Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Influence of exercise intensity and regional differences in the sudomotor recruitment pattern in exercising prepubertal boys and young men.

We investigated the influence of exercise intensities and regional differences in the sudomotor recruitment pattern in boys. Six prepubertal boys (age 11 ± 1 yr) cycled at light, moderate, and high exercise intensity (35%, 50%, and 65% VO2max) for 30 min in a temperate condition (28 °C, 40% relative humidity). Local sweat rate (ventilated capsule) and number of activated sweat glands (starch-iodine technique) at five body sites were assessed and sweat gland output was calculated. Responses in boys were compared with those in nine young men (23 ± 1 yr) tested under identical conditions. The forehead, chest, back, and forearm, but not thigh, sweat rate increased from light to moderate and at high intensities in boys (all p ≤ 0.005) but not from moderate to high (all p ≥ 0.071). The sweat rate on the forehead was relatively higher (p ≤ 0.045) and thigh was lower (p ≤ 0.050) than other sites in boys at moderate and high intensities. Exercise intensity-dependent sweating was associated with activating more sweat glands but not increasing glandular output in boys. The sweat rate in boys was attenuated versus men heterogeneously across body sites concurrent to low glandular outputs (all p ≤ 0.027). We conclude that exercise intensity modulates the sweat rate in boys by changing the number of activated sweat glands heterogeneously among skin sites. Age-related differences in the sudomotor pattern are evident at higher exercise intensities. Development of glandular output per gland occurring from boys to young men may play a key role in modulating sweat rate with respect to exercise intensity and regional differences.

In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients.

Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health.

Influenza A(H1N1)pdm09 virus exhibiting reduced susceptibility to baloxavir due to a PA E23K substitution detected from a child without baloxavir treatment.

Human-to-human transmission of PA I38 mutant influenza A(H3N2) viruses with reduced baloxavir susceptibility has been reported in Japan. In December 2019, we detected a PA E23K mutant A(H1N1)pdm09 virus from a child without baloxavir treatment. The PA E23K mutant virus exhibited reduced baloxavir susceptibility but remained susceptible to neuraminidase inhibitors. Epidemiological data suggest possible transmission of this PA E23K mutant virus among humans, although its growth capability relative to that of the wild-type virus was reduced. Therefore, baloxavir susceptibility monitoring of influenza viruses is essential.

G Protein Pathway Suppressor 1 Promotes Influenza Virus Polymerase Activity by Activating the NF-κB Signaling Pathway.

Influenza virus relies heavily on cellular machinery to replicate in host cells. Therefore, to better understand the influenza virus life cycle, it is important to identify which host proteins are involved and how they function in virus replication. Previously, we identified G protein pathway suppressor 1 (GPS1) to be a matrix protein 2 (M2)-interacting host protein. GPS1 is a component of the COP9 signalosome, which regulates the NF-κB signaling pathway. Here, we found that the downregulation of GPS1 expression reduced influenza virus replication by more than 2 log units. Although GPS1 was not involved in the early and late stages of virus replication, such as viral entry, uncoating, assembly, or budding, we found that viral polymerase activity was impaired in GPS1-downregulated cells. Moreover, our results suggest that M2 activates the NF-κB signaling pathway in a GPS1-dependent manner and that activation of NF-κB signaling leads to the upregulation of influenza virus polymerase activity. Our findings indicate that GPS1 is involved in the transcription and replication of influenza virus genomic RNA through the activation of the NF-κB signaling pathway.IMPORTANCE In the present study, we identified G protein pathway suppressor 1 (GPS1) to be a host cellular protein that is important for influenza virus replication. We also found that GPS1 plays a role in viral genome transcription through the NF-κB signaling pathway. Moreover, downregulation of GPS1 also affected the growth of vesicular stomatitis virus. Therefore, GPS1 may be a host target for antiviral drugs against influenza virus and possibly other viruses.

Human-to-Human Transmission of Influenza A(H3N2) Virus with Reduced Susceptibility to Baloxavir, Japan, February 2019.

In 2019, influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic gene, which confers reduced susceptibility to baloxavir, were detected in Japan in an infant without baloxavir exposure and a baloxavir-treated sibling. These viruses' whole-genome sequences were identical, indicating human-to-human transmission. Influenza virus isolates should be monitored for baloxavir susceptibility.

