改良抗体结合实验检测灭活狂犬病疫苗效价

目的:建立抗体结合试验检测狂犬病疫苗(aG株)效价的方法。方法:将待检测疫苗与疫苗标准品梯度稀释后分别加入人抗狂犬病毒免疫球蛋白国家标准品中和1 h,之后加入80%感染剂量的狂犬病毒CVS-11,体外中和1h后接种BSR细胞,培养24 h后免疫荧光染色,在显微镜下观察结果,通过检测剩余病毒量计算待检疫苗的效价,同时与小鼠中和试验法(NIH法)测定狂犬病疫苗效价进行比较。结果:2种方法对8个样品效价的检测结果无显著统计学差异(P=0.997,配对t检验)。结论:初步建立了改良抗体结合试验,可用于狂犬病疫苗中间产品的质量控制。     

博尔纳病病毒对BALB/c小鼠感染性检测

目的分析博尔纳病病毒（Borna disease virus,BDV）H1766株对BALB/c小鼠的感染性。方法选择病毒滴度为2.0×107FFU/ml的BDV病毒液分别对新生和成年BALB/c小鼠进行脑内接种,并用相同病毒液对原代培养的新生BALB/c小鼠脑细胞进行接种。经过一定时间的病毒作用后分别提取总RNA,采用巢式RT-PCR方法检测BDV-p40基因,并通过免疫组化方法检测脑内接种脑组织中BDV-P40蛋白。结果脑内接种病毒的小鼠脑组织中可以检测到BDV-p40基因和BDV-P40蛋白,培养的小鼠脑细胞中可以检测到BDV-p40基因。结论BDVH1766株可以感染新生和成年的BALB/c小鼠。     

小鼠体内SEG蛋白靶向运送shRNA抑制狂犬病毒复制

【目的】本试验前期已经证实,用单链抗体(sc Fv)-绿脓杆菌跨膜区(ETA)-酵母DNA结合结构域(GAL4)表达的蛋白(简写为SEG蛋白),SEG能与含sh RNA(short hairpin RNA)的质粒(p RNATU6.3-sh RNA)结合形成复合物SEG-sh RNA,并靶向运送该质粒进入感染狂犬病毒(Rabies virus,RV)的细胞,抑制RV复制。本研究用感染狂犬病病毒的小鼠模型,进行SEG-sh RNA复合物小鼠体内靶向性运送si RNA(short interfering RNA)和抑制RV复制的研究。【方法】用已建立RV CVS-24株小鼠肌肉注射模型进行试验。取50 LD_(50) CVS-24攻毒,在攻毒后12 h尾静脉注射SEG-sh RNA,流式细胞仪检测SEG-sh RNA的体内靶向性;同样方法攻毒后,尾静脉注射SEG-sh RNA,连续4 d,攻毒后第5天小鼠脑组织用q RT-PCR、RT-PCR、Western blot、免疫荧光染色法检测其中RV的含量;统计小鼠存活率;并检测小鼠体内IFN-α含量,从而分析SEG-sh RNA在体内的抗病毒作用。【结果】结果表明仅在RV攻毒小鼠的注射部位检测到绿色荧光蛋白(green fluorescent protein,GFP)的表达,未注射RV的腿部及脑、肝、脾、肾均无GFP表达,说明SEG-sh RNA可靶向RV感染细胞运送sh RNA。攻毒后第5天脑组织q RT-PCR结果表明靶向药物组比病毒对照减少4.88倍(3.9/0.8);RT-PCR、Western blot、免疫荧光染色试验结果表明使用SEG-sh RNA组病毒量明显少于病毒对照组;且攻毒后13 d,动物存活率达50%,而病毒对照100%死亡。检测小鼠体内IFN-α未见升高。【结论】以上试验表明SEG蛋白在小鼠体内靶向运送含sh RNA的质粒到感染组织细胞;对小鼠体内RV有明显抑制作用,因此可以用于狂犬病毒感染的特异辅助性救治研究。     

