It is not excluded that identical isolates may be the result of a live BVDV vaccine usage on those farms but this hypothesis has yet to be verified. Conclusion: Our results indicated that the BVDV infection is widespread in cattle herds in the Kharkiv region. Better characterization of viral isolates, as well as the introduction of the biosecurity programs on farms, are in progress under the ongoing Swiss-Ukraine-Slovak SCOPES project.

It was shown that PCR is a more specific, sensitive, and accurate diagnostic method compared to RID, as 40.6% of RID positive samples did not contain genetic material of leukemia virus when analyzed with PCR. It was also proven that PCR analysis detected positive samples at the earlier stages of disease than RID was able to. PCR screening for leukemia virus can help to prevent the spread of infection between animals or begin the recovery of farms as soon as possible. In our work, it was proven that selective molecular genetic testing of cattle at the initial and final stages of a farm’s recovery from leukemia is necessary.

Background: West Nile fever is a disease of horses, humans, and several avian species caused by a Flavivirus. Wild birds act as a reservoir for West Nile virus (WNV). Wildlife populations should be monitored for WNV using modern surveillance techniques. Ukraine is located in the migration pathways of wild birds connecting Asia, Europe, and Africa. These birds pose a risk for the introduction of WNV into Ukraine. The goal of this work was to develop a protocol for the detection of viral RNA using conventional PCR. Materials: Specific primers were developed for a 280 bp region of the gE gene of WNV based on GenBank published sequences of viral cDNA. The amplification reaction was optimized based on a gE recombinant template derived from the WNV vaccine seed. PCR master mix reagents from a Russian Federation manufacturer were used. The protocol was validated according to the OIE requirements for diagnostics techniques. Results: The gE gene sequences from the GenBank were analyzed for the conserved regions, and four of them were selected for the primer design. The forward and reverse primer pair, flanking 280 bp region of the gE gene, was designed. The protocol for the conventional PCR was a 40-cycle reaction with an annealing temperature of 57 ̊C. The reaction mix volume was 30 ml with 5 U of Taq polymerase, 1.5 mM Mg, and 20 pM of each primer. The PCR was able to detect viral cDNA in samples of vaccine virus and plasmid DNA (5-7 pg/ml). The PCR was validated with cDNA test panels and recognized as 95 % sensitive, 100 % specific, repeatable, and accurate. Screening of the blood and blood sera panels from wild birds, collected in Ukraine (n = 63), demonstrated the absence of the WNV in the specimens. Conclusion: A conventional PCR for WNV detection was developed and validated. Screening of field samples from wildlife demonstrated the absence of the virus. Further work is needed for the development of surveillance systems for WNV in wildlife and synanthropic birds in Ukraine.

Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites,
viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular
approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially.
The increase in aquaculture operations worldwide has provided new approaches for the
transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the
virus and the host for control of the cell’s death machinery is crucial for survival. Viruses are obligatory
intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their
host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of
action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated,
mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses
exploit the apoptotic pathways of their hosts may provide great opportunities for the development of
future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases.

The worldwide emergence of infectious diseases and high cost of synthetic drugs has warrants new agents for treatment of its infections. The present study investigated the Ethyl acetate, Methanol and aqueous extract of Ulva lactuca for its ability to inhibit the pathogens Staphylococcus aureus and Escherichia coli. The methanol extract of Ulva lactuca showed maximum (15 mm) anti-bacterial activity. Ulva lactuca exhibited high anti-bacterial activity with the inhibition zone of 13mm to 17mm compared with the standard antibiotic Ampicillin. The phytochemical evaluation of these extracts deciphered the presence of Cardiac glycosides, Terpenoids, Carbohydrates. Further studies needed to isolate and to characterize the active principle to establish its mode of action.

Staphylococcus aureus is one of the major causes of community and hospital-acquired infections. Bacteriophage considered as a major risk factor acquires S. aureus new virulence genetic elements. A total number of 119 S. aureus isolated from different specimens obtained from (RKH) were distinguished by susceptibility to 19 antimicrobial agents, phage typing, and PCR amplification for mecA gene. All of MRSA isolates harbored mecA gene, except three unique isolates.