Influenza A(H3N2) virus exhibiting reduced susceptibility to baloxavir due to a polymerase acidic subunit I38T substitution detected from a hospitalised child without prior baloxavir treatment, Japan, January 2019.

In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans.

Detection of influenza A(H3N2) viruses exhibiting reduced susceptibility to the novel cap-dependent endonuclease inhibitor baloxavir in Japan, December 2018.

The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.

Susceptibility of Influenza Viruses to the Novel Cap-Dependent Endonuclease Inhibitor Baloxavir Marboxil.

The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza A and B virus infections in February 2018 in Japan. Because of the need to monitor influenza viruses for reduced susceptibility to this drug, we used two cell-based screening systems - a conventional plaque reduction assay and a focus reduction assay - to evaluate the susceptibility of influenza viruses to baloxavir. First, we generated a reference virus possessing an I38T substitution in the polymerase acidic subunit (PA), which is known to be associated with reduced susceptibility to baloxavir, and demonstrated the validity of our systems using this reference virus. We then determined the susceptibility of a panel of neuraminidase (NA) inhibitor-resistant viruses and their sensitive counterparts to baloxavir. No significant differences in baloxavir susceptibilities were found between the NA inhibitor-resistant and -sensitive viruses. We also examined seasonal influenza viruses isolated during the 2017-2018 influenza season in Japan and found that no currently circulating A(H1N1)pdm09, A(H3N2), or B viruses had significantly reduced susceptibility to baloxavir and none of the viruses possessed an amino acid substitution at PA residue 38. Use of a combination of methods to analyze antiviral susceptibility and detect amino acid substitutions is valuable for monitoring the emergence of baloxavir-resistant viruses.

Age-related attenuation of conduit artery blood flow response to passive heating differs between the arm and leg.

PURPOSE: Little is known about why the attenuation of heat loss responses with aging begins in the lower limbs. This study sought to determine whether passive heating causes the age-related decrease and limb-specific difference of blood flow (BF) responses between conduit brachial and femoral arteries, which are related to differences of cutaneous vascular conductance (CVC) between the upper and lower limbs. METHOD: In 15 older and 12 younger males, BF in the brachial and femoral arteries was ultrasonically measured and CVC in the forearm and thigh was assessed during lower leg immersion in hot water at 42 °C (ambient temperature: 30 °C, relative humidity: 45%) for 40 min. RESULTS: The increased BF of brachial artery at the end of passive heating was similar between both age groups (older: 140 ± 4%; younger: 146 ± 11%), while that of femoral artery was smaller in older than younger group (119 ± 4% vs. 166 ± 11%, P < 0.01). Moreover, the increased CVC in the forearm was similar between the age groups (older: 356 ± 50%; younger: 308 ± 46%), although CVC in the thigh was significantly lower in older than younger group (303 ± 33% vs. 427 ± 51%, P < 0.05). These results corresponded to the BF responses of the brachial and femoral arteries, respectively. CONCLUSION: These results indicate that age-related decrease and limb-specific difference occur also in conduit arteries of arm and leg, which might be related to the different reduction in CVC between forearm and thigh.

Isolation of an Egg-Adapted Influenza A(H3N2) Virus without Amino Acid Substitutions at the Antigenic Sites of Its Hemagglutinin.

Antigenic changes in the hemagglutinin protein of recent A(H3N2) viruses often arise when these viruses adapt to their egg host. By serial egg passages of a cell-propagated virus, we successfully isolated an egg-adapted influenza A(H3N2) virus, A/Saitama/103/2014, without amino acid substitutions at the antigenic sites of its hemagglutinin protein but with multiple substitutions in its neuraminidase protein. Antigenic analysis of this egg-adapted A/Saitama/103/2014 virus indicated that its antigenicity did not differ from that of the World Health Organization prototype cell-propagated vaccine virus: A/Hong Kong/4801/2014. Our results suggest that this strategy may facilitate egg-based vaccine production without antigenic alterations in hemagglutinin by egg adaptation.

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015.

We characterized influenza A(H1N1)pdm09 isolates from large-scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread.

The carotid baroreflex modifies the pressor threshold of the muscle metaboreflex in humans.

