Some of the newly emerging corona viral variants show high numbers of mutations. This is unexpected for a virus with a low mutation rate due to an inherent proof-reading system. Could such a variant arise under very special conditions occurring in a host where the virus replicates and mutates in a rather unlimited fashion, such as in immune compromised patients? The virus was shown to replicate in an immunosuppressed cancer patient for more than 105 days and might be a source of new variants. These patients are asymptomatic and the virus may therefore escape detection and attention and be high-risk. Similarly, HIV-infected individuals may be immunocompromised and support coronavirus replication with increased mutation rates. The patients may promote “within-host evolution”. Some of the viruses present in such a highly mutagenic swarm or quasispecies within one patient may become founders and cause a pandemic by further “between-host evolution”. B.1.1.7 with 23 mutations may be such a case. Immunosuppressed patients can be identified and treated by the synthetic antibody cocktails as passive immunization and kept under control. Immunosuppressed patients can be easily identified and supervised by healthcare workers—once they become aware of the risk—to avoid new variants with pandemic potential. Viruses mutate. Especially single-stranded RNA viruses have a high mutation rate because their genome is not stabilized by a second matching strand as we know it from the DNA double-helix. It is an inherent viral property and survival strategy to mutate, as it guarantees a large number of progeny viruses even under restrictive conditions. Environmental obstacles can be overcome by escape mutations. Viruses have one major feature: to multiply and generate progeny.What we know about the single-stranded RNA viruses such as influenza or HIV is not true for coronaviruses—that was the hope of scientists. In contrast to all other known RNA viruses, coronaviruses do not mutate as much. They have a correction system for genome replication, a proofreading enzyme, which removes mismatched nucleotides during replication and transcription ,2. The nNature Communications which found that, among 46,723 isolated and characterized CoV-2 virus genomes from 99 countries, nothing alarming was observed, no mutations associated with increased transmissibility and no indication of viral adaptation to faster spread. Recurrent mutations were evolutionarily neutral and likely induced by the human immune system. There was no matter for concern [The error frequency of CoV-2 is about two mutations per month . This is concern .Most people have now forgotten about a mutation which was even briefly described as the “German” mutant, which, although started in China, was then detected in Germany and went around the world almost a year ago . The mutMost scientists did not expect many further mutations. The virus appeared to be so “fit”, as Charles Darwin defined the success of a species. There did not seem to be a capacity for further increase and selection for higher fitness. No selective pressure or selective advantage seemed to exist to push the virus to change with so many hosts available around the world. However, with so many virus particles spreading around the world, even a slow mutation rate can lead to new viral variants, most of which were characterized by several mutations.Now, we are witnessing almost a repetition of the previous situation. A new CoV-2 variant was discovered and announced on 4 January 2021, B.1.1.7, which stirred concern and led to shutdowns. This mutant is indeed very highly infectious and may increase the probability of severe disease. It could become a new pandemic.The B.1.1.7 lineage, also known as N501Y.V1, was first discovered in Kent county in England in December 2020 and within six weeks became the dominant CoV-2 strain in London . The existence of this variant is very surprising, because, in light of the low mutation rate of coronaviruses, one must find an explanation why it exhibits a total of 17 unique mutations . Six mutations are located in the S protein, which is in total about 1300 amino acids long, with the RBD spanning a region from amino acids 333 to 527 . Not allHow can one possibly explain so many mutations and such dramatic changes within one virus isolate?Could such a variant with as many as 23 mutations arise under very special conditions occurring in a host where the virus replicates and mutates in a rather unlimited fashion as in the absence of an immune system in an immunocompromised individual? Cell, in December of 2020, which describes rather unrestricted virus production in an immunocompromised individual with cancer [A recent paper discusses exactly this, it was published by the American National Institute of Health in the Journal h cancer . Here, aThe doctors treated the patient with the serum of a patient who had survived the disease and had antibodies in his blood, the convalescent serum. This treatment is an indirect or passive immunization ,14. TheyPrevention of virus replication could be better achieved by synthetic monoclonal antibody therapy instead of convalescent serum. These are supplied by companies such as Regeneron or Eli Lilly which have received emergency approval for treatment of people early during infection or even as prevention of infection. These synthetic antibody cocktails have proven to reduce the viral load early during infection and for patients undergoing organ transplants or chemotherapy. The reduction of hospitalization and death after early treatment was described as 70% ,14. The This patient showed several deletions in the virus isolates. Deletions had also been observed in the pandemic coronavirus from 2003 and correlated with disappearance of the virus and termination of the pandemic . Thus, tAnother report also described an immunocompromised host with persistent viral infection and accelerated viral evolution for 150 days. He was treated with synthetic antibodies leading to three viral reductions and new peaks. He developed 4 new mutations within the spike region and over time 8 additional ones, some disappeared, others were found in structural proteins and the polymerase region . Immunocompromised patients may accumulate more mutations than regular patients where the viruses are more constant and may have to be controlled more carefully. High numbers of mutations were also detected in the South African Variant, B.1.351, among them the mutation E484K, which was meanwhile also taken over by the British variant, and there HIV-infected people may also be endangered by some compromised immunity where long-term shedding and altered infection dynamics could result. Recently, an unusual variant with 40 mutations was detected in Angola, where HIV infection rates are high. The South African variants raise the question to what extend HIV/AIDS contribute to their evolution. A potential risk needs to be analyzed. A model shows the immunocompromised patient with viral “within-host” evolution and many variants .A characteristic property of all viruses is their occurrence as quasispecies, viruses form swarms of closely related members which are, however, distinct. This is due to the above-mentioned error frequency of the polymerase during replication. The high number of mutants may contain members which are more infectious and replication competent than others and will enable one of them to become the winner. In an immune compromised patient, no immune system counteracts the virus, it could freely mutate without immune restrictions. The fastest replicating virus will be the winner. Perhaps B.1.1.7 is such a winner. Furthermore, the selection of the fastest virus is independent of the later disease, which would probably not be of a selective advantage for the virus. Immunosuppressed patients are numerous. An estimated 3 million people in the United States have some form of immunocompromising condition, including individuals with HIV infection, solid organ transplant recipients, hematopoietic stem cell transplant recipients and individuals receiving chemotherapy or corticosteroids. The immunocompromised population is furthermore at higher risk of respiratory infections and disease complications .Virologists know from studies in the laboratory that if one grows a virus in cell culture, the viruses will change and mutate and adjust to the experimental or selective conditions. To give an example, in 2015, a coronavirus was engineered in the laboratory with the goal to study viral evolution, determine future emerging viruses and evaluate the disease potential of SARS-like viruses. They adapted the virus to grow efficiently in primary human lung cells with high titers and it gained higher pathogenicity. The research and further studies were restricted by government-mandated pause as too risky. This kind of adaptation is defined as gain-of-function (GoF). It is performed to investigate which mutations could make a virus dangerous and a potential threat. “Further testing in nonhuman primates is required”, but was forbidden and must be outweighed against potential risks . Nature and Science did not want to publish the details on how to generate such viruses and judged they could be bioweapons. This research was defined as “dual use”, use for learning about dangerous mutants and abuse for bioterrorism as biological weapon. One of the studies was transiently stopped by a moratorium but was ultimately published in reduced form. New regulatory guidelines evaluating risk versus benefits were issued [The gain of function mutants have previously been generated by two research groups led by Y. Kawaoka and R.A. Fouchier, with influenza virus H5N1 and the goal to define and predict which mutations are potentially dangerous and may give rise to a pandemic. The journals e issued ,21. The No more than biosafety level BSL3 laboratories are required for these GoF studies including coronavirus and more potentially pandemic viruses. Only monkey studies require BSL4. These rules should be changed. Meanwhile, as of February 2021, the British variant, characterized by N501Y, has acquired an additional eighths mutation within the spike protein, the same mutation E484K as present in the South African variant B.1.351, V2, which also contains N501Y and others, K417N, N439K-in total nine mutations in the spike region. The mutations E484K and N501Y are important for binding to the ACE2 Receptor. N501Y in the British variant is thought to be responsible for the high infectivity, worldwide fast spread and increased pathogenicity, while the E484K mutation leads to reduction of the effectiveness of some of the vaccines. A third variant in Brazil, P1 or B.1.1.248, also contains these two mutations and K417T in the spike region and in total 17 mutations ,23.In the city of Manaus, Brazil, achievement of herd immunity had been assumed with about three-quarters of the population seroconverted. This was estimated as above the theoretical threshold for herd immunity (60–67%) ,24. For In addition, in southern California a new mutant showed up, CAL.20C, which is spreading faster and may cause more severe disease that the wild-type virus. It was first detected in July 2020 in Los Angeles and from September to January increased to a prevalence of above 50% in California. It also occurs in other States of the US. It is more infectious, associated with more severe illness and partially resistant against neutralizing antibodies. It is a variant “of concern”. There are two