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA h to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus -DNA h complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus -DNA h complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings. D

ABSTRACT
In this study, the normal electrocardiogram of apparently normal, conscious pigeons was described. Standard bipolar and
augmented unipolar limb leads were recorded in a total of apparently healthy 30 pigeons. Waveforms were analyzed in all the
leads at 50 mm/s and 10 mm/mV. P-wave was monophasic and positive in leads I, II, III, aVL and aVF but negative in aVR.
Heart rate ranged from 205 to 342 beats/min with a mean (± SD) of 276.0±42.9 beats/min. The MEA ranged between -99 to -90
degrees. The predominant QRS pattern in lead in lead I was rs while the rS was predominant in leads II, III and aVR. The r and
Qr morphologies were observed in leads aVF and aVL respectively. ST slurring was found in all the leads. The ECG values and
patterns recorded in these clinically healthy pigeons will facilitate better understanding of ECG changes that are seen in diseases
and are expected to provide a comparison to aid clinical diagnosis
Keywords: Electrocardiogram, Pigeons, Limb leads.

"Relatively little is known about the distribution of avian paramyxoviruses (APMVs) among wild birds in Japan.  Surveillance of APMV in migratory waterfowl was conducted in the San-in region of western Japan during winters of 2006 to 2012.  A total of 16 avian paramyxoviruses consisting of three lentogenic Newcastle disease viruses (NDVs), 12 APMV-4, and one APMV-8 were isolated from 1,967 wild-bird fecal samples.  The results showed that NDV and APMV-4 are relatively widely distributed among wild waterfowl that migrate to Japan from northern regions. Phylogenetic analysis revealed that there was no genetic relationship between the isolates from wild birds and domestic poultry in Japan.  However, surveillance of APMVs in wild waterfowl needs to be conducted due to the pathogenic potential of these isolates in domestic poultry.
"

Епізоотичний моніторинг останніх років свідчить про те, що високопатогенний вірус грипу А птахів (H1N1) та (H7N9) активно циркулює на території Євразійських держав. За 2016 – 2019 рр. було зафіксовано1,6 тис. випадків спалахів. З них у Європі 872 випадки. Питання моніторингу за інфікованою як перелітною так і свійською птицею в місцях перехресного контакту в Україні є актуальним для запобігання виникнення спалахів епізоотії.  Мета дослідження. Розробити експрес – метод ідентифікації та визначення вірусу пташиного грипу А Н1N1 та Н7N9 штамів на основі полімеразної ланцюгової реакції з аналізом поліморфізму довжин рестрикційних фрагментів (ПЛР-ПДРФ) РНК вірусу. Результати та обговорення. Проведений аналіз in silico ампліконів генів HA, NA та NP дозволив in silico розрахувати праймери до варіабельних локусів досліджуваних генів, розрахувати умови проведення реакції, визначити сайти рестрикції до добраних рестриктаз отримати теоретичні електрофореграми ПЛР – ПДРФ аналізу. Розроблено методику експрес – методу для виявлення та ідентифікації вірусу грипу А H1N1 та H7N9 за трьома генами (HA, NA та NP) РНК H1N1 та H7N9 у полімеразній ланцюговій реакції ПЛР суміщеній з ПДРФ аналізом. Метод експрес – діагностики здатний виявляти вірус пташиного грипу А H1N1 та H7N9 і диференціювати його від зразків інших збудників вірусних інфекцій птахів і тварин.  Висновки. Розроблений метод експрес – ідентифікації на основі ПЛР суміщений з ПДРФ аналізом дає можливість значно спростити метод ідентифікації за рахунок специфічної ампліфікації ділянки РНК, що має поліморфний сайт рестрикції. Тестування стану цього локуса можливе шляхом попереднього проведення ПЛР та рестрикції ампліфікованого фрагменту. Встановлено, що метод експрес – діагностики ПЛР-ПДРФ здатний виявляти РНК вірус грипу А високопатогенних штамів H1N1 та H7N9 з високими показниками чутливості (100 % чутливість) Ключові слова: високопатогенний вірус пташиного грипу А H1N1, H7N9, експрес-метод діагностики ПЛР-ПДРФ, консервативні мотиви, варіабельні локуси, поліморфізм

In this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheic calves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII) were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested. Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1 and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while one was related to the Bo/Newbury1/76/UK strain.