小鼠感染细小病毒的临床特征分析

目的分析小鼠细小病毒(MVM)在人工感染小鼠体内的分布规律、排毒和抗体变化,以及自然条件下小鼠感染MVM的情况。方法对33只BALB/c小鼠腹腔接种0.2 m L MVM悬浮液,每天观察动物的外观、行为、饮食和精神状态,在接种第0~60天共12个时间点各对2~3只动物进行安乐死,并采取组织、粪便和血清样本进行检测。荧光定量PCR(QPCR)方法检测组织和粪便的病毒核酸,ELISA方法检测血清抗体。同时,采取100只SPF小鼠、76只开放饲养小鼠检测MVM核酸,采取1463只SPF小鼠、82只开放饲养小鼠检测MVM抗体。结果实验小鼠接种MVM后外观、行为、饮食和精神状态未见异常,剖检无明显病变。各组织在接种后都能检出病毒核酸,并在接种后4~7 d达到峰值,且到60 d仍可检出病毒核酸。各组织间比较,病毒峰值最高的组织是肝,其次是肾、脾、胃、心脏、肺、盲肠和脑。粪便排毒在接种后11 d可达到峰值,之后迅速下降,但到60 d还可检到。血清抗体在病毒接种后7 d开始产生,之后抗体效价逐渐升高,21 d就可以达到32倍左右,之后至60 d一直处于64倍左右。临床样本中,核酸检测SPF小鼠未检出,开放饲养小鼠的检测阳性率为14.5%,经过测序比较确认为MVM病毒感染;抗体检测SPF小鼠有较低的检出率(0.3%),开放饲养小鼠检出率为68.3%。结论 MVM感染小鼠后一般呈隐性感染状态,可长期排毒,主要组织脏器、粪便、血清都能用于MVM的检测,实验小鼠感染MVM可以通过病毒核酸和血清抗体检测方法进行病原检测。     

狂犬病毒滴度检测噬斑法(PFU)的建立及应用

目的建立狂犬病毒滴度的快速、经济的检测方法——噬斑法(PFU),验证其与小鼠脑内攻击法(计算LD50)的相关性,并用于PM株狂犬病毒的滴度测定。方法狂犬病毒做10倍系列稀释,然后接种于单层BHK21细胞上,37℃、5%CO2吸附1h后,加入覆盖液,33℃、5%CO2培养7~10天后用1.5%的结晶紫染色。用建立的噬斑法和小鼠脑内攻击法同时测定狂犬病毒的滴度,以比较两种方法的相关性和重复性。结果两种方法测定狂犬病毒滴度的结果差异无统计学意义,相关系数为0.939,两种方法的检测结果呈良好的正相关性。结论噬斑法可替代小鼠脑内滴定法检测狂犬病毒滴度。     

鸡产蛋下降综合征病毒NE4毒株对KM小鼠感染特性观察

目的初步探究鸡产蛋下降综合征病毒NE4（EDSV NE4）毒株对昆明小鼠的感染特性。方法用106TCID50攻毒量的EDSV NE4株对4～6周龄的KM小鼠进行攻毒试验,同时以正常尿囊液接种作为阴性对照。用荧光定量PCR对小鼠组织及粪便中的EDSV进行检测,同时用HE染色及免疫组化法对小鼠组织切片进行病理组织学观察和抗原定位。结果 EDSV攻毒对小鼠的采食情况产生一定影响,但并未引起明显的临床症状;攻毒组小鼠可产生针对EDSV特异性的抗体,HI抗体滴度可高达212,阴性对照组小鼠体内抗体检测为阴性;攻毒后小鼠大部分组织器官如肝脏、子宫、肾脏、肺脏等与攻毒7 d后粪便中均可检测到EDSV,阴性对照小鼠所有受检组织中均未检测到病毒;EDSV攻毒未能引起小鼠组织的病理学变化,攻毒后不同时间内,可在子宫、肺脏、肝脏、肾脏中可检测到阳性信号,最为典型的定殖位置是子宫腺体上皮细胞及肌层细胞的胞质中。结论 EDSV NE4株可以感染KM小鼠。     

猪圆环病毒3型感染BALB/c小鼠的实验观察

目的研究猪圆环病毒3型(porcine circovirus3,PCV3)对BALB/c小鼠的感染情况。方法将雌性BALB/c小鼠分为两组,实验组感染PCV3组织毒,对照组接种同样剂量的PBS。感染后每天观察小鼠状态,并在第0,3,7,11和14天采血进行荧光定量PCR检测和ELISA检测。实验结束后,对所有动物进行安乐死和剖检,对心脏、肝、脾、肺、肾、脑和淋巴结取样进行荧光定量PCR检测,并制片进行组织病理学检查。选择PCR检测阳性组织进行PCV3CAP基因测序分析。结果PCV3组织毒感染小鼠不引起明显的临床症状和病理变化。病毒可在感染早期的血清中检测到,病毒含量最高的器官是肝脏和脾脏,PCV3感染小鼠后核酸序列未发生变化。随着感染时间的增加血清中抗体水平逐渐升高。结论PCV3可以感染BALB/c小鼠,并在小鼠体内增殖。本研究结果为猪圆环病毒3型致病性的研究以及防控提供了参考。     