forms, B.1.427 and B.1.429 with slightly different mutations, both are also referred to as 20C/L452R. It has three mutations, one of them being L452R in the RBD. None of these three spike mutations is found in the three other main variants, from the UK, South Africa and Brazil ,25. Furthermore, a new variant appeared in New York recently described on Feb. 23rd, 2021, B.1.526 with seven spike mutations  [The new Indian variant B.1.617 first detected in Oct 2020 and in Feb. 2021 in England, has 13 mutations, two of them in the RBD such as E484Q and L452R.Some of the mentioned variants exhibit some identical mutations. It is assumed that they arose independently. These mutations in the RBD and other regions must have been independently selected for by their higher fitness, increased virus reproduction rates and provided a selective advantage by escape from the immunity of a host against a previous variant. This raises some hope, that the various escape mutations are identical. These variants are either spontaneous mutants, due to “between-host evolution”, or they are escape mutations . In contThere is some immunity even against the common seasonal coronaviruses in the population, against the four coronaviruses we encounter every winter. Immunological memory after infection with seasonal human coronaviruses was shown to contribute to cross-protection against CoV-2 . This isEven though influenza and coronaviruses differ in many respects, they share some features. They differ in duration of the disease and the onset of the disease after infection. However, similarly, the influenza viruses evolve rapidly at a global scale. Interestingly, Xue et al.  from SeaInfluenza virus infections typically last for 5–7 days. Acutely infecting viruses peak with viral production after 2–3 days and this is a short period to develop many mutations. Higher diversity can be caused by infection with multiple related viral strains. Chronic infections, in contrast, can favor extensive evolution and antigenic variants can reach high within-host frequencies. In addition, drug-resistant variants may arise. An important parameter for intra-host variations may also be attributed to the initial viral dose, the multiplicity of infection (MOI). The new variants may become founders of one or several global variants. The within-host arising variations may parallel global evolutionary trends.Beginning in December 2020, the van Dorp group at the University College, London, mentioned above, found 12,706 mutations in CoV-2, 398 of these occurring frequently and repeatedly and 185 have occurring at least three times. The authors ask whether they happened to occur and what the chance may be for higher transmission by a so-called founder effect, leading to more efficient transmission and possibly the start of a pandemic. To them, this did not appear to be the case [We may have to become more aware of newly emerging variants and we may have to watch out specifically for immunosuppressed people and try to reduce the risk of evolution of new virus variants in such specific cohorts. A selected number of people hot-spots, rather than random screening, would be sufficient watch out for these; they need a focused observation, testing and follow-up. The newly emerging viruses can be detected by new software tools, e.g., the Variant Data Base (VDB) founded at the California Institute of Technology, with a focus on the spike sequence . Perhaps


Mutations in ABCD4 or LMBRD1, which encode the ABC transporter ABCD4 and lysosomal membrane protein LMBD1, respectively, lead to errors in cobalamin metabolism, with the phenotype of a failure to release cobalamin from lysosomes. However, the mechanism of transport of cobalamin across the lysosomal membrane remains unknown. We previously demonstrated that LMBD1 is required for the translocation of ABCD4 from the endoplasmic reticulum to lysosomes. This suggests that ABCD4 performs an important function in lysosomal membrane cobalamin transport. In this study, we expressed human ABCD4 and LMBD1 in methylotrophic yeast and purified them. We prepared ABCD4 and/or LMBD1 containing liposomes loaded with cobalamin and then quantified the release of cobalamin from the liposomes by reverse-phase HPLC. We observed that ABCD4 was able to transport cobalamin from the inside to the outside of liposomes dependent on its ATPase activity and that LMBD1 exhibited no cobalamin transport activity. These results suggest that ABCD4 may be capable of transporting cobalamin from the lysosomal lumen to the cytosol. Furthermore, we examined a series of ABCD4 missense mutations to understand how these alterations impair cobalamin transport. Our findings give insight into the molecular mechanism of cobalamin transport by which ABCD4 involves and its importance in cobalamin deficiency.Vitamin B The ABC transporters comprise a superfamily of membrane-bound proteins that exist in almost all organisms from eubacteria to mammals. In humans, there are 48 members classified into the seven subfamilies A to G, based on structural organization and amino acid homology. These proteins catalyze the ATP-dependent transmembrane transport of a wide variety of substrates in order to sustain cellular homeostasis. Defect in their functions results in various inherited metabolic diseases , 2.2-terminal hydrophobic region responsible for peroxisomal targeting. As a result, ABCD4 is recognized by signal recognition particles and integrated into the endoplasmic reticulum (ER) membrane  from the lysosomal lumen to the cytosol, since the dysfunction of ABCD4 results in the failure of lysosomal cobalamin efflux  and reconstituted purified ABCD4 and/or LMBD1 in liposomes, as reported previously  and then purified by affinity chromatography (A). Since ABCD4 functions as a homodimer in mammalian cells .ABCD4 and LMBD1 form a complex on lysosomal membranes and are involved in the transport of cobalamin transport across the lysosomal membrane , 14. ABCablished . His-ABCtography A. Since an cells , 20, we eviously A.Figure A, approximately 40 kDa band emerged. This band seems to be derived from ABCD4 existing in inside-out orientation. Approximately 70% of ABCD4 reconstituted into liposomes existed in right-side-out orientation calculated from the signal intensities . The 110 kDa band is a nonspecific protein derived from the yeast cells during His-tag affinity chromatography. This protein is not an ABCD4-associated protein because it was not coimmunoprecipitated with ABCD4 (A). Since this protein is also reconstituted in liposomes, the liposomes containing only this protein were used as a negative control (B).To examine proper insertion of ABCD4 on liposomes, we evaluated the proportion of the right-side-out orientation. ABCD4-liposomes were treated with trypsin and subjected to immunoblot analysis using anti-ABCD4 antibody recognizing C-terminal 149 amino acid of ABCD4. As shown in Fig. S2th ABCD4 A. Since  control B.B, ABCD4 displayed a ∼140 kDa band consistent in mass with dimeric ABCD4 when SDA was photoactivated by UV irradiation. Subsequently, the ATPase activity of liposome-reconstituted ABCD4 was assayed based on the release of organic phosphate from ATP. The reconstituted ABCD4 exhibited ATPase activity, and the activity was increased in a time- and dose-dependent manner (C and D). These results indicate that ABCD4 was successfully reconstituted in liposomes in the active form.As mentioned previously, ABCD4 functions as a homodimer in mammalian cells , 20. We Figure 1t manner , C and DA). On the other hand, we prepared ABCD4-liposomes without cobalamin and then measured the ATPase activity with cobalamin and ATP in the reaction mixture. As shown in Figure 2A, the addition of cobalamin did not stimulate the ATPase activity of ABCD4. Subsequently, we evaluated the ATPase activities of ABCD4-liposomes containing various concentrations of cobalamin. The stimulation of ABCD4 ATPase activity was increased in proportion to the concentration of cobalamin inside of the ABCD4-liposomes (B). These results indicate that the cobalamin inside liposomes is recognized by ABCD4 as substrate and stimulates the ATP hydrolysis of ABCD4.As is well known, the ATPase activity of most ABC transporters is stimulated in the presence of transport substrate. We examined whether cobalamin stimulates the ATPase activity of ABCD4. As ABCD4 exits liposomes in both an inside-out and right-side-out orientation, we initially prepared ABCD4-liposomes with cobalamin and measured ATPase activities by the addition of ATP from outside the liposomes. The ATPase activity of ABCD4 was stimulated when cobalamin was present inside of the ABCD4-liposomes A. On theiposomes B. These A, cobalamin was transported in a time-dependent manner by ABCD4. Kinetic properties were determined using ABCD4 containing liposomes loaded with various concentrations of cobalamin. Lineweaver–Burk plot was used to calculate the Km and Vmax values, which came out as 426 μM and 667 pmol/min/mg, respectively  was purified and reconstituted in liposomes using the same procedure as used for His-ABCD4 (A). It was confirmed that this mutant had lost ATPase activity (B). In the transport assay, cobalamin was not released from ABCD4 (K427A)-liposomes loaded with cobalamin at any time point (A). This indicates that ABCD4 (K427A) had lost transport activity in addition to ATPase activity. These results demonstrated that ABCD4 transports cobalamin from the inside to the outside of liposomes in a manner that is dependent on ATPase activity.Next, to test whether cobalamin transport depends on ATPase activity, we prepared ABCD4 (K427A), a Walker A lysine mutant deficient in ATPase activity. We also expressed His-ABCD4 (K427A) under the control of the is-ABCD4 A. It wasactivity B. In theme point A. This iLMBRD1, which encodes the lysosomal membrane protein LMBD1, result in the failure of cobalamin release from lysosomes, which is the same phenotype as seen in ABCD4 dysfunction. We previously demonstrated that LMBD1 interacts with ABCD4 and regulates the translocation of ABCD4 from the ER to lysosomes in mammalian cells (K. phaffii strain expressing glutathione-S-transferase (GST)–tagged LMBD1. The expressed LMBD1–GST was solubilized with β-DDM and then purified by GST-tag affinity chromatography . In addition, LMBD1 was not coprecipitated with His-ABCD1 unlike ABCD4 (B), suggesting that interaction of ABCD4 with LMBD1 is specific, and it is not nonspecific interactions between two misfolded membrane proteins. Furthermore, the binding stoichiometry between purified ABCD4 and LMBD1 was analyzed by crosslinking. As shown in Fig. S9C, ∼300 kDa band consistent in mass with one dimeric His-ABCD4 plus one dimeric LMBD1–GST was detected in addition to dimeric His-ABCD4 or dimeric LMBD1–GST when cross-linking reagents were photoactivated by UV irradiation. These results indicate that purified ABCD4 and LMBD1 interact each other with a 1:1 stoichiometry.As described previously, mutations in an cells , suggesttography . Then we agarose A. In addke ABCD4 B, suggesC). ABCD4/LMBD1-liposomes also exhibited both ATPase and cobalamin transport activity. There was no difference in either the ATPase or the cobalamin transport activities compared with the ABCD4-liposomes (B and C). LMBD1 itself had neither ATPase nor cobalamin transport activities (B and C).Subsequently, we prepared ABCD4/LMBD1-liposomes. It was confirmed that ABCD4 and LMBD1 maintained the same binding stoichiometry in ABCD4/LMBD1-liposomes C. ABCD4/iposomes , B and Ctivities , B and CABCD4 gene that result in cobalamin deficiency have been reported  and the lysosomal side of transmembrane helix 6 (TM6), respectively, as shown by cryo-EM  and ABCD4 (Y319C) in mammalian cells. We previously established CHO cells stably expressing LMBD1–GFP and demonstrated that the ABCD4–hemagglutinin (HA) transiently expressed in these cells is localized to lysosomes . ABCD4 , indicatK. phaffii strain expressing His-ABCD4 (N141K) or His-ABCD4 (Y319C). Both types of mutant ABCD4 were purified and reconstituted in liposomes using the same procedure as used for His-ABCD4 (A). Moreover, it was confirmed that these mutants were reconstituted in liposomes as a dimer (B). Subsequently, the ATPase and transport activities of these mutants were examined. As shown in A and B, ABCD4 (N141K) exhibited ATPase activity corresponding with the wildtype but lost cobalamin transport activity. On the other hand, ABCD4 (Y319C) lost both the ATPase and transport activities.Next, to evaluate the function of ABCD4 (N141K) and ABCD4 (Y319C), we prepared a is-ABCD4 A. Moreov a dimer B. Subseq141 is located on the cytosolic side of TM3 and faces the transmembrane cavity and is deduced to form a hydrogen bond with Asp221, which is located on the cytosolic side of TM4  and ABCD4 (N141D) also exhibited ATPase activity equal to that of the wildtype. Interestingly, the cobalamin transport activity was recovered to the same level as the wildtype in both ABCD4 (N141A) and ABCD4 (N141D) (B). These results indicate that an increase in the anionic charge in this area by substitution of Asn141 to Lys141 might disturb the cobalamin transport of ABCD4.Asn (N141D) B. These 319, substitution to Cys resulted in the loss of the ATPase activity as well as cobalamin transport activity. Surprisingly, Tyr319 is located on the lumen side of TM6 far away from the NBD that is indispensable for ATPase activity  using an ATP probe for photoaffinity labeling (A), since ABCD4 (Y319C) lacks ATPase activity. As a result, ABCD4 (Y319C) was labeled by the ATP probe under UV irradiation, and this labeling was inhibited by the presence of adenylyl imidodiphosphate, a nonhydrolyzable analog of ATP, in a dose-dependent manner (B). These results suggest that the conformational change coupled to the hydrolysis of ATP was disturbed in the Y319C mutant. Recently, in Cyanidioschyzon merolae ABCB1, the van der Waals contacts and hydrogen-bonding network in the cluster of aromatic hydrophobic side chains in the upper part of the transmembrane helices were shown to play an important role in the interactions among the transmembrane helices  and ABCD4 (Y319F) and evaluated both the ATPase and cobalamin transport activities. As shown in A and B, neither the ATPase activity nor the transport activity was recovered in ABCD4 (Y319A). In contrast, ABCD4 (Y319F) recovered both the ATPase and transport activities (A and B). These results indicate that Tyr319 in ABCD4 plays an important role in the conformational change related to cobalamin transport.In the case of Tyractivity . To evallabeling A, since t manner B. These  helices , 24. Thetivities , A and BABCD4 and LMBRD1, which encode ABCD4 and LMBD1, respectively, result in a failure to release cobalamin from lysosomes . It is predicted that the COOH-terminal half of ABCD4, including the NBD, is exposed to the cytosolic surface , SUR1 (ABCC8), and SUR2 (ABCC9) are categorized as importers  channels built from four SUR subunits . These results indicate that LMBD1 is not directly involved in cobalamin transport on the lysosomal membrane, although the dysfunction of LMBD1 did result in a failure in lysosomal cobalamin release. Lysosomes contain more than 60 hydrolases to degrade extracellular materials as well as intracellular components. Many of the lysosomal membrane proteins are highly glycosylated within the lysosomal lumen in order to let them escape lysosomal proteolysis  exhibits much lower ATPase activity than the wildtype (A), the defect of cobalamin metabolism in the R432Q mutant is almost certainly because of the loss of ATPase activity.There are eight clinically relevant mutations in wildtype . Since iwildtype A, the de141 and Tyr319 are located on the cytosolic side of TM3 and the lysosomal side of TM6, respectively. ABCD4 (N141K) lacks only cobalamin transport activity, whereas ABCD4 (Y319C) lacks both ATPase and cobalamin transport activity (A and B). From the amino acid coordinates of ABCD4 deposited in the Protein Data Bank (6JBJ), Asn141 faces the transmembrane cavity  and ABCD4 (N141D) (B), an increase in the cationic charge around Asn141 by substitution to Lys141 might disturb the cobalamin transport of ABCD4. Asn141 is believed to form a hydrogen bond with Asp221 located on the cytosolic side of TM4. However, the substitution of Asn141 to Ala141, which does not form a hydrogen bond with Asp221, did not influence the cobalamin transport activity of ABCD4. It is thus suggested that the hydrogen bond between Asn141 and Asp221 is not important for cobalamin transport. From the cryo-EM structure, the distance between Asn141 and Asp221 is very close (2.5 Å)  B, an inc (2.5 Å) . Since t319 is located on the lysosomal side of TM6 and deduced to be involved in the hydrophobic entrance of the transmembrane cavity (A and B), disturbance in the accessibility of cobalamin to the cavity does not seem to be the exclusive reason for the impaired transport of cobalamin. It is speculated that in the Y319C mutant, the two Cys319 residues have a tendency to form a disulfide bond in a dimeric conformation (A and B). ABCD4 (Y319C) was able to bind but not hydrolyze ATP . These results indicate that the Y319C mutant has an additional conformational change impairment. Recently, the inward- and outward-facing X-ray crystal structure of C. merolae ABCB1 was reported, and the structural features during conformational realignment revealed based on high-resolution crystallographic data . Further crystallographic study of ABCD4 will be needed.Tyre cavity . Since te cavity , A and Bormation  and, as ormation , A and Bhic data . The aroIn this study, it was demonstrated for the first time that ABCD4 transports cobalamin from the lysosomal lumen to the cytosol as an “importer.” In contrast, other members of the ABC transporter subfamily D, ABCD1‒3, are involved in the transport of fatty acyl-CoAs from the cytosol to the peroxisomal lumen. Furthermore, it is recently reported that some bacterial ABC transporters, which possess the same transmembrane folding with mammalian ABC exporters, function as an importer , 33. EluK. phaffii strain SMD1168 his4 was used as the host strain to express the human ABCD4 and LMBD1. The strains were grown in 1% yeast extract, 2% peptone, and 2% glucose or buffered minimal (0.5% yeast extract and 0.5% methanol) medium.g for 10 min. The resulting supernatant (cell-free extract) was subjected to centrifugation at 14,000g for 30 min to obtain an organelle pellet. Membranes were solubilized by 0.5% β-DDM for 3 h on an end-over-end rotator. Insoluble material was removed by centrifugation at 100,000g for 30 min. The supernatant was incubated with cOmplete His-Tag Purification Resin (Roche) with 5 mM imidazole on an end-over-end rotator for 16 h. Subsequently, the resin was washed twice with Tris buffer containing 0.1% β-DDM and 50 mM imidazole, and His-ABCD4 was eluted with Tris buffer containing 0.1% β-DDM and 500 mM imidazole.The expressed proteins were purified according to the procedure previously described  with somTo purify LMBD1–GST, solubilization of LMBD1–GST was performed by the same procedure as used for His-ABCD4. The supernatant after β-DDM treatment was incubated with COSMOGEL GST-Accept agarose . The resin was washed with Tris buffer and eluted with Tris buffer containing 50 mM glutathione.Protein concentration in eluate fraction was determined by the method of Bradford. As His-ABCD4 and the nonspecific protein existed in the eluate fraction, the amount of His-ABCD4 was calculated from the ratio of the intensity of His-ABCD4 and the nonspecific protein in acrylamide gel after SDS-PAGE and Coomassie brilliant blue staining.l-α-phosphatidylcholine  was suspended in the suspension buffer (20 mM Tris–HCl at pH 7.5). The mixture was sonicated until clear in a bath-type sonicator and then frozen and thawed five times. Liposomes were stored at −80 °C until use. Aliquot of 50 μg of protein in eluate fraction was mixed with 500 μg of liposomes in a 1:10 ratio (W/W) and then frozen and thawed twice. The mixture was diluted 30-fold with the reconstitution buffer (20 mM Tris–HCl at pH 7.5 and 0.5 mM DTT). Reconstituted proteoliposomes were pelleted via centrifugation at 150,000g for 1 h at 4 °C and then suspended in 200 μl of 20 mM Tris–HCl at pH 7.5. To prepare proteoliposomes containing both ABCD4 and LMBD1, protein solutions of His-ABCD4 and LMBD1–GST were incubated for 1 h at 4 °C before being mixed with the liposomes. To prepare proteoliposomes containing cobalamin, we added cobalamin to both the suspension and the reconstitution buffer. The amount of ABCD4 incorporated into liposomes was calculated by immunoblot analysis of His-ABCD4 using His-ABCD4 in eluate fraction as standard. The signal strength of His-ABCD4 was quantified by the ImageJ image analysis software (NIH).Liposomes were prepared as previously described . Soybean2, 1.1 mM EGTA, 2.2 mM DTT, 10 mM sodium azide, and 2 mM ouabain). The reaction was started by the addition of ATP  and incubated at 37 °C. This reaction was stopped by the addition of 1 N HCl and supplemented with malachite green solution . Absorption at 620 nm was measured after incubation at 30 °C for 30 min.The ATPase activity of the reconstituted proteins in the liposomes was determined by measuring the release of inorganic phosphate from ATP, as previously described . Proteolm-maleimidobenzoyl-N-hydroxysuccinimide ester (Thermo Fisher Scientific)  was used in addition to SDA.ABCD4-liposomes were incubated with SDA (Thermo Fisher Scientific)  at room temperature for 30 min. The reaction was stopped by the addition of Tris–HCl at pH 8.8 at a final concentration of 200 mM and then incubated on ice for 15 min. SDA-labeled proteins were photoactivated by UV irradiation at 365 nm for 1 min. Samples were subjected to immunoblot analysis. To evaluate binding stoichiometry between ABCD4 and LMBD1, 2, 4 mM DTT, and 2 mM ATP) at 37 °C. Aliquots were removed at various time points and applied to a Sephadex G-50 column  to collect the proteoliposomes. The proteoliposomes were then disrupted with 1% Triton X-100 and applied on a COSMOSIL 5C18-MS-II column  at a flow rate of 1 ml/min equilibrated with 20 mM ammonium acetate buffer. Elution was performed with a linear gradient of an increasing acetonitrile concentration  in 20 mM ammonium acetate. Cobalamin was detected by UV 361 nm. The amount of cobalamin transported from inside of the liposomes at each time point was calculated by subtraction of the amount of cobalamin in the liposomes at each incubation from that without any incubation.Proteoliposomes containing 100 μM of cobalamin were incubated with the reaction buffer  were mixed and rotated at 4 °C for 15 min. The mixture was then incubated with cOmplete His-tagged Resin or COSMOGEL GST-Accept agarose overnight at 4 °C. The resin or agarose was collected by centrifugation and washed twice with wash buffer . The bound proteins were eluted in SDS contained in the sample buffer and subjected to immunoblot