Filoviruses are highly lethal in humans and nonhuman primates, likely due to potent antagonism of host interferon (IFN) responses early in infection. Filoviral protein VP35 is implicated as the major IFN induction antagonist, while Ebola virus (EBOV) VP24 or Marburg virus (MARV) VP40 are known to block downstream IFN signaling. Despite progress elucidating EBOV and MARV antagonist function, those for most other filoviruses, including Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV), Bundibugyo (BDBV) and Ravn (RAVV) viruses, remain largely neglected. Thus, using standardized vectors and reporter assays, we characterized activities by each IFN antagonist from all known ebolavirus and marburgvirus species side-by-side. We uncover noncanonical suppression of IFN induction by ebolavirus VP24, differing potencies by MARV and RAVV proteins, and intriguingly, weaker antagonism by VP24 of RESTV. These underlying molecular explanations for differential virulence in humans could guide future investigations of more-neglected filoviruses as well as treatment and vaccine studies.

We describe 2 bat-transmitted outbreaks in remote,
rural areas of Portel and Viseu Municipalities, Par  State,
northern Brazil. Central nervous system specimens were
taken after patients’ deaths and underwent immunofluorescent
assay and histopathologic examination for rabies antigens;
also, specimens were injected intracerebrally into
suckling mice in an attempt to isolate the virus. Strains
obtained were antigenically and genetically characterized.
Twenty-one persons died due to paralytic rabies in the 2
municipalities. Ten rabies virus strains were isolated from
human specimens; 2 other cases were diagnosed by
histopathologic examination. Isolates were antigenically
characterized as Desmodus rotundus variant 3 (AgV3).
DNA sequencing of 6 strains showed that they were genetically
close to D. rotundus–related strains isolated in Brazil.
The genetic results were similar to those obtained by using
monoclonal antibodies and support the conclusion that the
isolates studied belong to the same rabies cycle, the virus
variants found in the vampire bat D. rotundus.

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Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI ® Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI ® RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI ® RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI ® RVP-Plus assay in 33% of hMPV-positive patients.

Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically di...

Although human diseases of retrotransposition-derived etiology have been documented, retrotransposon RNA expression and the occurrence of retrotransposition events in the human oocyte are not studied. We investigated the RNA expression of L1 and HERV-K10 retrotransposons in human oocytes by RT-PCR analysis with designed primers. Using denucleated germinal vesicles (GVs), we detected RT-PCR products of expressed L1, HERV-K10 and, unexpectedly, SINE-R, VNTR and Alu (SVA) retrotransposons. Their transcript specificities were identified as such following RNA-FISH and their origin by cloning and sequence alignment analyses. Assessing the expression level in comparison with somatic cells by densitometry analysis, we found that although in normal lymphocytes and transformed HeLa cells their profile was in an order of L1 > HERV-K10 > SVA, remarkably this was reversed in oocytes. To investigate whether de novo retrotransposition events occur and reverse transcriptases are expressed in the human oocyte, we introduced in GVs either a retrotransposition active human L1 or mouse reverse transcriptase deficient-VL30 retrotransposon tagged with an EGFP-based retrotransposition cassette. Interestingly, in both the cases, we observed EGFP-positive oocytes, associated with an abnormal morphology for L1 and granulation for VL30, and the retrotransposition events were confirmed by PCR. Our results: (i) show that L1, HERV-K10 and SVA retrotransposons are transcriptionally expressed and (ii) provide evidence, for the first time, for retrotransposition events occurring in the human oocyte. These findings suggest that both, network of retrotransposon transcripts and controlled retrotranspositions, might serve important functions required for oocyte development and fertilization while the uncontrolled ones might explain the onset of genetic disorders.

Abbreviations: VL30s (viral-like 30 elements); NAC (N-acetylcysteine); ROS (reactive oxygen species); EGFP (enhanced green fluorescent protein); FACS (fluorescence-activated cell scanning); LTR (long terminal repeat); LINE-1 (L1) (long interspersed nuclear element-1); LT (large Tantigen); MOMP (mitochondrial outer membrane permeabilization); LMP (lysosomal membrane permeabilization)