Monkeypox virus diagnosis and laboratory testing

The multi-country outbreak of monkeypox virus (MPXV) infection, while the coronavirus disease 2019 pandemic is still an ongoing issue, has caused a new challenge. The re-emergence of MPXV and the rising incidence in non-endemic countries is turning into an upcoming threat to global health. Hence, rapid identification of the virus with appropriate methodology with the lowest false results plays a critical role in estimating the global extent of the crisis and providing preventive measures. This review summarised the main applicable strategies for primary detection and confirmation of MPXV and highlighted available data in biosafety, requirements, standard operating procedures, specimen collection, transportation and storage of clinical samples, and waste disposal of the viral agent. Also, various assays including molecular techniques, immunoassays, histopathological methods, electron microscopy, genomic sequencing, and cell culture have been illustrated. Moreover, we reflected on current knowledge of the advantages and disadvantages of each approach.     

Mpox diagnostics: Review of current and emerging technologies

Mpox is a zoonotic disease caused by monkeypox virus (MPXV) from the Orthopoxvirus genus. Unprecedented transmission events have led to more than 70 000 cases reported worldwide by October 2022. The change in mpox epidemiology has raised concerns of its ability to establish endemicity beyond its traditional geographical locations. In this review, we discuss the current understanding of mpox virology and viral dynamics that are relevant to mpox diagnostics. A synopsis of the traditional and emerging laboratory technologies useful for MPXV detection and in guiding “elimination” strategies is outlined in this review. Importantly, development in MPXV genomics has rapidly advanced our understanding of the role of viral evolution and adaptation in the current outbreak.     

The pathophysiological and immunological background of the monkeypox virus infection: An update

In addition to the COVID-19 waves, the globe is facing global monkeypox (MPX) outbreak. MPX is an uncommon zoonotic infection characterized by symptoms similar to smallpox. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus that belongs to the genus Orthopoxvirus (OPXV). MPXV, which causes human disease, has been confined to Africa for many years, with only a few isolated cases in other areas. Outside of Africa, the continuing MPXV outbreak in multiple countries in 2022 is the greatest in recorded history. The current outbreak, with over 10 000 confirmed cases in over 50 countries between May and July 2022, demonstrates that MPXV may travel rapidly among humans and pose a danger to human health worldwide. The rapid spread of such outbreaks in recent times has elevated MPX to the status of a rising zoonotic disease with significant epidemic potential. While the MPXV is not as deadly or contagious as the variola virus that causes smallpox, it poses a threat because it could evolve into a more potent human pathogen. This review assesses the potential threat to the human population and provides a brief overview of what is currently known about this reemerging virus. By analyzing the biological effects of MPXV on human health, its shifting epidemiological footprint, and currently available therapeutic options, this review has presented the most recent insights into the biology of the virus. This study also clarifies the key potential causes that could be to blame for the present MPX outbreak and draw attention to major research questions and promising new avenues for combating the current MPX epidemic.     

Monkeypox: Clinical Considerations,Epidemiology, and Laboratory Diagnostics

Monkeypox virus (MPXV) has garnered recent attention as outbreaks are continually reported outside historic regions of endemicity in Africa. Consequently, MPXV is becoming routinely included in the differential diagnosis of rash illnesses, requiring clinicians and laboratorians alike to quickly adapt to a new public health emergency. This review discusses the epidemiology, clinical presentation, and laboratory testing of MPXV in the context of recent outbreaks.     

Loop‐mediated isothermal amplification‐based diagnostic assay for monkeypox virus infections

Monkeypox virus (MPXV) causes a smallpox‐like disease in non‐human primates and humans. This infection is endemic to central and western Africa. MPXV is divided into two genetically different groups, Congo Basin and West African MPXV, with the former being the more virulent. A real‐time quantitative MPXV genome amplification system was developed for the diagnosis of MPXV infections using loop‐mediated isothermal amplification (LAMP) technology. Primers used for genome amplification of Congo Basin (C‐LAMP), West African (W‐LAMP), and both Congo Basin and West African (COM‐LAMP) MPXV by LAMP were designed according to the nucleotide sequences of the Congo Basin‐specific D14L gene, the West African‐specific partial ATI gene, and the partial ATI gene that is shared by both groups, respectively. The sensitivity and specificity of the LAMP were evaluated with nested PCR using peripheral blood and throat swab specimens collected from Congo Basin MPXV or West African MPXV‐infected monkeys. The sensitivity and specificity of COM‐LAMP, C‐LAMP, and W‐LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively. The viremia level determined by LAMP assays increased with increases in the severity of the monkeypox‐associated symptoms. The newly developed LAMP assay was confirmed to be a rapid, quantifiable, and highly sensitive and specific system effective in the diagnosis of MPXV infections. The LAMP assays made it possible to discriminate between Congo Basin and West African MPXV. The LAMP developed in this study is useful not only for diagnosis of but also for the assessment of MPXV infections. J. Med. Virol. 81:1102–1108, 2009. © 2009 Wiley‐Liss, Inc.     