analysis.All data are given in the main article or supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Lyssavirus genus, was the first rabies-related virus described in 1956. This virus is endemic to the African continent and is rarely encountered. There are currently four lineages, although the observed genetic diversity exceeds existing lyssavirus species demarcation criteria. Several exposures to rabid bats infected with LBV have been reported; however, no known human cases have been reported to date. This review provides the history of LBV and summarizes previous knowledge as well as new detections. Genetic diversity, pathogenesis and prevention are re-evaluated and discussed.Lagos bat virus (LBV), one of the 17 accepted viral species of the  Lyssavirus genus (n = 8) from passive surveillance in South Africa. The genetic diversity is re-evaluated, and the pathogenicity and implications for prevention and control are discussed.Until the 1950s, it was thought that rabies virus (RABV) was the only causative agent of the disease known as rabies, but this changed when the first rabies-related virus, Lagos bat virus (LBV), was detected in 1956 in Nigeria , and 17 us genus  3]. Of . Of LyssEidolon helvum bats in 1956 on Lagos Island in Nigeria via the mouse inoculation test. This virus, LBV, was not neutralized by any rabies immune sera, indicating that it was not a strain of RABV [Surveillance efforts focused on encephalitic and tropical fevers isolated a virus from male  of RABV ,8. DurinE. helvum again 29 years later in Senegal [E. helvum bat. Several organs were also positive for viral RNA, including the brain, salivary glands, tongue, bladder, lung, stomach, adrenal glands, liver, heart, ovaries and kidneys [E. helvum bat [A total of four LBV detections have been reported. After the first detection in 1956, LBV was only detected in  kidneys . In 2013lvum bat .Epomophorus wahlbergi have been restricted to the KwaZulu-Natal (KZN) province in South Africa and were first detected in the early 1980s [E. wahlbergi, although it is believed that all individuals were of the same species. Of the 13 positive bats, only three were subsequently isolated and confirmed to be LBV with monoclonal antibody tests [E. wahlbergi bat found dead in 1990 [E. wahlbergi again over a decade later in 2003 after implementing a passive surveillance study [E. wahlbergi was submitted for rabies testing after being caught by a domestic cat [E. wahlbergi bat on her lawn one morning. She reported hearing squeaking noises around her house the previous evening. The FAT was positive for both bats with subsequent successful virus isolation, monoclonal antibody typing, RT-PCR and sequencing [E. wahlbergi bat with her pup still attached on the lawn. The caretaker placed the bats in a nearby tree. However, they were later found on the ground again, where a cat was toying with them. The bats were then submitted to a local bat rehabilitator, though the adult bat had died. The FAT performed for the adult bat was negative; however, RT-PCR detected LBV RNA in the brain [E. wahlbergi bat was submitted for rabies diagnosis from a bat rehabilitator in 2006. The bat was initially very calm but appeared to be dehydrated and had trouble breathing. The bat was given water and liquidized fruit; although the bat drank the liquids, the bat rehabilitator noted that the throat was inflamed with severe swelling. After consumption of the liquids, the bat started producing excessive amounts of saliva and shaking its head. The eyes of the bat appeared to be opaque, and its breathing deteriorated. The bat eventually started choking and died shortly after. This bat’s brain material was positive with FAT and identified as LBV with RT-PCR and sequencing [E. wahlbergi bat submitted for rabies diagnosis by a local veterinarian. The veterinarian initially treated the bat for hyperthermia; however, the veterinarian decided to euthanize the bat after it started to display neurological signs, including difficulty swallowing [All LBV detections in ly 1980s . The prody tests ,13. The ce study  during wce study ,15. In 2stic cat . In 2004quencing . The folhe brain . The puphe brain . An E. wquencing . Shortlyallowing .Pteropus sp. but was later corrected to more likely be Rousettus sp. [Rousettus aegyptiacus in Kenya [7.5 MICLD50  was detected in a salivary gland [In 1999, a bat imported from Africa (possibly Togo or Egypt) died in the department du Gard, France, and was shown to be infected with LBV. The bat species involved was initially reported as ttus sp. ,19. Duriin Kenya ,21. All ry gland ,22.Micropterus pusillus (current taxonomic status: Epomophorus pusillus [The first and only detection of LBV in pusillus ) occurrepusillus . It was pusillus .Nycteris gambianus) during a survey in 1985 in Guinea [Lagos bat virus was detected for the first and only time in an insectivorous bat  [The first report of LBV in a spillover host  was in nfection . During nfection . In 2003nfection . The firdinosus) . The aniR. aegyptiacus, all cases occurred in the KZN province of South Africa.Through passive surveillance networks, which include national diagnostic laboratories, bat rehabilitation centres, and bat interest groups, eight new cases of LBV have been identified in South Africa since 2013. Except for one case in E. wahlbergi bat was found grounded at a private residence in Scottburgh by children in 2013. They first thought that the bat was dead, but they provided the bat with water after discovering that it was alive. It was noted that the bat behaved abnormally. A local bat rehabilitator collected the bat, and it was observed that the bat was listless and had lost the use of its back legs. The bat’s condition deteriorated , but it continued to drink water. The bat died shortly after that. The brain material tested positive with FAT and quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) [A large male qRT-PCR)  . The veterinarian and a veterinary nurse at the clinic cared for the bat for approximately two weeks before it suddenly died. During this time, the bat appeared healthy, with no clinical signs of illness. The veterinary nurse was also bitten on the finger by the bat, and as such, post-exposure prophylaxis was initiated. The bat carcass was submitted for rabies diagnosis, and brain material tested positive with FAT and qRT-PCR [4 RNA copies), intestine (1.62 × 104 RNA copies) and the salivary glands (5.62 × 103 RNA copies) [In January 2014, a juvenile  qRT-PCR  (Allerto copies) .E. wahlbergi bat was found on a path  by children in Umhlanga. The bat was submitted to a local veterinarian and appeared to be healthy. However, due to the human exposures involved, post-exposure prophylaxis was initiated, and the bat was euthanized and submitted for rabies diagnosis. The brain material tested positive with FAT and qRT-PCR [In April 2016, an adult male  qRT-PCR  . The bat was submitted to a local bat rehabilitator that noted a pneumonia-like disease. The bat died approximately one month later and tested positive with FAT and qRT-PCR [During the same time, another male  qRT-PCR  . The reported clinical signs included profuse salivation, panting, general weakness, teeth grinding and the inability to open its eyes. The brain material tested positive for viral RNA with qRT-PCR [In March 2018, an adult female  qRT-PCR  . His two dogs attempted to retrieve the bats, during which one of the dogs received a bite from one of the bats. Both bats were submitted for rabies testing. One of these bats tested positive for viral RNA with qRT-PCR  , when sampled as part of a broader biosurveillance program on viruses. The brain material tested positive for viral RNA with qRT-PCR [During the same time, an adult female  qRT-PCR   and the fluorescent antibody virus neutralization test (FAVN) are most frequently used ,36. BothE. helvum in Ghana could neutralize a lineage A virus  but could not neutralize a lineage B virus  [However, the interpretation of serological results is significantly influenced by the cross-reactivity of antibodies to different lyssavirus species and the cut-off thresholds used in the assay . For lysa, 1956) .The antibody prevalence in a specific population is determined by comparing the results obtained to a reference cut-off value . TherefoLyssavirus genus was initially divided into four serotypes, i.e., RABV (serogroup 1), LBV (serogroup 2), MOKV (serogroup 3) and DUVV (serogroup 4), based on reaction patterns with monoclonal antibodies [R. aegyptiacus as the only known associated host , no clinical signs were observed before the bats were found dead [Experimental infections  have mosstigated ,64. Simistigated ,64. Howeund dead . This phund dead .E. helvum with LBV indicated that bats that survived the challenge had developed virus neutralizing antibodies (53%), suggesting that bats can develop an immune response without clinical disease development and thus not excrete virus [The mechanisms of virus maintenance and transmission of lyssaviruses in bats are not well understood and could be influenced by various factors ,26. The te virus . This woCommercial rabies biologics (rabies immunoglobulin (RIG) and vaccines) are based on RABV , and it Lagos bat virus is a unique endemic African lyssavirus, and detections remain rare and sporadic, mostly linked to where specific surveillance programs have been implemented. Surveillance efforts usually include only limited species diversity, mainly focused on those species previously implicated in infection. Viral nucleic acid detection has indicated an association of LBV with specific bat species, predominantly frugivorous bats. LBV lineages also appear to be associated with particular bat species. Serological surveillance has implicated a much broader geographical and species range, highlighting the lack of surveillance. Lyssaviruses are neurotropic and only disseminate late in infection to other organs and potential excretions; however, virus shedding may be intermittent. As such, the most reliable sample for viral nucleic acid detection will be post-mortem brain material. Detection rates using this strategy are very low (below 1%). Considering the vital part bats play in ecosystems, combined with reduced population sizes and increased habitat destruction ,80, thisSerology on serum collected from healthy bat populations can be used as a surveillance tool through non-destructive sampling to obtain an estimate of the circulating diversity in specific species in a geographical space. However, serological studies’ inherent limitations, including the potential cross-reactivity of lyssaviruses, should be considered. Seroprevalence in natural bat populations is significantly higher than the rate of virus detection. Specific bat species are regarded as reservoirs of lyssavirus infection and can generate an antibody response after initial infection. This mechanism is still not understood and potentially attributed to an abortive infection outside the central nervous system . SpillovThere is currently a lack of knowledge on the host–pathogen interactions at the level of the pathogen  and the host . Our understanding of the LBV infection dynamics, factors and mechanisms necessary for LBV transmission and maintenance, spillover mechanisms and information regarding bat population dynamics remains incomplete. Only once surveillance efforts are aligned with bat ecology and conservation using a one-health approach can we begin to address these fundamental but inherently complicated questions.