中枢神经系统隐球菌感染动物模型的研究

目的 构建中枢神经系统隐球菌感染的动物模型。方法 给予小鼠脑内接种隐球菌构建中枢神经系统隐球菌感染的动物模型。小鼠被随机地分为实验组和对照组,给予实验组小鼠脑内接种隐球菌菌悬液,对照组小鼠脑内接种生理盐水。结果 从组织病理方面观察到,实验组小鼠脑组织中的蛛网膜下腔、软脑膜表面、脑实质内、侧脑室脉络丛组织内均可见隐球菌菌体,脑膜轻度增生,侧脑室轻度扩大,脉络丛血管轻度扩张充血。对照组小鼠脑组织可见侧脑室轻度扩大,蛛网膜下腔血管、脑实质内血管、脉络丛血管均有轻度扩张充血,而蛛网膜下腔、软脑膜表面、脑实质内及侧脑室和室旁均未见隐球菌浸润。从组织病理观察结果两组具有一定的对比性。结论 小鼠脑内接种隐球菌构建其中枢神经系统隐球菌感染的模型,为研究人中枢神经系统隐球菌病提供了一个工具。     

小鼠接种百日咳鲍氏杆菌疫苗后产生对腺病毒感染的抵抗力

<正>百日咳鲍氏杆菌菌苗(BPV)具有多种免疫调节活性。从百日咳鲍氏杆菌(BP)提取的某些无菌体成分具有其中某些免疫调节活性。小鼠接种BPV或BP提取物后其病毒感染的发病机理发生改变。腹腔内接种BPV后5～7天的小鼠对鼻内流感病毒攻击的易感性增强。脑内狂犬病毒攻击的同时,经皮下、静脉或腹腔内接种BP提取物,则试验动物对该病毒攻击的抵抗力增强。腹腔     

森林脑炎病毒对鸡胚单层细胞的致病变作用及其实际应用

从人尸脑组织或蜱分离出的森林脑炎病毒株感染鸡胚皮肤肌单层细胞时,均可出现明显的细胞病变TCID50滴度与小白鼠脑内毒力LD50相一致,可达log 7.0—9.0。病毒如加有被确诊的森林脑炎病人恢复期血清或者免疫动物血清则不出现病变,证明细胞病变作用的特异性。病毒经鸡胚细胞传代后仍保持稳定的致病变作用。应用鸡胚细胞作血清中和试验与小鼠腹腔接种法中和试验测定的抗体结果相一致,而其敏感性和特异性都高于用小鼠法所测结果。本文对森林脑炎病毒在鸡胚细胞引起病变的条件和影响因素及其实际应用价值加以讨论。     

Intracerebral vaccination suppresses the spread of rabies virus in the mouse brain

To investigate the efficacy of intracerebral (IC) immunization in preventing viral spread in the brain, we immunized mice with inactivated rabies virus via the subcutaneous (SC) or IC route, followed by administration of a lethal dose of rabies virus (challenge virus standard strain), directly into the brains of immunized mice. Progressive paralytic neurological signs were observed in control and 75% of SC immunized mice, whereas only 20% of IC immunized mice exhibited symptoms. Neutralizing antibody titers in blood plasma were significantly elevated in SC and IC immunized mice, with the highest levels seen in IC immunized mice. Analysis of whole brain lysates revealed a strong induction of immunoglobulin in the brains of IC immunized mice that had virus neutralizing activity. Histopathological examination of brain tissue revealed mild encephalitis and disseminated viral antigen in control and SC immunized mice, but rare in IC immunized mice. These results suggest that IC immunization induces a preventive humoral immune response against intracerebrally inoculated rabies virus. Induction of neutralizing antibody in cerebrospinal fluid represents a putative therapeutic measure for the treatment of rabid animals and humans.     