Experimental infection of an African dormouse (Graphiurus kelleni) with monkeypox virus

Suitable animal models are needed to study monkeypox virus (MPXV) as human monkeypox clinically resembles smallpox and MPXV is a zoonotic and potential bioterroristic agent. We have demonstrated that a species of African dormice, Graphiurus kelleni, is susceptible to a lethal infection of MPXV and that MPXV replicated in multiple organs of this species. Following intranasal administration, MPXV replicated locally in the nasal mucosa causing necrosis and hemorrhage with subsequent systemic spread to lymph nodes, spleen, liver, and other tissues where it caused severe necrosis and/or hemorrhage leading to death. The dormouse model was validated for testing prophylactic (Dryvax vaccine) and therapeutic (cidofovir) test articles against intranasal challenges with MPXV.     

Clinical presentation,viral kinetics,and management of human monkeypox cases from New Delhi,India 2022

We describe the clinical and demographic characteristics, virological follow-up, and management of five confirmed monkeypox cases from New Delhi, India without any international travel history. The viral load kinetics and viral clearance were estimated in oropharyngeal swabs (OPS), nasopharyngeal swabs (NPS), EDTA blood, serum, urine, and various lesion specimens on every fourth day of follow-up ranging from 5 to 24 post onset day (POD) of illness. All five cases presented with mild to moderate-grade intermittent fever, myalgia, and lesions on the genitals, groins, lower limb, trunk, and upper limb. Four cases had non-tender firm lymphadenopathy. No secondary complications or sexually transmitted infections were recorded in these cases except for the presence of viral hepatitis B infection marker hepatitis B virus surface antigen (HBsAg) in one case. All the cases were mild and had a good recovery. A higher viral load was detected in lesion fluid (POD 9), followed by lesion roof (POD 9), urine (POD 5), lesion base (POD 5), and OPS/NPS (POD 5). The monkeypox virus (MPXV) DNA was detected in clinical samples from 5th to 24th POD. These monkeypox cases without international travel history suggest the underdiagnosed monkeypox infection in the community. This emphasizes the need for active surveillance of MPXV in the high-risk population such as men having sex with men and female sex workers.     

Dosage comparison of Congo Basin and West African strains of monkeypox virus using a prairie dog animal model of systemic orthopoxvirus disease

The prairie dog is valuable for the study of monkeypox virus (MPXV) virulence and closely resembles human systemic orthopoxvirus disease. Herein, we utilize a variable dose intranasal challenge with approximately 10³, 10⁴, 10⁵, and 10⁶ PFU for each clade to further characterize virulence differences between the two MPXV clades. A trend of increased morbidity and mortality as well as greater viral shedding was observed with increasing viral challenge dose. Additionally, there appeared to be a delay in onset of disease for animals challenged with lower dosages of virus. Mathematical calculations were used to determine LD₅₀ values and based on these calculations, Congo Basin MPXV had approximately a hundred times lower LD₅₀ value than the West African clade (5.9 × 10³ and 1.29 × 10⁵ respectively); reinforcing previous findings that Congo Basin MPXV is more virulent.     

Detection of monkeypox virus with real-time PCR assays.

BACKGROUND: Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak. OBJECTIVES: We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak. STUDY DESIGN: A TaqMan-based assay (E9L-NVAR) targets the orthopoxvirus DNA polymerase gene and detects Eurasian orthopoxviruses other than Variola. A hybridization assay, utilizing a MGB Eclipsetrade mark (Epoch Biosciences) probe, targets an envelope protein gene (B6R) and specifically detects monkeypox virus (MPXV). Assays were validated using coded orthopoxvirus DNA samples and used to evaluate lesion samples from five confirmed US monkeypox cases. RESULTS: E9L-NVAR did not detect variola (48 strains), North American orthopoxviruses (2), or DNA derived from non-poxviral rash illnesses. The assay reproducibly identified various concentrations of 13 Eurasian orthopoxvirus strains and was sensitive to 12.5 vaccinia genomes. The B6R assay recognized 15 different MPXV strains, while other orthopoxvirus (9) and bacteria (15) strains did not cross-react. Of the 13 human samples tested from confirmed cases, both assays identified 100% as containing MPXV DNA. CONCLUSIONS: E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections.     