I conducted this study to develop an improved method for glycome detection using fluorescent magnetic beads, whose surfaces were modified using lectins, for the highly sensitive detection of saccharides or glycoproteins via fluorescence quenching using a novel fluorescence emitter and quencher pair. The emitter (Cy3 fluorophore) was incorporated into magnetic beads, and a fluorescence quencher (cyanopyranyl group) was bound to glycomes via covalent bonding. The fluorescence intensities of fluorescent magnetic beads containing lectins decreased specifically in the presence of glycomes, which was a result of fluorescence quenching from Cy3 to cyanopyranyl groups due to the formation of a stable complex between lectins and glycome. Fluorescence intensities were plotted as a function of glycoprotein concentration, and good linear relationships were observed. This method enabled the fluorescent reading-out of a series of lectin-glycome interactions on the basis of recognition selectivity and affinity of immobilized lectins without tedious washing processes. Moreover, a simple profiling process was performed using this assay for diverse glycoconjugates, which not only included simple saccharides but also glycoproteins and glycome in cell lysates. These results clearly indicate that the combination of magnetic beads with the novel emitter-quencher pair enabled the highly sensitive detection of lectin-glycome interactions. The glycome, which is the repertoire of glycans, such as oligosaccharides, glycolipids, and glycoproteins, produced by individual biological systems such as cells, tissues, and organisms, has been widely studied to modulate numerous physiological and pathological states ,2. GlycoRecently, optical biosensors based on nanomaterials have been designed and synthesized for the analysis of various biological substances based on fluorescence spectroscopic methods ,20,21,22I previously developed fluorescent sensors for the detection of various saccharides based on lectin-saccharide interactions using fluorescent magnetic beads . LectinsTherefore, I conducted this study to develop an improved method for glycome detection that utilizes the properties of nanometer-sized fluorescent magnetic beads, whose surfaces were modified using lectins via avidin-biotin interactions (FMB-Lectin), as shown in All chemicals used were of analytical grade, and they were all purchased from Tokyo Chemical Industry , Wako Pure Chemical Industries, Ltd. , or Sigma-Aldrich. Fluorescent magnetic beads modified with streptavidin were purchased from Tamagawa Seiki Co., Ltd. . Absorption spectra were recorded at 25 °C using a UV/visible spectrophotometer , and fluorescence spectra were recorded at 25 °C using a JASCO FP-6500 fluorophotometer.(E)-2--2-oxoethoxy)styryl)-6-methyl-4H-pyran-4-ylidene)malononitrile (Quen-1) was labeled to glycoproteins as follows: The activated-esterized compound Quen-1 was dissolved in 50 μL dimethylsulfoxide and the mixture was added to 500 μL of PBS buffer (pH 7.4) in the presence of 1.0 mg glycoprotein. The reaction mixture was stirred at 4 °C for 12 h, and it was purified via gel permeation chromatography (eluent: PBS buffer). Based on the UV-VIS spectra, the first fraction, which was yellow in color, was identified as a glycoprotein modified using Quen-1. The optimal experimental conditions for the immobilization of biotinylated lectins onto fluorescent streptavidin magnetic beads were as follows: 1 mg of fluorescent streptavidin magnetic beads (50 µL of 20 mg/mL beads) were added to each of the microtubes, and subsequently, PBS buffer solution was added. After spinning down, the beads were magnetically separated, and PBS buffer solution was then added. After the addition of biotinylated lectin (100 μL) at a concentration of 0.5 mg/mL in PBS, the reaction mixture was stored at 4 °C for 1 h. After spinning down, the reacted magnetic beads were magnetically separated, and they were then washed with HEPES buffer solution (pH 7.9) twice. After addition of preservative buffer, the fluorescent magnetic beads modified using lectins were stored at 4 °C.Fluorescent magnetic beads labeled with lectin were added to HEPES buffer solution (pH 7.0) at a final concentration of 1.0 μg/100 μL. After the addition of 50 μL of glycoprotein to 50 μL of fluorescent magnetic beads, the reaction mixture was incubated. After spin down, magnetic separation, and brief washing, the fluorescence spectra of reacted fluorescent magnetic beads were recorded to analyze the amount of lectin-glycoprotein complexes.Carbohydrate inhibitors were added to the solution of fluorescent magnetic beads labeled using lectin and glycoprotein in PBS buffer solution (pH 7.0), and the changes in fluorescence intensities were monitored over time. The carbohydrate inhibitors used were as follows: α-lactose (0.1 M), melibiose (0.1 M), d-mannose (0.1 M), N-acetyl-d-glucosamine (0.05 M), and α-L-fucose (0.05 M).The activated-esterized compound Quen-1 was dissolved in 50 μL dimethylsulfoxide and the mixture was added to 50 μL of cell lysates. The reaction mixture was stirred at 4 °C for 12 h, and was used for the measurement using lectin-modified fluorescent magnetic beads without further purification. The biotinylated lectin concanavalin A (Con-A) was immobilized on the surface of streptavidin-based magnetic beads containing a fluorophore (Cy3) via avidin-biotin interactions, as shown in The fluorescence intensities of FMB-ConA at 560 nm were plotted as a function of RNase B concentration, and the results are shown in To investigate the selectivity of lectin-labeled fluorescent magnetic beads against various glycoproteins, biotinylated lectins, such as Con A, wheat germ agglutinin (WGA), and aleuria aurantia lectin (AAL), were modified on the surface of streptavidin-based fluorescent magnetic beads (FMB-WGA and FMB-AAL), and fluorescence intensities were monitored after the addition of RNase B modified by Quen-1. To observe the dependence of pH on the fluorescence from FMB-ConA, the fluorescence intensities of FMB-ConA were monitored at different pH values ranging from 4 to 9. The data are shown in The reaction time between FMB-Con A and RNase B attached to Quen-1 was investigated by monitoring the fluorescence intensities of FMB-Con A, and the results are shown in Inhibitor sugars, such as D-glucose and α-L-fucose, were used for the recovery of glycoproteins adsorbed on the lectin column. To take advantage of this phenomenon, the glycoproteins adsorbed on fluorescent magnetic beads were recovered, and the elution process was monitored by assessing the changes in fluorescence intensities with time after the addition of inhibitor sugars. To investigate the lectin-glycoprotein interactions and release of glycoproteins from fluorescent magnetic beads by the addition of inhibitor sugars using this method, WGA and Ulex europaeus agglutinin I (UEA-1) were immobilized on the surface of fluorescent magnetic beads . The interactions between FMB-WGA and Ovalbumin and FMB-UEA-1 and Mucin, and the effect of the addition of inhibitor sugars, such as N-acetyl-D-glucosamine for FMB-WGA and α-L-fucose for FMB-UEA-1, were monitored by observing the changes in fluorescence intensities over time. The results are shown in This method, constructed from the magnetic bead-based assay for the detection of glycoproteins, was compared with enzyme-linked immunosorbent assay (ELISA) methods from the viewpoint of quantitative analysis of glycoproteins. Fluorescent magnetic beads were modified using lens culinaris agglutinin (LCA) lectin via avidin-biotin bonding for the detection of human transferrin (FMB-LCA), whereas commercially available ELISA kits were used for the detection of human transferrin. Using these analytical methods, the unknown concentrations of human transferrin samples were detected, and the observed concentrations from calibration graphs were compared. As an application of this method using real samples, cell lysates originating from MCF-7 were profiled. Fluorescent magnetic beads were modified using Con A, WGA, AAL, UEA-1, and GSL-1, and they were reacted with cell lysates from MCF-7, which were monitored using fluorescence spectrometry. In conclusion, fluorescent magnetic beads containing lectins were synthesized, and lectin-saccharide and lectin-glycoprotein interactions were detected with the use of “on-off”-based sensing systems. Moreover, the sensing system could fluorescently respond to the types and concentrations of sugars or glycoproteins using a high-throughput and convenient sensing of various saccharide derivatives. The present lectin beads are very promising for many objectives, particularly for the throughput analysis of all glycoconjugates in cell lines and organisms. Future studies will focus on investigating possible indicators for the detection of glycome and other biological substances in combination with various analytical methods.


Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from China exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors  and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China. Cryptococcus gattii is one of the life-threatening fungal pathogens that can infect immunocompetent individuals. C.gattii can be invaded through the respiratory tract and can be spread to the brain, causing meningoencephalitis and even death. In 1999, an outbreak of C. gattii VGII occurred on Vancouver Island in Canada and spread in the Pacific Northwest region, which attracted more attention. Compared with other nations, the data of C.gattii VGII in China was limited. To better understand the molecular epidemiology and microbiological characteristics of C.gattii VGII isolates from China, bioinformatics analysis and in vivo and in vitro experiments were performed for clinical strains. Multi-locus Sequence Typing and whole-genome sequencing reveal the genetic diversity of the isolates and genetic relationships with isolates from other countries. The antifungal drug susceptibility and virulence differences of the isolates were also investigated. The authors expect that our work can provide useful data for the prevention and control of C. gattii VGII in China and to draw more attention to C. gattii. C. gattii is a specific pathogenic fungus that can infect immunocompetent individuals [C. gattii and spread to the Pacific Northwest (PNW) of the United States [C. gattii infections had been reported in six continents.ividuals ,2. It coividuals ,4. In 19d States ,6. SubseC. gattii was currently classified into four distinct subtypes: VGI, VGII, VGIII, and VGIV [C. gattii VGII showed the difference in virulence, and their clinical manifestations also vary with the virulence. Current knowledge supports that the virulence of VGIIa is stronger than that of VGIIb [Using the International Society for Human and Animal Mycology consensus MLST scheme, and VGIV . MLST reand VGIV . The WGSand VGIV . The genof VGIIb . We founof VGIIb . Moreoveof VGIIb .C. neoformans, the ability to grow at 37°C, capsule size, and production of melanin were considered as the three classical virulence factors in C.gattii. The adaptability and survivability at 37°C are key virulence factor for C.gattii, which shows the ability of the strain to infect the host. A lack of thermotolerance limits their ability to cause infection of the host as well as cause disease [C. gattii VGII infection was reported in China [C. gattii VGII in China. Therefore, C.gattii VGII was collected from 19 hospitals and analyzed the genetic relationships among strains. In vitro and in vivo experiments were performed to characterize the differences in virulence of C. gattii VGII isolates. Finally, possible virulence factors were screened to explain the differences in virulence among isolates.Similar to  disease . Its cap disease . Melanin disease . In 2008in China , followein China –19. WhilThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Experimental Ethics Committee of Beijing Shijitan Hospital. The study was also approved by the Ethics Committee of Beijing Shijitan Hospital, and all participants provided written informed consent. (protocol code sjtky11-1x-2020 (20))C. gattii were collected, of which all isolates have been identified as C. gattii VGII by molecular typing of URA5-RFLP analysis [A total of seven strains of analysis . The cliC.gattii using liquid nitrogen grinding, and the DNA of all isolates was extracted by CTAB (cetyl trimethyl ammonium bromide) method [Breaking the cell wall of ) method . DNA of CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, and URA5) were performed independently amplified and sequenced. The STs of the clinical isolates identified were determined by comparing the sequences with the MLST database (http://mlst.mycologylab.org/), and reference MLST sequences were also downloaded from the MLST database. In addition, we searched all related publications in PubMed to collect another 16 strains of C. gattii VGII information reported in China. Finally, a maximum likelihood tree was constructed based on all sequences using RAxML 8.2.12 [As described in the previous studies , seven uL 8.2.12 .http://picard.sourceforge.net) AddOrReplaceReadGroups and MarkDuplicates were used for pre-processing, followed by GATK HaplotypeCaller 4.1.8.1 [The 7 clinical isolates were performed by WGS, and 117 additional WGS data were collected from BioProject PRJNA244927. Each Illumina data was aligned to VGII reference genome R265 by bwa-mem2 . To call 4.1.8.1  with 100C.gattii, we used epidemiological cutoff values (ECVs) as reference values for VGII, FCZ: 32mg/L; VCZ: 0.25mg/L; ICZ: 0.5mg/L; POS: 0.5mg/L; AMB: 1mg/L; 5-FC: 8mg/L.The Sensititre YeastOne broth microdilution system  was used to perform the antifungal susceptibility testing for six antifungal drugs, fluconazole (FCZ), voriconazole (VCZ), itraconazole (ICZ), Posaconazole (POS), amphotericin B (AMB) and 5-flucytosine(5-FC). Follow the instructions to perform antifungal sensitivity testing. Since there were no clinical breakpoints (CBPs) for the 7cells/ml by counting cells on a hemocytometer, gradient diluted to 107cells/ml, 2×106cells/ml, 4×105cells/ml, 8×104cells/ml, and 1.6×104cells/ml, and then performed the following assays.All clinical isolates were placed in yeast extract-peptone dextrose (YPD) medium and cultured overnight at 30°C. The cells were washed twice with sterile saline, centrifuged at 4000 rpm for 2 min, and the supernatant was discarded. The strains were uniformly adjusted to 5×10The ability to grow at 37°C: The cells (3 μl from each concentration) were respectively plated onto agar-based YPD medium, and cultured at 30°C, 37°C, and 39°C for three days to observe the growth at distinct temperatures.Production of melanin: For the melanin production assay, induction of melanin using minimal medium containing L-3,4-dihydroxyphenylalanine (L-DOPA). The cells (3μl from each concentration) were respectively cultured on a minimal medium containing L-DOPA at 30°C for three days, and photographed for melanin production .Capsule size: Using the method described in the previous article to measure capsule diameter of isolate strains , cells w6cell/ml. Female C57BL/6 mice aged 8–10 weeks were used and anesthetized with 10% chloral hydrate, 50μl fungal suspension was slowly dripped into the nasal cavity of each mouse [All clinical isolates and reference strains (R265 and R272) were selected for virulence assay. The strains were cultured overnight at 37°C in YPD medium, after centrifugation, washed twice with sterile saline, and the final concentration of the cells was adjusted to 2×10ch mouse .Ten mice per group were tested for survival test. Observe the death of mice every day and continuously for 90 days, recorded the death of mice.Five mice per group were tested for fungal burden, the mice were euthanized 14 days after infection. After dissection, the lungs and brains were separately placed in a 15 ml centrifuge tube containing 4 ml PBS. The tissue was smashed and homogenized in a biosafety cabinet, and then tissue suspension was serially diluted (10 times) and spread on the YPD plate. Culture at 30°C and then count Colony-Forming Unit (CFU) after two days.Four mice per group were used for pathological studies, mice infected with the different strains were euthanized at 14 days or when showing signs of poor health. All surviving mice were euthanized at the end of the experiment (90 days). After dissection, lung tissue was fixed in formalin, embedded in paraffin, and sectioned, stained sections with Hematoxylin solution & Eosin dye (H&E), then dehydrated and sealed with neutral gum. Finally, observe with microscope inspection, image acquisition, and analysis.All data were analyzed using SPSS software , and a descriptive statistical method was applied to summarize the clinical characteristics of patients. Continuous variables between two groups by the Manne Whitney U test. A p-value of 0.05 was considered significant. Using GraphPad Prism version 8.0  for plotting.C. gattii VGII patients , ST21 (1 strain), ST44 (2 strains), ST173 (1 strain), and ST126 (1 strain). To better study the genetic relationships of C. gattii VGII in China, sixteen strains reported so far in China were collected and combined to construct a phylogenetic tree. The result shows a high genetic variability amongst C. gattii VGII isolates from China, twelve STs were identified among 23 strains.ST7 was the most common ST (7/23), followed by ST44 (3/23). But no ST20 was found in China    .C. gattii VGII strains found in China and other countries. Illumina Hi-Seq platform was used to perform the whole genome sequencing of clinical isolates, and WGS data of VGII strains published from the database was also collected. R265 was used as an outgroup to construct the phylogenetic tree. We found that the phylogenetic tree constructed based on the whole genome had similar clustering to the phylogenetic tree based on MLST. The VGII population was highly diverse, where 35492 SNPs were identified. The WGS results revealed two VGIIb strains  but no VGIIa strain in China. G1 showed a close genetic relationship with strains from the United States and Australia, and G14 was clustered with strains from Venezuela  range was shown in All isolates showed similar tolerance to 37°C and showed growth well, but at 39°C, the growth of the strains exhibited remarkable differences. Compared to R265, only strain G17 was less tolerant to high temperature, while all other clinical strains showed higher resistance to high temperature . The capTo evaluate the virulence difference of clinical isolates, the reference strain and all clinical strains were chosen to establish a mouse inhalation model. Fourteen days post-infection, the highest CFU counts were G14 in the lung, followed by G1 and R265, and the lowest was R272, while the highest CFU counts were G10 in the brain, followed by G17, as shown in Fig .Survival curves showed that R265 from Vancouver Island was the most virulent strain with the minimum survival time. Each clinical strain presented a different survival time and was ordered as follows: G14, G10, G1. G17 and G13 have killed one and two mice, respectively, while R272 and G26 survived until the end of the experiment, and were all considered to be less virulent strains. In contrast, all the mice had not been killed by the G6 strain at the end of the experiment and thus could neither be considered as a virulent strain nor a less virulent strain . CombineSubsequently, histopathological analysis of lung tissue was performed. Interestingly, the degree of lung tissue damage was different between the stronger and less virulent strains. At 14 days, the less virulent strains showed normal lung tissue structure, while the highly virulent strains showed the initiation of pathological injury until a late stage when obvious millet-like edema can be observed on the surface of lung tissue. H&E staining showed obvious damage and change of lung structure, and a large number of non-uniform fungal cells were dispersed in the alveolar cavity . We beliC. gattii VGII in China. In line with previous reports [C. gattii VGII is more susceptible to infecting males and immunocompetent patients. In this study, most patients had symptoms of CNS involvement, unlike previous PNW reports [In this study, we investigated the clinical characteristics of  reports , C. gatt reports ,31, and C. gattii VGII isolates in the current study, with 6 different STs. The most common ST was ST7, which was widely distributed in other parts of the world and had been reported in Canada, Brazil, Thailand, and other places [A high genetic diversity amongst r places ,32. Accor places , while GC. gattii VGII show the highest geometric mean values [The azoles MICs differ among different genotypes, among which the isolates of n values . Clinican values . InteresIn vivo experiments, R265 from Vancouver Island was the most virulent strain, which was stronger than R272 ,9 and otWe performed the three classic virulence factor assays on all strains and found that all clinical isolates were able to grow well at 37°C, which showed the ability of the strains to consistently infect animals. However, the ability to grow at 39°C did not correlate with differences in virulence, and the results also showed that melanin production capacity and capsule size did not show a correlation with virulence either, like previous studies . This alC. gattii VGII in China, mainly due to the lack of awareness and conditions in many laboratories to identify and isolate Cryptococcus [gattii VGII, and provides useful data for the prevention and control of C. gattii VGII in China.Nonetheless, the limited number of strains available makes it difficult to fully reflect the prevalence of tococcus . In summ