Collaboration of Antibody and Inflammation in Clearance of Rabies Virus from the Central Nervous System

To investigate the involvement of various cellular and humoral aspects of immunity in the clearance of rabies virus from the central nervous system, (CNS), we studied the development of clinical signs and virus clearance from the CNS in knockout mice lacking either B and T cells, CD8⁺ cytotoxic T cells, B cells, alpha/beta interferon (IFN-α/β) receptors, IFN-γ receptors, or complement components C3 and C4. Following intranasal infection with the attenuated rabies virus CVS-F3, normal adult mice of different genetic backgrounds developed a transient disease characterized by loss of body weight and appetite depression which peaked at 13 days postinfection (p.i.). While these animals had completely recovered by day 21 p.i., mice lacking either B and T cells or B cells alone developed a progressive disease and succumbed to infection. Mice lacking either CD8⁺ T cells, IFN receptors, or complement components C3 and C4 showed no significant differences in the development of clinical signs by comparison with intact counterparts having the same genetic background. However, while infectious virus and viral RNA could be detected in normal control mice only until day 8 p.i., in all of the gene knockout mice studied except those lacking C3 and C4, virus infection persisted through day 21 p.i. Analysis of rabies virus-specific antibody production together with histological assessment of brain inflammation in infected animals revealed that clearance of CVS-F3 by 21 days p.i. correlated with both a strong inflammatory response in the CNS early in the infection (day 8 p.i.), and the rapid (day 10 p.i.) production of significant levels of virus-neutralizing antibody (VNA). These studies confirm that rabies VNA is an absolute requirement for clearance of an established rabies virus infection. However, for the latter to occur in a timely fashion, collaboration between VNA and inflammatory mechanisms is necessary.     

Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.     

Rabies diagnosis for developing countries

Background

Canine rabies is a neglected disease causing 55,000 human deaths worldwide per year, and 99% of all cases are transmitted by dog bites. In N''Djaména, the capital of Chad, rabies is endemic with an incidence of 1.71/1,000 dogs (95% C.I. 1.45–1.98). The gold standard of rabies diagnosis is the direct immunofluorescent antibody (DFA) test, requiring a fluorescent microscope. The Centers for Disease Control and Prevention (CDC, Atlanta, United States of America) developed a histochemical test using low-cost light microscopy, the direct rapid immunohistochemical test (dRIT).

Methodology/Principal Findings

We evaluated the dRIT in the Chadian National Veterinary Laboratory in N''Djaména by testing 35 fresh samples parallel with both the DFA and dRIT. Additional retests (n = 68 in Chad, n = 74 at CDC) by DFA and dRIT of stored samples enhanced the power of the evaluation. All samples were from dogs, cats, and in one case from a bat. The dRIT performed very well compared to DFA. We found a 100% agreement of the dRIT and DFA in fresh samples (n = 35). Results of retesting at CDC and in Chad depended on the condition of samples. When the sample was in good condition (fresh brain tissue), we found simple Cohen''s kappa coefficient related to the DFA diagnostic results in fresh tissue of 0.87 (95% C.I. 0.63–1) up to 1. For poor quality samples, the kappa values were between 0.13 (95% C.I. −0.15–0.40) and 0.48 (95% C.I. 0.14–0.82). For samples stored in glycerol, dRIT results were more likely to agree with DFA testing in fresh samples than the DFA retesting.

Conclusion/Significance

The dRIT is as reliable a diagnostic method as the gold standard (DFA) for fresh samples. It has an advantage of requiring only light microscopy, which is 10 times less expensive than a fluorescence microscope. Reduced cost suggests high potential for making rabies diagnosis available in other cities and rural areas of Africa for large populations for which a capacity for diagnosis will contribute to rabies control.     

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth Curve and Distribution of Rous Sarcoma Virus (Bryan) in Japanese Quail

On primary infection with the Bryan strain of Rous sarcoma virus (RSV), the growth curve of the virus in the brain of Japanese quail was similar to that observed in chicks and turkey poults. Infectious virus disappeared from the brain after inoculation. After an eclipse period during which no virus was detectable, infectious virus began to appear at 2 days and reached maximal titers in the brain samples at 7 days after inoculation. When Japanese quail were infected intracerebrally with RSV, relatively high titers of virus were recovered from brain tissue but not from liver, lung, kidney, or blood of moribund birds. Only tumors produced in the wing web of quail infected subcutaneously yielded high titers of virus. Other tissues yielded no virus, even though wing web tumors appeared as early as in chicks similarly infected. RSV could be propagated in the wing web of quail for at least 14 passages without any loss of infectivity. On the other hand, serial passage in quail brain resulted in a progressive loss of infectivity until virus was completely lost.     

Comparison of Biotinylated Monoclonal and Polyclonal Antibodies in an Evaluation of a Direct Rapid Immunohistochemical Test for the Routine Diagnosis of Rabies in Southern Africa

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.     

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.     

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.