Oropharyngeal samples versus lesion specimens at diagnosis in patients infected with monkeypox virus in Northern France

The ongoing outbreak of monkeypox virus (MPXV) is the largest one in historically non-endemic countries. Early reports described atypical epidemiological and clinical presentations. We investigated MPXV DNA detection in oropharyngeal samples (OPS), and compared the viral load to that in lesion samples at diagnosis in patients infected with MPXV. We retrospectively included patients suspected to have monkeypox in Northern France, who underwent a MPXV PCR in the Virology Laboratory, University Hospital of Lille, from May 23 to August 18, 2022. Overall, a total of 228 patients (376 samples) were included. A positive result in at least one sample was found in 138 patients (60.5%). We compared PCR results between OPS and lesion samples (i.e., cutaneous or anal/rectal samples) in patients with both samples. A positive result in OPS was observed in 54 out of 60 patients (90%). The viral load in OPS (median C_t value = 29.5; interquartile range [IQR] = 24.7−34) was significantly lower than that in lesion samples (median C_t value = 17.8; IQR = 16.3 and 19.7) (p < 0.0001). This report shows that pharyngeal sampling does not bring additional information for the initial diagnosis in patients presenting with typical lesions.     

Susceptibility to winter vomiting disease: a sweet matter

Norovirus, the cause of winter vomiting disease, has emerged in recent years to be a major cause of sporadic and epidemic gastroenteritis worldwide. The virus has been estimated to cause >200,000 deaths each year in developing countries. Although the virus is highly contagious, volunteer and field studies have shown that a subset of individuals appears resistant to infections. A single nucleotide mutation (G428A) in the fucosyltransferase gene (FUT2) on chromosome 19 provides strong protection from infection in 20% of the white population. Histo-blood group ABO(H) antigens with terminal fucose are believed to function as receptors for human norovirus in the gastrointestinal tract, but also negatively charged potential receptors have been identified. Norovirus infection is a unique example where a single nucleotide mutation in a fucosyltransferase gene plays a crucial role in susceptibility to one of the most common viral diseases. This review discusses the role of host genetics and carbohydrate structures in susceptibility to winter vomiting disease.     

Dendritic cells in viral pathogenesis: protective or defective?

Dendritic cells (DC) are potent antigen-presenting cells that are critical in the initiation of immune responses to control and/or eliminate viral infections. Recent studies have investigated the effects of virus infection on the biology of DC. This review summarizes these changes, focusing on both the DC parameters affected and the viral factors involved. In addition, the central role of DC biology in the pathogenesis of several viral families, including herpesviruses, paramyxoviruses and retroviruses, is explored. The field of pathogen recognition by DC is addressed, focusing on its role in protecting the host from viral infection, as well as the ability of viruses to exploit such host receptor ligation and signalling to their replicative advantage. The hypothesis is proposed that virus and host have evolved a symbiotic relationship to ensure both viral transmission and host survival.     

The multifarious roles of heterogeneous ribonucleoprotein A1 in viral infections

Viruses are obligate parasites known to interact with a wide variety of host proteins at different stages of infection. Current antiviral treatments target viral proteins and may be compromised due to the emergence of drug resistant viral strains. Targeting viral‐host interactions is now gaining recognition as an alternative approach against viral infections. Recent research has revealed that heterogeneous ribonucleoprotein A1, an RNA‐binding protein, plays an essential functional and regulatory role in the life cycle of many viruses. In this review, we summarize the interactions between heterogeneous ribonucleoprotein A1 (hnRNPA1) and multiple viral proteins during the life cycle of RNA and DNA viruses. hnRNPA1 protein levels are modulated differently, in different viruses, which further dictates its stability, function, and intracellular localization. Multiple reports have emphasized that in Sindbis virus, enteroviruses, porcine endemic diarrhea virus, and rhinovirus infection, hnRNPA1 enhances viral replication and survival. However, in others like hepatitis C virus and human T‐cell lymphotropic virus, it exerts a protective response. The involvement of hnRNPA1 in viral infections highlights its importance as a central regulator of host and viral gene expression. Understanding the nature of these interactions will increase our understanding of specific viral infections and pathogenesis and eventually aid in the development of novel and robust antiviral intervention strategies.     

Dengue virus infection activates cellular chaperone Hsp70 in THP-1 cells: downregulation of Hsp70 by siRNA revealed decreased viral replication

The pathogenic mechanism of dengue virus infection is related to the host responses within target cells, and therefore we assessed intracellular changes in stress proteins following dengue virus infection. This study provides evidence that Hsp70 helps in viral multiplication by suppressing the type 1 interferon response. Dengue virus infection in human monocytic THP-1 cells led to overexpression of Hsp70, which also acts as a chaperone. The functional role of Hsp70 in dengue virus multiplication was identified by downregulating the Hsp70 gene with its specific siRNA duplexes, which led to a decrease in viral RNA copy numbers in cellular supernatants and intracellular viral load. It also resulted in an increased IFN-α level, which mediates its antiviral effect through double-stranded RNA-induced protein kinase-PKR. Collectively these results suggest that an increased level of Hsp70 expression in dengue-virus-infected THP-1 cells assists in viral replication by escaping the antiviral effect of type 1 interferon.     