The purpose of the present study was to test our hypothesis that unloading the carotid baroreceptors alters the threshold and gain of the muscle metaboreflex in humans. Ten healthy subjects performed a static handgrip exercise at 50% of maximum voluntary contraction. Contraction was sustained for 15, 30, 45, and 60 s and was followed by 3 min of forearm circulatory arrest, during which forearm muscular pH is known to decrease linearly with increasing contraction time. The carotid baroreceptors were unloaded by applying 0.1-Hz sinusoidal neck pressure (oscillating from +15 to +50 mmHg) during ischemia. We estimated the threshold and gain of the muscle metaboreflex by analyzing the relationship between the cardiovascular responses during ischemia and the amount of work done during the exercise. In the condition with unloading of the carotid baroreceptors, the muscle metaboreflex thresholds for mean arterial blood pressure (MAP) and total vascular resistance (TVR) corresponded to significantly lower work levels than the control condition (threshold for MAP: 795 ± 102 vs. 662 ± 208 mmHg and threshold for TVR: 818 ± 213 vs. 572 ± 292 kg·s, P < 0.05), but the gains did not differ between the two conditions (gain for MAP: 4.9 ± 1.7 vs. 4.4 ± 1.6 mmHg·kg·s-1·100 and gain for TVR: 1.3 ± 0.8 vs. 1.3 ± 0.7 mmHg·l-1·min-1·kg·s-1·100). We conclude that the carotid baroreflex modifies the muscle metaboreflex threshold in humans. Our results suggest the carotid baroreflex brakes the muscle metaboreflex, thereby inhibiting muscle metaboreflex-mediated pressor and vasoconstriction responses.NEW & NOTEWORTHY We found that unloading the carotid baroreceptors shifts the pressor threshold of the muscle metaboreflex toward lower metabolic stimulation levels in humans. This finding indicates that, in the normal loading state, the carotid baroreflex inhibits the muscle metaboreflex pressor response by shifting the reflex threshold to higher metabolic stimulation levels.

Sweating responses to isometric hand-grip exercise and forearm muscle metaboreflex in prepubertal children and elderly.

NEW FINDINGS: What is the central question of this study? Non-thermal factors (e.g. muscle metaboreflex) contribute to the sweating response during exercise. Although it is well recognized that the sweating responses caused by core temperature elevation in prepubertal children and the elderly are attenuated compared with young adults, it is unknown whether non-thermal sweating is also attenuated in these populations. What is the main finding and its importance? The non-thermal sweating response during isometric hand-grip exercise and isolated muscle metaboreflex were attenuated in prepubertal children compared with young adults in a non-uniform manner over the body, but only during the muscle metaboreflex in the elderly. This may explain the maturation- and ageing-related decline of sweating during exercise. The purpose of the present study was to investigate sweating responses to isometric hand-grip (IH) exercise and muscle metaboreflex in prepubertal children and the elderly. In hot conditions (ambient temperature, 35°C; relative humidity, 45%), 13 healthy young adults, 10 prepubertal children and 10 elderly subjects (aged 20.4 ± 1.2, 11.4 ± 0.5 and 63.5 ± 3.1 years, respectively) repeated a three hand-grip exercise protocol that consisted of 1 min IH exercise at 15, 30 or 45% of maximal voluntary contraction (MVC) followed by 2 min postexercise forearm occlusion. Local sweat rates (SRs) on the forehead, chest, forearm, thigh and palm were continuously measured (ventilated capsule method). The forehead SR in prepubertal children during IH exercise at 45% MVC was significantly lower than that of young adults (0.26 ± 0.22 and 0.08 ± 0.15 mg cm-2  min-1 for young adults and children, respectively; P < 0.05) but not of the elderly at any exercise intensities. The SR on the chest (0.22 ± 0.22 and -0.01 ± 0.05 mg cm-2  min-1 for young adults and children, respectively), forearm (0.14 ± 0.12 and 0.03 ± 0.04 mg cm-2  min-1 ) and thigh (0.13 ± 0.10 and 0.02 ± 0.03 mg cm-2  min-1 ) during postexercise occlusion at 45% MVC was significantly lower in children than in young adults (P < 0.05). Elderly subjects showed a significantly lower SR on the forearm (0.04 ± 0.04 and 0.01 ± 0.02 mg cm-2  min-1 for young adults and elderly, respectively) and thigh (0.07 ± 0.07 and 0.01 ± 0.03 mg cm-2  min-1 ) at 15% MVC and on the thigh at 45% MVC (0.13 ± 0.10 and 0.04 ± 0.04 mg cm-2  min-1 ) during postexercise occlusion compared with young adults (P < 0.05). These results suggest that sweating responses to IH exercise and muscle metaboreflex were underdeveloped in prepubertal children and that ageing attenuates the response to the muscle metaboreflex in a way that is not consistent across the body.