Jeju black cattle, a type of native Korean cattle characterized by black fur covering the entire body, inhabit Jeju Special Self-Governing Province, a World Natural Heritage Site. Although this breed was state designated as a natural monument in 2013 due to its characteristics and genetic traits, it is on the verge of extinction and thus there is a need to preserve this breed and further improve its traits. Therefore, we evaluated sperm motility, vitality, and morphology, which have long been considered good predictors of fertility in the absence of female infertility factors. Our findings showed that the semen of the JBC-A bull was superior to the semen of four other JBC bulls. Due to the aging of the population of JBC breeding bulls, strategies should be devised to improve sperm production in vivo.To optimize the reproduction of Jeju black cattle (JBC), freezing conditions for sperm were established and sperm motility, vitality, morphology, and fertility were evaluated to select the optimal bull for breeding. Semen samples from five JBC bulls were individually mixed with freezing medium at a final concentration of 1 × 108 sperm/mL and frozen in liquid nitrogen vapor at a height of 3 or 7 cm . When the freezing conditions were compared, the motility of 7 cm sperm was significantly higher than that of 3 cm sperm for the JBC-A bull. The motility, curvilinear velocity, straight-line velocity, and average path velocity of fresh and frozen–thawed sperm were the highest for the JBC-A bull. The vitalities of fresh and frozen–thawed sperm were the highest for the JBC-A/E and JBC-A bulls, respectively. The percentage of normal cells in fresh sperm was the highest for the JBC-D bull. The rates of the normal formation of two pronuclei and total sperm penetration were the highest in zygotes fertilized with sperm from the JBC-A bull. The sperm from the JBC-A bull had superior qualities and are thus the most appropriate choice for the preservation and reproduction of these endangered cattle. Modern innovations in animal husbandry allow the establishment of animal genetic resources. Farmers depend on these innovations to obtain better-quality domestic animals. However, genetic materials from animals of economic merit, wild animals, and endangered species may be lost due to unexpected reproductive failure or death. Efforts are thus needed to avoid the loss of these genetic materials.Four cattle breeds distinguished by coat color patterns are native to Korea, namely, a brown or red breed (Hanwoo), a brown breed with black stripes , and two black breeds distinguished by their localities, with Korean black cattle living on the mainland and Jeju black cattle (JBC) on Jeju Island. There are an estimated 2.9 million Hanwoo cattle in Korea, whereas there are small populations of the other three breeds, especially JBC . JBC, whMale reproductive capacity is a major factor in mammalian reproduction and depends on the quality of germ cells produced by male animals. Sperm quality can be determined by measuring three important parameters: motility, morphology, and vitality. Vitality is the proportion of live spermatozoa in a sample, as determined by the evaluation of membrane integrity . DiffereAll chemicals and reagents were purchased from Sigma  unless stated otherwise.n = 5) were used. These animals were bred at the Livestock Promotion Agency in Jeju Special Self-Governing Province, Korea, with compound feed and roughage feed twice a day. The Livestock Promotion Agency distributes frozen semen to farmers; therefore, this study focused on frozen semen, and data for fresh semen were collected as a control to determine whether freezing should be performed.All animal studies were carried out in strict accordance with the recommendations in the Guide for the JBC Conservation Management Plan at the Jeju Special Self-Governing Province Livestock Promotion Agency and Guide for the Care and Use Committee (IACUC) of the Jeju National University (No. 2022-0010). Semen samples were collected from 38 ejaculates (6–10 samples per bull) from 5 JBC bulls using an artificial vagina in the morning hours every 2 weeks for 20 weeks. Semen from clinically healthy JBC bulls aged 2 to 7 years (Semen samples were mixed with freezing medium (20% egg yolk and 20% Triladyl) to yield a concentration of 1 × 108 sperm cells/mL. Sperm was cooled by placing 0.5 mL plastic straws filled with diluted semen samples in a refrigerator at 4 °C for 2 h. The straws were placed 3 or 7 cm  over liquid nitrogen (LN2) vapor for 10 min and then directly plunged into LN2 for storage. Straws were thawed by immersing them in a 37 °C water bath for 30 s.Sperm motility was assessed using the Sperm Analysis Imaging System . Semen samples (10 μL) were placed in a pre-warmed makler counting chamber and sperm motility characteristics were analyzed. At least 200 sperm from 5 fields were counted per reading. The software used for analysis was SAIS Advanced v.2008. Sperm motility  was assessed by measuring the percentage of sperm cells showing forward progress. Other sperm parameters measured included curvilinear velocity , straight-line velocity , average path velocity , linearity , straightness , beat cross frequency , and amplitude of lateral head displacement . Semen samples from five JBC bulls were analyzed 6–10 times each.The vitality of spermatozoa was assessed by the eosin–nigrosin  staining technique, with some modifications. This method was based on the degree of membrane permeability of dead spermatozoa, the heads of which are colored pink or red, whereas the low permeability of live gametes excludes eosin, with their heads remaining whitish. Briefly, a small amount of 2% eosin solution was mixed with a similar volume (10 μL) of each sample. Following incubation for 60 s, 40 μL of 10% nigrosin was added, the solution was mixed, and 5 μL of each stained sample was smeared onto a clean slide. Each slide was dried by placement on a warm (37 °C) plate, followed by an examination of 200 spermatozoa per slide at a magnification of 1000× with a bright field microscope. Sperm stained red or pink were classified as dead, except for sperm with light pink coloration of the neck region, which were classified as live  11]. Se. Se11]. Sperm morphology and staining features were assessed using Diff-Quik kits , which contain a fixative (methanol), an anionic/acidic dye (eosin) that stains positively charged/basic proteins red, and a cationic dye (methylene blue and its derivatives) that stains nuclei and negatively charged molecules blue. Briefly, 5 µL of a sperm suspension was smeared onto each slide, covered with a coverslip, and allowed to dry on a warm plate (40 °C). The slides were immersed in methanol for 60 s, allowed to air dry, immersed sequentially for 60 s each in each kit solution, and rapidly dipped in water to remove excess dye. The slides were allowed to air dry, and 200 spermatozoa on each slide were observed at a magnification of 1000× with a bright field microscope. Based on their morphological characteristics, the spermatozoa were classified into seven categories: normal, abnormal head, detached head, abnormal midpiece, bent midpiece, bent tail, and coiled tail . Semen sw/v) bovine serum albumin (BSA). Sets of ten COCs were matured in 50 μL droplets of TCM-199  supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 0.5 µg/mL follicle-stimulating hormone, 1 μg/mL estradiol-17β, and 0.1 μg/mL gentamycin under mineral oil for 21 h at 38.8 °C in air containing 5% CO2.Bovine ovaries were collected from a local slaughterhouse, immersed in 0.9% saline supplemented with 75 μg/mL penicillin G and 50 μg/mL streptomycin sulfate, and transported to the laboratory within 2 h at 30–33 °C. Generally, there are many cows over 10 years old. Cumulus–oocyte complexes (COCs) were aspirated from follicles with a diameter of 2–6 mm using an 18-gauge needle and a disposable 10 mL syringe. COCs were washed three times in tissue culture medium (TCM)-199–HEPES containing 0.1%  containing 6 mg/mL fatty acid-free (FAF) BSA and inseminated with 2 μL of highly motile sperm (2.5 × 107 spermatozoa/mL), recovered from the thawed semen of JBC bulls. Two microliters each of heparin (2 µg/mL) and PHE  was added to the 44 µL IVF drop. Egg yolk was removed by slowly adding thawed semen to 500 µL of 20% Triladyl solution (Triladyl: distilled water = 1:4) and incubating the sample at room temperature for 5 min. A 1 mL aliquot of sperm in 20% Triladyl solution was gently layered on top of the Sperm Filter  and incubated at 38 °C for 5 min. The suspension was centrifuged at 200× g (2000 rpm) for 5 min, the supernatant was aspirated, and the pelleted sperm were counted using a hemocytometer and diluted to 2.5 × 107 cells/mL by adding SP-TALP  containing 6 mg/mL FAF-BSA.At 21 h after IVM, COCs were transferred to TL-STOCK medium  and/or a sperm head in an oocyte at 18 h after IVF. To remove cumulus cells, IVF embryos were treated with TL-HEPES containing 0.1% hyaluronidase. Denuded embryos were washed with TL-HEPES and fixed for 2–3 min in 2% formaldehyde. Fixed embryos were stained with 25 μg/mL bisbenzimide (Hoechst 33258) for 10 min, washed three times, mounted onto glass slides, and examined by fluorescence microscopy at a magnification of ×200. Two JBC bulls were analyzed three times each.p <  0.05 was considered statistically significant.Significant differences among bulls were analyzed using paired Tukey’s multiple range tests. Data were analyzed using the general linear model procedure within the Statistical Analysis System . p < 0.05). However, the motility of 3 and 7 cm sperm did not significantly differ for the other four bulls . Therefore, we compared 7 cm sperm in subsequent experiments.The motility of sperm from individual JBC bulls was analyzed using the SAIS. To check the effect of the height at which sperm were exposed to LN2 vapor, the optimal freezing conditions were determined at a height of 3 or 7 cm . The motp < 0.05), but sperm motility did not significantly differ between the JBC-A bull and the three other bulls. VCL, VSL, and VAP were significantly higher for the sperm from the JBC-A bull than for the sperm from the JBC-D bull (p < 0.05), but did not differ significantly between the sperm from the JCB-A bull and the sperm from the JBC-B, JBC-C, and JBC-E bulls.The motility of fresh sperm  ranged fp < 0.05). VCL, VSL, and VAP were significantly higher for the sperm from the JBC-A bull than for the sperm from the JBC-B, JBC-D, and JBC-E bulls, but did not significantly differ between the sperm from the JBC-A bull and the sperm from the JBC-C bull. Overall, these findings show that the sperm from the JBC-A bull had the highest motility, whereas the sperm from the JBC-B bull had the lowest motility.The motility of frozen-thawed sperm  ranged fp < 0.05). The vitality of frozen–thawed sperm from the JBC-A bull was significantly higher than that of frozen–thawed sperm from the JCB-B, JCB-C, and JBC-E bulls (p < 0.05). Overall, these findings show that sperm from the JBC-A bull had the highest vitality, whereas sperm from the JBC-B bull had the lowest vitality.Sperm vitality was assessed using fresh and frozen–thawed sperm samples from the five bulls by eosin–nigrosin staining . The vitp < 0.05).The morphology of fresh  and frozFrozen–thawed sperm from the JBC-D bull had the highest percentage of normal spermatozoa, but this percentage did not significantly differ from the corresponding percentages for frozen–thawed sperm from the other bulls. An evaluation of abnormal sperm showed that the percentage of spermatozoa with a bent midpiece was higher than the percentages of spermatozoa with other abnormalities.p < 0.05). In addition, the rate of normal 2 PN formation was significantly higher for zygotes fertilized by sperm from the JBC-A bull than for zygotes fertilized by sperm from the