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The human gut is colonised by a vast array of microbes that include bacteria, viruses, fungi, and archaea. While interest in these microbial entities has largely focused on the bacterial constituents, recently the viral component has attracted more attention. Metagenomic advances, compared to classical isolation procedures, have greatly enhanced our understanding of the composition, diversity, and function of viruses in the human microbiome (virome). We highlight that viral extraction methodologies are crucial in terms of identifying and characterising communities of viruses infecting eukaryotes and bacteria. Different viral extraction protocols, including those used in some of the most significant human virome publications to date, have introduced biases affecting their a overall conclusions. It is important that protocol variations should be clearly highlighted across studies, with the ultimate goal of identifying and acknowledging biases associated with different protocols and, perhaps, the generation of an unbiased and standardised method for examining this portion of the human microbiome.

Genetic variants of the COVID-19 causative virus have been arising and circulating globally. In many countries, especially in developing ones with a huge population, vaccination has become one of the major challenges. SARS-CoV-2 variants’ fast transmission rate has an upsurge in the COVID cases, leading to more stress on health systems. In the current COVID-19 scenario, there is the requirement of more adequate diagnostic approaches to check the COVID-19 spread. Out of many diagnostic approaches, a magnetic nanoparticle-based reverse transcription polymerase chain reaction could be nontrivial. The use of magnetic nanoparticles is to separate nucleic acid of SARS-CoV-2 from the patient samples and apply for SARS-CoV-2 detection in an easy and more effective way. Herein, the magnetic nanoparticles are synthesized using the solgel autocombustion methods and then successfully coated with biopolymer (chitosan) using ultrasonication. Chitosan-coated nanoparticles are successfully integrated into the graphene oxide sheets to introduce carboxyl groups. Crystallite size calculation, morphological and magnetic studies of synthesized magnetic nanoparticles, and multifunctional magnetic nanoparticles are done using XRD, SEM, TEM, and VSM, respectively. Besides, the potentiality of the fabricated nanocomposites in RNA extraction protocol is also discussed with schematic representation.

Human noroviruses (HuNov) are the major cause of foodborne illness globally. Several HuNoV outbreaks have been linked to contaminated ready-to-eat seaweed products. Standard protocols such as the ISO 15216 show limited efficiency in extracting foodborne viruses from seaweed products. Therefore, we evaluated the efficiency of an extraction protocol based on porcine gastric mucin conjugated magnetic beads (PGM-MBs) to recover HuNoVs from Wakame seaweed salad. Compared to other HuNoV extraction methods, the PGM-MB method was more efficient. We then aimed to further improve this protocol by modifying several factors such as the buffers, pH, bead concentration, centrifugation and incubation time. The optimized PGM-MB method yielded 19 ± 3% and 17 ± 4% recovery, for HuNoV GI and GII, respectively. The limit of detection (LOD95) for Wakame seaweed salad was 131 and 56 genomic equivalents per 25 g for HuNoV GI and GII. Although some variability in recovery efficiency was observed between the PGM sources, the optimized PGM-MB protocol effectively extracts HuNoVs from Wakame seaweed salads of various brands and other commodities such as dates, green onions, and salted seaweed. These results support the implementation of the optimized PGM-MB method as a viable alternative for HuNoV surveillance in complex food matrices.

Foodborne viruses such as human norovirus (HuNoV) and hepatitis A virus (HAV) are the major causes of foodborne illnesses worldwide. These viruses have a low infectious dose and are persistent in the environment and food for weeks. Ready-to-eat (RTE) low moisture foods (LMFs) undergo minimal pathogen reduction processes. In recent years, multiple foodborne HAV outbreaks involving hundreds of individuals were associated with the consumption of dates, indicating that they could be important vehicles for foodborne infection. There is no standard method for the extraction and detection of foodborne viruses from dates, but herein we have compared the efficiency of three different protocols based on the ISO 15216 method in the extraction of murine norovirus (MNV) from whole Medjool dates and successfully employed the best performing method in the extraction of HAV, HuNoV GI, and GII and determined the limit of detection (LOD95) of 61, 148, and 184 genomic equivalent (gEq) per 25 g, respectively. Finally, we tested the adopted method on various varieties of dates including pitted ones and reported the detection of HuNoV GI and GII from four naturally contaminated date varieties. This ISO 15216 protocol could be employed for surveillance purposes and outbreak management related to dates.

Background Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly. Results In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis. Conclusions The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

In March 2019, the Finnish Institute for Health and Welfare and Finnish Food Authority started an outbreak investigation after a notification of food business operators’ recall of frozen bilberries due to a norovirus finding. A retrospective search was conducted in the food and waterborne outbreak notification system to identify the notifications linked to norovirus and consumption of bilberries in January–March 2019. Five outbreaks were found in which norovirus GII or GII.17 had been detected in patient samples. A pooled retrospective cohort study was performed for those four in which a questionnaire study had been done. A case was defined as a person with diarrhoea or vomiting within 2 days after consuming a meal studied at one of the outbreak locations. Of 79 participants, 45 (57%) cases were identified. Persons that had consumed foods containing unheated bilberries were three times more likely to get ill than those who had not consumed them (RR 3.1, CI 95% 1.2–8.1, p  = 0.02). Norovirus GII.17 was found in 16/17 patient samples sent for further typing. Identical norovirus GII.17 was detected in frozen Finnish bilberries and patient samples. At the berry packaging premises, signs of norovirus GII contamination were found in packaging lines. A new procedure for extracting viral nucleic acid from food and environmental samples was used during the outbreak investigation. Consumption of industrially packed frozen berries as heated would be one of the means to prevent norovirus infections.

Dengue virus RNA purification from human plasma is useful for research and clinical purposes. Dengue is endemic in the Espirito Santo State, Brazil, and it is progressively becoming a hard-to-control public health problem [ 1 ]. Dengue virus types 1, 2 and 3 are currently found in Brazilian territory, and recently Dengue virus type 4 has been reported to enter Brazilian borders. This virus spreads rapidly during epidemic outbreaks, and thousands of patients are infected annually, with an underestimated number of deaths in consequence of hemorrhagic Dengue. Because this disease affects mainly developing countries, it is imperative that a robust, rapid and low cost method for viral nucleic acid purification is found. In this manuscript we compare two RNA extraction methods from serum/plasma of patients with clinical diagnosis of dengue. The QIAamp ® UltraSens Virus Kit (Qiagen Inc., Valencia, USA) and the less expensive Chomczynski–Sacchi method were used to analyze a total of 47 samples. After nucleic acid purification, reverse transcription and polymerase chain reaction amplification with dengue virus type 2 specific primers were performed. This subtype is the most prevalent in our geographical location. Thirty-four samples were positive when RNA was extracted by the Chomczynski–Sacchi technique, whereas only 27 of these were positive when the QIAamp ® UltraSens Virus Kit was used. These results favor the utilization of the more affordable technique for the purification of viral RNA, which is especially important for developing countries [ 2 ].

Avian influenza is a serious threat to both animal and human health. To enable cutting-edge research in this field, we developed a molecular test on the basis of real-time polymerase chain reaction (real-time PCR), which detects influenza virus RNA. The test enables highly sensitive detection of influenza virus A and B strains, including H5N1, and specific identification of influenza A virus H5 subtypes. The kit was tested against a broad panel of influenza A and B subtypes and other respiratory viruses in collaboration with worldwide authoritative laboratories and shows a very high specificity and sensitivity. An internal control verifies RNA extraction as well as real-time PCR success. With this kit, rapid and reliable detection of influenza A and B viruses and identification of H5 subtypes can be achieved.