Influenza A(H1N1)pdm09 virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual H275Y/G147R substitution, Japan, March 2016.

An influenza A(H1N1)pdm09 virus carrying a G147R substitution in combination with an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir, was detected from an immunocompromised inpatient in Japan, March 2016. This dual H275Y/G147R mutant virus exhibited enhanced cross-resistance to both drugs compared with the single H275Y mutant virus and reduced susceptibility to zanamivir, although it showed normal inhibition by laninamivir.

Sex differences in age-related changes on peripheral warm and cold innocuous thermal sensitivity.

Cutaneous thermal sensitivity to a warm and cold stimulus was compared amongst 12 older (OF, 65.2±1.0year) and 29 younger (YF, 21.6±0.2years) female participants, and 17 older (OM, 66.2±1.5years) and 13 younger (YM, 21.2±0.4years) male participants to examine the effects of ageing and sex. In a neutral condition (27.5°C, 50% RH) during rest, warm and cold thermal sensitivity was measured on eight body regions (forehead, chest, back, forearm, hand, thigh, calf, and foot). Using the method of limits, a thermal stimulator was applied to the skin at an adapting temperature and either increased or decreased at a constant rate (0.3°C/s) until the participants detected the temperature with a push button. Thermal sensitivity declined with ageing to both a cold (older: 1468.6±744.7W/m(2), younger: 869.8±654.7W/m(2), p<0.001) and warm (older: 2127.0±1208.3W/m(2), younger: 1301.7±1055.2W/m(2), p<0.001) innocuous stimulus. YF and OF were more sensitive than YM and OM to both a warm and cold stimulus (p<0.05). There was no interaction between age and sex suggesting that whilst thermal sensitivity decreases with age the decrease is similar between the sexes (p>0.05). There was an interaction between temperatures, age and location and it seemed that cold thermal sensitivity was more homogenous for young and older participants however warm thermal sensitivity was more heterogeneous especially in the younger participants (p<0.05). Although the pattern was not similar between ages or sexes it was evident that the forehead was the most sensitive region to a warm and cold stimulus. Interestingly the decline in sensitivity observed with ageing occurred for all locations but was attenuated at the forehead in both males and females (p>0.05).

Increasing blood flow to exercising muscle attenuates systemic cardiovascular responses during dynamic exercise in humans.

Reducing blood flow to working muscles during dynamic exercise causes metabolites to accumulate within the active muscles and evokes systemic pressor responses. Whether a similar cardiovascular response is elicited with normal blood flow to exercising muscles during dynamic exercise remains unknown, however. To address that issue, we tested whether cardiovascular responses are affected by increases in blood flow to active muscles. Thirteen healthy subjects performed dynamic plantarflexion exercise for 12 min at 20%, 40%, and 60% of peak workload (EX20, EX40, and EX60) with their lower thigh enclosed in a negative pressure box. Under control conditions, the box pressure was the same as the ambient air pressure. Under negative pressure conditions, beginning 3 min after the start of the exercise, the box pressure was decreased by 20, 45, and then 70 mmHg in stepwise fashion with 3-min step durations. During EX20, the negative pressure had no effect on blood flow or the cardiovascular responses measured. However, application of negative pressure increased blood flow to the exercising leg during EX40 and EX60. This increase in blood flow had no significant effect on systemic cardiovascular responses during EX40, but it markedly attenuated the pressor responses otherwise seen during EX60. These results demonstrate that during mild exercise, normal blood flow to exercising muscle is not a factor eliciting cardiovascular responses, whereas it elicits an important pressor effect during moderate exercise. This suggests blood flow to exercising muscle is a major determinant of cardiovascular responses during dynamic exercise at higher than moderate intensity.