JBC-B bull (p < 0.05). By contrast, the rate of polyspermy was significantly lower for zygotes fertilized by sperm from the JBC-B bull than for zygotes fertilized by sperm from the JBC-A bull.The rates of polyspermy and formation of 2 PN at 18 h after insemination were analyzed . Sperm fLittle is currently known about the qualities of semen from JBC bulls, a breed of native Korean cattle living on Jeju Island. This breed is endangered because only about 1400 of these animals remain in Jeju Special Self-Governing Province. Elite breeding bulls are thus needed to preserve this breed and to further improve its traits. Spermatozoa must be sufficiently viable and motile for fertilization. The present study therefore evaluated the functional qualities of sperm samples from JBC bulls, including the motility, vitality, and morphology of spermatozoa, as the latter must be sufficiently viable and motile for successful fertilization. This analysis provided more precise information about the characteristics of sperm from individual JBC bulls and the use of these samples in assisted reproductive technologies (ARTs).p < 0.05). These results suggest that JBC sperm were less damaged when they were frozen by exposure to LN2 vapor at a height of 7 cm and subsequent experiments were conducted based on these results.The present study was conducted to select the optimal JBC bulls for breeding. To determine the appropriate LN2 vapor exposure height for JBC bulls, experiments were conducted at heights of 3 cm and 7 cm, referring to 3 cm and 6.5 cm used in previous studies ,13. The The ability of mammalian spermatozoa to swim toward an oocyte is important for successful fertilization. Sperm propulsion is caused by the active motion of their flagella. Sperm quality has been assessed by measuring sperm motility, including measurements of VCL, VSL, and VAP ,16,17,18Sperm vitality is estimated by assessing membrane integrity. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in non-vital (dead) cells, allow the entry of membrane-impermeant stains. In these assays, eosin is used to stain cells with damaged plasma membranes and nigrosin is used as a background stain . The perFresh sperm from the JBC-D bull had the highest percentage of morphologically normal spermatozoa. After freeze–thawing, however, the percentage of normal sperm from this bull significantly decreased, whereas the percentages of sperm with abnormal heads and bent tails significantly increased. The percentage of human sperm with abnormal tails has also been found to increase after freeze–thawing . The morThese analyses of the motility, vitality, and morphology of spermatozoa in fresh semen samples from five JBC bulls showed that sperm from the JBC-A, JBC-C, and JBC-E bulls were superior. After freeze–thawing, however, these parameters decreased in samples from the JBC-C and JBC-E bulls, suggesting that samples from these two bulls are more vulnerable to cryopreservation than samples from the JBC-A bull. Taken together, these results showed that motility and vitality were the highest for sperm from the JBC-A bull and lowest for sperm from the JBC-B bull, and these differences are reflected in the fertilization rates of semen samples from these two bulls.Although the rate of normal 2 PN formation was the highest in zygotes fertilized with sperm from the JBC-A bull, the rate of polyspermy was also high. Monospermic fertilization occurs only when fully mature oocytes with a normal zona pellucida are fertilized with an optimal sperm concentration under optimized conditions . An abnoThe morphology, motility, vitality, and total penetration rate of sperm differed among samples obtained from five JBC bulls. Sperm morphology, motility, and vitality have long been regarded as good predictors of fertility in the absence of female infertility factors ,30,31. O


Flaviviridae. The patient in this case is from the municipality of São Bernardo do Campo, an area endemic for arboviruses. He presented symptoms of fever, myalgia and headache.Arboviruses primarily consist of RNA, which favours greater genetic plasticity, with a higher frequency of mutations that allow the virus to adapt to different hosts. The initial symptomatology is nonspecific, in that the patient can present fever, myalgia, arthralgia, rash and headache. This makes a clinical diagnosis using laboratory tests difficult and time-consuming. In Brazil, the main arboviruses involved in epidemics belong to the family The multiplex assay for arboviruses detected genetic material from the dengue 2 and yellow fever viruses.This result confirms the importance of molecular tests showing high sensitivity and specificity that can assist clinical diagnosis, particularly in endemic areas during periods of outbreak for other arboviruses, like the epidemiological picture in Brazil in 2018, when significant co-circulation of dengue virus and yellow fever virus occurred. The presence of co-circulating arboviruses increases the chance of coinfection and demonstrates the importance of differential diagnosis. Arboviruses primarily consist of RNA, which favours greater genetic plasticity, with a higher frequency of mutations that allow the virus to adapt to different hosts . TransmiFlaviviridae [In Brazil, the main arboviruses involved in epidemics belong to the family iviridae . There aiviridae . In the iviridae .In the State of São Paulo, 538 cases of yellow fever (YF)were confirmed, resulting in 184 (34.2 %) deaths . The mosin vitro that serum samples from convalescent patients for dengue have demonstrated neutralizing activity against yellow fever virus (YFV) (55.6 %) and St Louis encephalitis virus (SLEV) (33.4 %). Serum samples from patients convalescent for Zika virus (ZIKV) were able to neutralize West Nile virus (WNV) (40 %), Ilheus virus (ILHV) (40 %) and Rocio virus (ROCV) (20 %), confirming cross-reactivity between sera and difficulty in serological diagnosis [The laboratory diagnosis of flavivirus is complex. Rapid tests show sensitivity between 40–70 %, while those using the ELISA methodology have sensitivity up to 95 %, although these tests show greater cross-reactivity [iagnosis .Aedes aegypti. In the last two decades, YFV transmissions were registered outside the limits of the area considered to be endemic (the Amazon region).YFV is epidemiologically important due to its clinical severity and its potential for dissemination in urban areas infested by the mosquito The patient is from the municipality of São Bernardo do Campo and was admitted to a public hospital of the same city in July 2018, showing clinical symptomatology compatible with an arbovirus: fever, myalgia and headache.Biochemical, serological and molecular tests were requested to detect arboviruses. Serum RNA was extracted using the QIAamp Viral RNA Mini kit , and the RNA was then converted into cDNA using the QuantiNova Reverse Transcription kit  and multiplex qRT-PCR composed of two platforms to determine the virus  was performed in an ABI 7500 thermal cycler. Primers and probes were designed with the assistance of the IDT First Design program .−3, an increase in direct bilirubin of 0.9 mg dl−1, alanine aminotransferase (ALT) of 68 μ l−1, aspartate transaminase (AST) of 60 μ l−1, gamma-glutamyl transferase (GGT) of 301 μ l−1 and C-reactive protein (C-PR) of 240.59 mg dl−1 (Laboratory tests revealed a leukocytosis of 20.9 103 mm mg dl−1 . HaemocuThe patient was hospitalized for 15 days, during which time he was given 0.9 % saline and 1.0 g of novalgine; hepatic enzyme stabilization occurred and clinical recovery was observed; there was no sequel.This result confirms the importance of molecular tests showing high sensitivity and specificity that can assist clinical diagnosis, particularly in endemic areas during periods of outbreak of other arboviruses, like the epidemiological picture in Brazil in 2018, when significant co-circulation of DENV and YFV occurred. The presence of co-circulating arboviruses increases the chance of coinfection and demonstrates the importance of differential diagnosis and vector control.In a study in Bali involving travellers who showed at least one clinical symptom for dengue, laboratory tests showed that 66.2 % were contaminated with DENV. In this same study they were unable to detect Japanese encephalitis, Zika and chikungunya, indicating that other viruses are circulating in that location .A study of 652 patients in the Democratic Republic of Congo detected 3 cases of co-infection by DENV1 and 2 using qRT-PCR. The data obtained indicated the importance of molecular differential tests in directing health actions .Dengue is the disease with the highest incidence in travellers, and the relative risk of severe dengue in the second infection can increase by sevenfold . In addiIn addition, a country’s socio-economic conditions can cause a large influx of immigrants from endemic areas to other countries. The search for better living conditions for immigrants from countries in the African and South American continents may incur an increase in cases of some arboviruses . Hence, Broad differential testing for arboviruses has a relevant epidemiological role, as carried out in Colombia between 2015–2016, during the ZIKV epidemic. Molecular tests were performed on 23871 samples that detected 1.84 % DENV, 1.07 % CHIKV and 42.38 % ZIKV, with 0.14 % CHIKV–ZIKV coinfection cases [The use of two molecular platforms to investigate eight viral types has been shown to be effective and useful, given that Brazil is an endemic country for DENV and there are several different arboviruses in co-circulation. This is especially important, since these arboviruses present similar clinical features and laboratory tests are required to achieve a diagnosis.The proposed platforms were capable of detecting the arboviruses studied. It is important that such tests increase the detection of different viruses, that the use of such platforms enables rapid laboratory diagnosis, the detection of endemic viruses, viruses involved in recent outbreaks and viruses of low incidence when routine tests are negative.


We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells. By February 1, 2021, Hong Kong had recorded 10,453 reverse transcription PCR (RT-PCR)–confirmed COVID-19 cases, and many of those occurred during the last 2 waves. The third wave occurred during late June to early September 2020 and was caused by a single introduction of GISAID    Figure 2We also noted 4 imported cases (patients A1–A4) in a nearby hotel (hotel A), which is ≈350 m walking distance from bar/building X and hotel C, during late September to early October 2020  Table 1.Patient A4, who was quarantined in hotel A during September 13–27, 2020, also traveled from Nepal to Hong Kong on a separate flight. The viral genome of case A4 is identical or closely related to sequences from patients A1–A3 and B1 and B2. Patient A4 had consecutive negative RT-PCR results upon arrival and on day 12 during quarantine . Patienthttps://doi.org/10.1101/2020.10.27.357558), including mutations in the RNA-dependent RNA polymerase (RdRp[L323P]), Spike(D614G), open reading frame 3a (ORF3a[Q57H]), ORF3b(E14*), and nucleocapsid (N[S194L]) proteins. The ORF3a(Q57H) mutation leads a major truncation of ORF3b protein, ORF3b(E14*)  . Our seqWe first studied the virus replication kinetics by using human bronchus and lung ex vivo cultures  Figure 3We previously demonstrated that the wave 1 virus is not a potent proinflammatory cytokine and chemokine inducer in infected human cells . The cytDespite the major ORF3b deletion, our results demonstrate that the wave 4 virus does not have an enhanced ability to replicate ex vivo and retains potent innate immune evasion capacity in our experimental models. We noted that the wave 3 virus replicates slightly better than isolates from wave 1 and 4, and it can induce higher innate immune responses. The wave 3 virus has several unique mutations not found in the other 2 viruses . Many ofIn summary, we found the virus causing the fourth COVID-19 epidemic wave in Hong Kong does not have enhanced replication kinetics and is not a potent cytokine or chemokine inducer. However, our work highlights the need for stringent COVID-19 control policy in quarantine settings.Additional information on SARS-CoV-2 viruses from COVID-19 epidemic waves 1, 3, and 4, Hong Kong, China.