Infection of cynomolgus macaques with a recombinant monkeypox virus encoding green fluorescent protein

Monkeypox virus (MPXV) causes a vesiculopustular rash illness resembling smallpox in humans and produces a similar disease in nonhuman primates. To enhance the ability of researchers to study experimental MPXV infections, we inserted a gene encoding green fluorescent protein (GFP) into Monkeypox virus Zaire-79. Wild-type and MPXV-GFP replicated with similar kinetics in cell culture and caused a similar disease when injected intravenously into cynomolgus macaques. In MPXV-GFP-infected animals, examination under fluorescent light facilitated the identification of skin lesions during disease development and internal sites of replication at necropsy. MPXV-GFP could improve the quantitative assessment of antiviral therapy and vaccine efficacy.     

Immune control of HSV-1 latency

A hallmark of the herpes family of viruses is their ability to cause recurrent disease. Upon primary infection, Herpes Simplex virus (HSV) establishes a latent infection in sensory neurons that persists for the life of the individual. Reactivation of these latent viral genomes with virion formation is the source of virus for most HSV recurrent disease. This review details recent exciting findings supporting a role for the host immune system, particularly CD8+ T cells in maintaining HSV-1 in a latent state.     

The role of galectins in virus infection - A systemic literature review

BackgroundGalectins are β-Galactose binding lectins expressed in numerous cells and play multiple roles in various physiological and cellular functions. However, few information is available regarding the role of galectins in virus infections. Here, we conducted a systemic literature review to analyze the role of galectins in human virus infection.MethodsThis study uses a systematic method to identify and select eligible articles according to the PRISMA guidelines. References were selected from PubMed, Web of Science and Google Scholar database covering publication dated from August 1995 to December 2018.ResultsResults indicate that galectins play multiple roles in regulation of virus infections. Galectin-1 (Gal-1), galectin-3 (Gal-3), galectin-8 (Gal-8), and galectin-9 (Gal-9) were found as the most predominant galectins reported to participate in virus infection. The regulatory function of galectins occurs by extracellularly binding to viral glycosylated envelope proteins, interacting with ligands or receptors on immune cells, or acting intracellularly with viral or cellular components in the cytoplasm. Several galectins express either positive or negative regulatory role, while some had dual regulatory capabilities on virus propagation based on the conditions and their localization. However, limited information about the endogenous function of galectins were found. Therefore, the endogenous effects of galectins in host-virus regulation remains valuable to investigate.ConclusionsThis study offers information regarding the various roles galectins shown in viral infection and suggest that galectins can potentially be used as viral therapeutic targets or antagonists.     

Intracellular RNA recognition pathway activates strong anti‐viral response in human mast cells

Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti‐viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double‐stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti‐viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF‐α. In contrast, extracellular Toll‐like receptor 3 (TLR)‐3‐activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti‐viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up‐regulated the expression of melanoma differentiation–associated gene 5 (MDA‐5) and retinoic acid‐inducible gene‐1 (RIG‐1), and TLR‐3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti‐viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti‐viral proteins human myxovirus resistance gene A (MxA) and interferon‐induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.     

Hepatitis C virus spontaneous clearance: immunology and genetic variance

Abstract Hepatitis C virus (HCV) infection is one of the most common chronic viral infections in the world. Approximately 80-90% of acutely infected individuals develop persistent infection, which is a major risk for liver cirrhosis and liver cancer. However, a small portion of patients (10-20%) clear the virus. Clinical outcomes of HCV infection are determined by the interplay between the host immune response, and viral and environmental factors. In regulating immune responses, cytokines play an indispensable role that controls the underlying pathogenesis and the resulting outcome of HCV infection. Cytokines themselves are manipulated by polymorphisms in their genes. In fact, the majority of genetic variants that apparently confer a significant risk for chronic HCV infection have been localized in genes involved in cytokine synthesis and the ultimate immune response. So far, treatment strategies for HCV infection have remained controversial. Genotyping of different polymorphisms will aid clinical decision making for both current standard and personalized care. Genotyping can potentially be useful for future integration of other agents, which provides an opportunity for clinicians to personalize treatment regimens for HCV patients. This review summarizes findings of different studies on host immune responses after HCV infection and the association between cytokine gene polymorphisms and the likelihood of HCV clearance.